WO2018172540A1

WO2018172540A1 - Method to predict the progression of alzheimer's disease

Info

Publication number: WO2018172540A1
Application number: PCT/EP2018/057511
Authority: WO
Inventors: Carole ELBIM; Yuan Dong; Guillaume Dorothee; Marie SARAZIN
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Sorbonne Université; Université Paris-Sud; Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives (Cea); Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2017-03-24
Filing date: 2018-03-23
Publication date: 2018-09-27

Abstract

The present invention relates to Alzheimer's and method to determine its progression and its severity. The inventors managed a study to phenotypically and functionally characterize human circulating PMNs in whole-blood at different disease stages in Alzheimer's patients. They also investigated associations between these indicators and rate of disease progression. They also showed (by luminex assays) that some proteins and particularly some cytokines could be used to predicting the severity of Alzheimer's disease. Thus, the present invention relates to methods for predicting the rapidity of progression of Alzheimer's or the severity of the disease in a patient by determining the activation state of neutrophils or the level of expression of different proteins (particularly cytokines).

Description

METHOD TO PREDICT THE PROGRESSION OF ALZHEIMER'S DISEASE

FIELD OF THE INVENTION:

The present invention relates to a method for predicting the rapidity of progression of

Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the neutrophils are not hyperactivated as compared to the predetermined reference.

BACKGROUND OF THE INVENTION:

Alzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by a triad of pathological features: extracellular amyloid deposits predominantly composed of amyloid-β (Αβ) peptides, intracellular neurofibrillary tangles chiefly comprised of abnormally folded tau protein, and gliosis consisting of reactive microglia and astrocytes surrounding β-amyloid plaques. Significant variability and overlap exists in the extent of amyloid-β and Tau pathology in AD and non-demented populations and it is clear that other factors must influence progression of cognitive decline, perhaps independent of effects on amyloid pathology. There are now many clinical studies indicating that infections and systemic inflammation are associated with clinical AD [Holmes, 2009]. Importantly, the impact of acute inflammatory events on cognitive decline has also been prospectively verified in AD patients, demonstrating that carer-reported acute systemic inflammatory events accelerate cognitive decline [Holmes, 2009; Cunningham, 2015]. Finally, emerging evidence supports the concept that microglia respond to pathological changes in the CNS by becoming primed and that systemic inflammation leads to exaggerated responses of primed microglia, contributing to disease progression in several pathological conditions, including neurodegenerative diseases [Perry, 2014].

Polymorphonuclear neutrophils (PMN) are the most abundant circulating leukocytes and are key components of the early innate response. PMN act as first line of host defense and kill pathogens through various strategies, including phagocytosis, degranulation, rapid production of reactive oxygen species (ROS) in oxidative burst, and release of neutrophil extracellular traps (NETs), a process called NETosis [Nauseef, 2014]. PMNs are also important - - mediators of inflammation-induced injury and their improper activation may lead to an oxidative stress and exaggerated inflammatory reactions [Nemeth, 2012; Nathan, 2006]. In addition, increasing evidence over the last decade has demonstrated an unexpected phenotypic heterogeneity and functional versatility of the neutrophil population. In particular, subsets of PMNs differ markedly in their proinflammatory activity. Senescent PMNs express elevated levels of CXCR4 and represent an overly active subset [Zhang, 2015]. Conversely, an immunosuppressive PMN subset CD16^bnght/CD62L^dim exhibit decreased in pro-inflammatory activity [Kamp, 2012; Pillay, 2012; Sauce, 2017].

Of note, it has been recently reported in transgenic models of AD that PMN depletion reduced AD-like neuropathology and improved memory in mice already showing cognitive dysfunction. These results strongly highlight the contribution of PMNs to AD pathogenesis and cognitive impairment [Zenaro, 2015]. Otherwise, PMNs are exceptionally sensitive to external stimuli such as pro-inflammatory cytokines i.e. TNFa, the circulating levels of which have been reported to be increased in AD [Holmes, 2011]. Increased activation of circulating PMNs might amplify the systemic inflammatory process by an excessive and inappropriate production of toxic mediators such as damaging ROS and intravascular NETs [Pun, 2009]. However, the few published studies on circulating PMN functions in AD patients yielded conflicting results. In fact, some authors reported normal PMN oxidative burst [Song, 1999; Vitte, 2004] and normal phagocytosis [Song, 1999] in AD patients while others demonstrated an increased degranulation [Scali, 2002] and ROS production [Licastro, 1994] or a decreased phagocytosis [Davy do va, 2003]. Importantly, an issue raised by many of these studies is the analysis of PMN isolated from their blood environment through various procedures that may differently modulate cell responses [Rebecchi, 2000; Pallister, 2006]. In addition, the modulation in AD patients of PMN NETosis as well as of the distribution of the senescent and immunosuppressive PMN subsets has never been investigated.

The aim of the present study was to phenotypically and functionally characterize human circulating PMNs at different disease stages in AD. This study was performed in whole-blood conditions in order to minimize activation due to isolation procedures. This presents the advantage of taking into account the proinflammatory cytokine environment. We also investigated associations between these indicators and rate of disease progression. This investigation was justified, since link between consequences of PMN activation and the rapidity of AD progression has never been elucidated. Indeed the only known information concerning the rate of disease progression was presented by Scali and colleagues and was restricted to the token test, a verbal comprehension test. In addition, Scali et al analyzed only one parameter of PMN activation (CD l ib expression) and did not explore PMN homeostasis. Thus, understanding the impact of PMN phenotype on the rate of cognitive decline may constitute an innovative prognostic blood biomarkers in patients with AD. SUMMARY OF THE INVENTION:

The aim of the present study was to phenotypically and functionally characterize human circulating PMNs at different disease stages in AD. This study was performed in whole-blood conditions in order to minimize activation due to isolation procedures. This presents the advantage of taking into account the proinflammatory cytokine environment. The inventors also investigated associations between these indicators and rate of disease progression.

Thus, the present invention relates to a method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the neutrophils are not hyperactivated as compared to the predetermined reference. Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

A first aspect of the invention relates to a method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the neutrophils are not hyperactivated as compared to the predetermined reference.

As used herein, the term "rapidity of progression of Alzheimer's disease (AD)" allows to discriminate AD patients into slow decliners (SD) or fast decliners (FD) based on the progression of the Clinical Dementia Rating (CDR) score after two years and/or the progression of the Mini-Mental State Examination (MMSE) score after one or two years. SD Patients with Alzheimer's disease remain stable, showing unchanged CDR score, while patients with Alzheimer's disease declin (FD), with an increase of 0.5 or more of the CDR score (see for example Solomon PR et al, 1998). - -

In a particular embodiment, the neutrophils are the polymorphonuclear neutrophils (PMN).

Thus, in a particular embodiment, the invention relates to a method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of polymorphonuclear neutrophils ii) comparing the activation state of the polymorphonuclear neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the polymorphonuclear neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the polymorphonuclear neutrophils are not hyperactivated as compared to the predetermined reference.

As used herein, the term "hyperactivated neutrophils" or "hyperactivated polymorphonuclear neutrophils" denote a state of activation of these cells characterised by different parameters like an increased oxidative stress comprising an increased constitutive ROS (free radicals) production by resting PMNs and an increased capacity of PMNs to produce ROS in response to various stimuli), a decrease in L-selectin (CD62L) or FCyRIIIB (CD 16b) expression on resting PMN, an increased degranulation characterized by an higher CD1 lb and CD1 lc expression on resting PMNs or an increased percentage of apoptotic and necrotic PMNs as well as a decreased PMN survival in response to stimuli such as TNFa or TLR agonists.

According to the invention, the measurement of the oxidative stress is realized as described in the Materials & Methods parts and for example in the article Bass DA et al 2016. The result of the oxydative stress will be expressed as mean fluorescence intensity (MFI) of Ethidium which is directly correlated to the amount of reactive oxygen species produced by PMNs.

According to the invention, the measurement of the decrease in L-selectin (CD62L) or FCyRIIIB (CD 16b) expression on resting PMN is realized as described in the Materials & Methods parts and for example in the article Campillo-Gimenez L et al, 2014. The result will be expressed as mean fluorescence intensity (MFI) which correlated with the number of molecules of CD62L or CD 16b per PMN.

According to the invention, the measurement of the increased degranulation is realized as described in the Materials & Methods parts and for example in the article Campillo-Gimenez L et al, 2014. The result will be expressed as mean fluorescence intensity (MFI) which correlated with the number of molecules of CD1 lb or CD1 lc per PMN.

According to the invention, the measurement of the increased percentage of apoptotic and necrotic PMNs is realized as described in the Materials & Methods parts and for example in the article Francois S. et al 2005.The results will be expressed as the percentage of apoptotic and necrotic PMNs among the total population of PMNs.

According to the invention, the measurement of the decreased PMN survival is realized as described in the Materials & Methods parts and for example in the article Francois S. et al 2005. The results will be expressed as the percentage inhibition of PMN apoptosis.

For the different measurements described above, the measured values will be interpreted by comparison with the reference interval (Rl ) defined as the central 95% of a reference population. The reference population includes age-matched well-defined healthy individuals (reference individuals) (see for example J. Graham and A. Barker. Reference intervals. Clin Biochem Rev 2008 Aug; 29(Suppl 1): S93-S97). Exclusion criteria in the reference population included acute or chronic inflammatory conditions, and corticoid or non-steroidal antiinflammatory drugs, known to modulate PMN phenotype and functions (see for example Campillo-Gimenez L et al, 2014).

For example, the values obtained in the reference population the different measurements are shown below in the table 1 :

Table 1: reference intervals and mean for each parameters used to evaluate the activation status of neutrophils.

Thus, according to the invention, at least one parameter indicated above can be used alone or combined with the others to determine the activation state of neutrophils. - -

As used herein and according to all aspects of the invention, the term "sample" denotes, blood, peripheral-blood, serum, plasma, urine or Cerebrospinal fluid (CSF).

As used herein, the term "patient" refers to an individual with symptoms of Alzheimer's disease. Patients with Alzheimer's disease are characterized by the following criteria: (i) progressive episodic memory impairment, characterized by a low free recall not normalized with semantic cueing [Sarazin et al., 2007 and Dubois et al., 2010]; (ii) absence of extrapyramidal signs; and (iii) CSF Alzheimer's disease profile, when available, defined as score 50.8, calculated with the formula amyloid- 42/[240 + (1.18 x T-tau)] [De Souza et al.,

2011]. In another embodiment, the invention relates to a method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive PMN subsets ii) comparing the ratio between the senescent and the immunosuppressive PMN subsets at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the ratio between the senescent and the immunosuppressive PMN subsets is higher than its predetermined reference or predicting a slow progression when the ratio between the senescent and the immunosuppressive PMN subsets did not differ from its predetermined reference.

As used herein, the term "senescent PMN subset" denote aged or senescent CXCR4^hlghPMNs and are characterized by lower expression of CD62L, increased expression of β2 integrins, as well as increased production of ROS and susceptibility of NET formation compared to the total circulating pool (see for example Zhang D. et al, 2015).

As used herein, the term "immunosuppressive PMN subset" denote CD16^bright/CD62L^dim PMNs, showing decreased adhesion properties and ROS production (see for example Pillay J. et al, 2012).

According to the invention, the measurement of the ratio between the senescent and the immunosuppressive PMN subsets is realized as described in the Materials & Methods parts and for example in the article Sauce D. et al, 2017.

The measured values will be interpreted by comparison with the reference interval (RI) defined as the central 95% of a reference population. The reference population includes age- matched well-defined healthy individuals (reference individuals). Exclusion criteria in the - - reference population included acute or chronic inflammatory conditions, and corticoid or nonsteroidal anti-inflammatory drugs, known to modulate PMN phenotype and functions (see for example Campillo-Gimenez L et al, 2014) (see table 2).

Table 2: reference intervals and mean for the ratio between the senescent and the immunosuppressive PMN.

The inventors also show that the activation state of the neutrophils (or polymorphonuclear neutrophils) could be used to determine the severity of Alzheimer's disease that is to say if the patient has an Alzheimer's disease with dementia or not.

Thus, in another embodiment, the invention relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the expression level determined at step i) is higher than its predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower than its predetermined reference value.

In another embodiment, the invention relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive PMN subsets ii) comparing the ratio between the senescent and the immunosuppressive PMN subsets at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the ratio determined at step i) is higher than its predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the ratio determined at step i) is lower than its predetermined reference value. - -

In another embodiment, the invention relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the level of circulating intravascular NETs ii) comparing the level of circulating intravascular NETs at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the level determined at step i) is higher than its predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the level determined at step i) is lower than its predetermined reference value.

According to the invention, the parameters described above (activation status of neutrophils, the ratio between the senescent and the immunosuppressive PMN subsets and the level of circulating intravascular NETs) can be used to stratify patients suffering from Alzheimer's disease. In function of the expression level of these biomarkers, the patients can be stratified as: Alzheimer's disease with mild cognitive impairment also called prodromal Alzheimer's disease or Alzheimer's disease with dementia.

The inventors also showed that some proteins and particularly some cytokines could be used for predicting the severity of Alzheimer's disease.

In another embodiment, the invention relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the expression level of MMP-9 and the expression level of TIMP-1 ii) comparing the ratio determined at step i) with its predetermined reference value and iii) determining that the patient has a severe Alzheimer's disease when the ratio determined at step i) is higher than its predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower than its predetermined reference value.

In another embodiment, the invention relates to a method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the expression level of at least one protein selected in the group consisting in IL-Ιβ, IL-6, TNF- , IL-18, IL-8, IL17, IL-22 and IL10 ii) comparing the expression level determined at step i) with their predetermined reference values and iii) determining that the patient has a severe Alzheimer's disease when the expression level determined at step i) is higher than their predetermined reference values for at least one of the proteins IL-Ιβ, IL-6, TNF-a, IL-8 and IL17 or when the expression level determined at step i) is lower than their predetermined reference values for at least one of the proteins IL-18, IL-22 and IL10 or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower than their predetermined reference values for at least one of the proteins IL-Ιβ, IL-6, - -

TNF-α, IL-8 and IL17 or when the expression level determined at step i) is higher than their predetermined reference values for at least one of the proteins IL-18 IL-22 and IL10.

As used herein, the term "severe Alzheimer's disease" denotes that the patient suffer from an Alzheimer's disease with dementia. A "not severe Alzheimer's disease is characterised by an Alzheimer's disease with mild cognitive impairment also called prodromal Alzheimer's disease.

As used herein the term "MMP-9" for "Matrix metallopeptidase 9" also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB), has its general meaning in the art and denotes a matrixin, a class of enzymes that belong to the zinc-metalloproteinases family involved in the degradation of the extracellular matrix. In humans the MMP9 gene encodes for a signal peptide, a propeptide, a catalytic domain with inserted three repeats of fibronectin type II domain followed by a C-terminal hemopexin-like domain. An exemplary sequence for human MMP-9 protein is deposited in the UniProt database under accession numbers P14780.

As used herein the term "TIMP-1" for "tissue inhibitors to the metalloproteinases-1 » denote a factor that forms complexes 1 : 1 with active MMP-9 and the MMP-9/TIMP-1 balance reflects the net proteolytic activity present in several physiological processes. An exemplary sequence for human TIMP-1 protein is deposited in the UniProt database under accession numbers P01033.

As used herein, the term "II- 1β" for "Interleukin 1 beta" also known as leukocytic pyrogen, leukocytic endogenous mediator, mononuclear cell factor, lymphocyte activating factor has its general meaning in the art and denotes a cytokine protein that in humans is encoded by the IL1B gene. There are two genes for interleukin- 1 (IL-1): IL-1 alpha and IL-1 beta (this gene). IL-Ιβ precursor is cleaved by cytosolic caspase 1 (interleukin 1 beta convertase) to form mature IL- 1 β. An exemplary sequence for human IL- 1 β protein is deposited in the UniProt database under accession numbers P01584.

As used herein, the term "IL-6" for "Interleukin 6" has its general meaning in the art denotes an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. Interleukin 6 is secreted by T cells and macrophages to stimulate immune response, e.g. during infection and after trauma, especially burns or other tissue damage leading to inflammation. IL-6 also plays a role in fighting infection, as IL-6 has been shown in mice to be required for resistance against bacterium Streptococcus pneumoniae. An exemplary sequence for human IL- 1 β protein is deposited in the UniProt database under accession numbers P05231. - -

As used herein, the term "TNF-a" for "Tumor necrosis factor" has its general meaning in the art and denotes a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. An exemplary sequence for human TNF-a protein is deposited in the UniProt database under accession numbers P01375.

As used herein, the term "IL-18" for "Interleukin-18", also known as interferon-gamma inducing factor, has its general meaning in the art and denotes a protein which in humans is encoded by the IL18 gene. The protein encoded by this gene is a proinflammatory cytokine. An exemplary sequence for human IL-18 protein is deposited in the UniProt database under accession numbers Q 14116.

As used herein, the term "IL-8" for "Interleukin 8" (has its general meaning in the art and denotes a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells and endothelial cells. Endothelial cells store IL-8 in their storage vesicles, the Weibel-Palade bodies. In humans, the interleukin-8 protein is encoded by the IL8 gene. IL-8 is initially produced as a precursor peptide of 99 amino acids long which then undergoes cleavage to create several active IL-8 isoforms. In culture, a 72 amino acid peptide is the major form secreted by macrophages. An exemplary sequence for human IL-8 protein is deposited in the UniProt database under accession numbers P10145.

As used herein, the term "IL-17" for "Interleukin 17" has its general meaning in the art and denotes the founding member of a group of cytokines called the IL-17 family. Known as CTLA8 in rodents, IL-17 shows high homology to viral IL-17 encoded by an open reading frame of the T-lymphotropic rhadinovirus Herpesvirus saimiri. According the invention, the IL-17 used is the IL-17 A. An exemplary sequence for human IL-17A protein is deposited in the UniProt database under accession numbers Q 16552.

As used herein, the term "IL-22" for "Interleukin-22" has its general meaning in the art and denotes a member of a group of cytokines called the IL-10 family or IL-10 superfamily (including IL-19, IL-20, IL-24, and IL-26), a class of potent mediators of cellular inflammatory responses. An exemplary sequence for human 11-22 protein is deposited in the UniProt database under accession numbers Q9GZX6.

As used herein, the term "IL-10" for "Interleukin 10" has its general meaning in the art and denotes an anti-inflammatory cytokine. In humans, interleukin 10 is encoded by the IL10 gene. IL-10 signals through a receptor complex consisting of two IL-10 receptor- 1 and two IL- 10 receptor 2 proteins. Consequently, the functional receptor consists of four IL-10 receptor - - molecules. IL-10 binding induces STAT3 signalling via the phosphorylation of the cytoplasmic tails of IL-10 receptor 1 + IL-10 receptor 2 by JAKl and Tyk2 respectively. An exemplary sequence for human IL- 10 protein is deposited in the UniProt database under accession numbers P22301.

According to the invention, the biomarkers described above can be used to stratify patients suffering from Alzheimer's disease. In function of the expression level of these biomarkers, the patients can be stratified as: Alzheimer's disease with mild cognitive impairment also called prodromal Alzheimer's disease or Alzheimer's disease with dementia.

Particularly, the ratio MMP-9/TIMP-1 and the cytokines IL17 and IL18 can be used to stratify patients suffering from Alzheimer's disease.

Measuring the expression level of the proteins of the invention (MMP-9, TIMP-1, IL- 1β, IL-6, TNF-a, IL-18, IL-8, IL17, IL-22 and IL10, defined as "the proteins of the invention" below) can be done by measuring the gene expression level of these proteins or by measuring the level of the proteins and can be performed by a variety of techniques well known in the art.

Typically, the expression level of a gene may be determined by determining the quantity of mR A in peripheral leucocytes populations. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell e¥ tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable - - label). A "detectable label" is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- - -

4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarninofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfiuorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap - - of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can be coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20132016, 1998; Chan et al, Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (puhlished May 27, 1999). Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors hased on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif).

Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.

Detectable labels that can be used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

Alternatively, an enzyme can be used in a metallographic detection scheme. For example, silver in situ hyhridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along - - with a water-soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.

For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al, Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al, Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al, Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929. - -

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.

In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in - - this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling - - may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the stage of the patient, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.

According to the invention, the level of the proteins of the invention may also be measured and can be performed by a variety of techniques well known in the art.

Typically protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.

Detection of protein concentration in the sample may also be performed by measuring the level of the proteins of the invention. In the present application, the "level of the proteins" or the "proteins level expression" means the quantity or concentration of said proteins. In another embodiment, the "level of the proteins" means the level of fragments of the proteins. In still another embodiment, the "level of the proteins" means the quantitative measurement of the proteins expression relative to a negative control.

Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS). etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. - -

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.

Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of the proteins of the invention" refers to an amount or a concentration of a transcription product, for instance the proteins. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the proteins. Thus, in a particular embodiment, fragment of the proteins may also be measured.

In a particular embodiment, the detection of the level of the proteins of the invention can be performed by flow cytometry.

In a particular embodiment, the detection of the level of the proteins of the invention can be performed by luminex assays.

In a particular embodiment, the detection of the level of the proteins of the invention can be performed by MesoScale Discovery assays.

In a particular embodiment, the detection of the level of the proteins of the invention can be performed by nano-ELISA assays, or other ultrasensitive single molecule immunoassays, including Simoa technology.

Predetermined reference values used for comparison for the proteins of the invention may comprise "cut-off or "threshold" values that may be determined as described herein. Each - - reference ("cut-off) value for the proteins expression of the invention may be predetermined by carrying out a method comprising the steps of

a) providing a collection of samples from patients suffering of Alzheimer's disease; b) determining the expression level of at least one of the proteins of the invention (the protein itself or the gene) for each sample contained in the collection provided at step a); c) ranking the tissue samples according to said expression level;

d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level;

e) providing, for each sample provided at step a), information relating to the actual clinical status for the corresponding patient (severe AD or not);

f) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets

g) selecting as reference value for the expression level, the value of expression level for which the p value is the smallest.

The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.

In routine work, the reference value (cut-off value) may be used in the present method to discriminate AD samples and therefore the corresponding patients.

The man skilled in the art also understands that the same technique of assessment of the expression level of a gene or the protein itself should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a gene or the protein itself of a patient subjected to the method of the invention.

Such predetermined reference values of expression level may be determined for any gene defined above.

A further object of the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of the proteins of the invention in the sample obtained from the patient.

The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise - - hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. Alternatively the kit of the invention may comprise amplification primers that may be pre- labelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.

A second aspect of the invention relates to a method for monitoring the efficacy of a treatment of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the treatment is effective when the neutrophils are not hyperactivated as compared to the its predetermined reference or predicting that the treatment is not effective when the neutrophils are hyperactivated as compared to the its predetermined reference.

In another embodiment, the invention relates to a method for monitoring the efficacy of a treatment of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive PMN subsets ii) comparing the ratio between the senescent and the immunosuppressive PMN subsets at step i) with its predetermined reference and iii) predicting that the treatment is not effective when the ratio between the senescent and the immunosuppressive PMN subsets is higher than its predetermined reference or predicting that the treatment is effective when the ratio between the senescent and the immunosuppressive PMN subsets did not differ from its predetermined reference.

Compounds used or foreseen for treating to treat Alzheimer's disease are described below.

Another aspect of the invention relates to a method for stratifying patients as fast or slow progressor or as having a severe or not a severe Alzheimer's disease for designing a clinical trials to test new compound for the treatment of Alzheimer's disease by using the activation status of the neutrophils as determined in the invention and/or the biomarkers of the invention (the proteins of the invention).

A third aspect of the invention relates to a compound that reduces the presence or the activity of neutrophils in blood and/or in brain for use in the treatment of Alzheimer's disease in a patient with a rapid progression and/or a severe disease as described above.

As used herein, a "compound that reduces the presence or activity of neutrophils in blood and/or in brain" modulates or interferes with at least one pathway of neutrophil trafficking - - or activation and is well described in the patent application WO 2015051152. For example, depending on the specific pathway, said compound may be an inhibitor, blocking or depleting agent, e.g. an antibody that depletes a targeted cell population, an agonist, or an antagonist of one or more of these pathways. For example said compound may block the activity of a protein that acts in one or more of these pathways, e.g. a chemoattractant, a protein tyrosine kinase, an adhesion molecule, etc.

Pathways of interest for intervention by the methods of the invention include (i) depletion of neutrophil/myeloid cell populations systemically or locally in the brain; (ii) blocking neutrophils/myeloid cell adhesion and crawling; (iii) blocking transmigration and infiltration of neutrophils/myeloid cells into the brain; (iv) blocking cell-cell interactions between neutrophil/myeloid cells and endothelial cells and/or neural cells; (v) blocking neutrophil/myeloid cell extracellular-matrix interactions; (vi) reducing motility of neutrophils/myeloid cells in the brain parenchyma; (vii) blocking Αβ-induced activation and adhesion of neutrophils/myeloid cells; (viii) blocking intracellular signalling controlling adhesion and activation; (ix) blocking neutrophil activation and/or degranulation; (x) blocking release of reactive oxygen species, proteases, cytokines, lipid mediators or other damaging agents from myeloid cells and/or neutrophils; (xi) blocking neutrophil/myeloid cell activation leading to increased affinity and valency resulting from clustering of integrin receptors that increases binding; (xii) blocking formation of neutrophil extracellular traps (NETs) in brain vessels or parenchyma and/or in peripheral blood; (xiii) blocking neurodegenerative processes including synaptic dysfunction and/or degradation; (xiv) reducing activation and/or number of microglial cells.

As used herein, compound that targets adhesion molecules involved in neutrophils trafficking or extravasation, including but not limited to: integrins and their ligands, e.g. ICAM- 1, LFA-1, CDl la, CDl lb, CDl lc, CD18, alpha-4 integrins and their ligands VCAM-1 and MAdCAM-1; CD49; E-, P- and L- selectin and their ligands, e.g. including but not limited to PSGL-1 , CD44, CD43, hyaluronan, glycolipids,

In one embodiment, the compound inhibits the interaction between an adhesion molecule involved in neutrophils trafficking to the brain, and its ligand.

As used herein, compound targeting protein tyrosine kinases included but not limited to, Syk, Abl, JAK3, Jak2, and BTK and MAPK; and/or PI3K.

In another embodiment, further therapeutic active agent can be administered to the patient. This active agent can be a cholinesterase inhibitor, the memantine, a N-methyl D- aspartate antagonist, a BACE1 inhibitor, a γ secretase inhibitor, passive anti-Αβ - - immunotherapy in the form of monoclonal anti-Αβ antibodies, active anti-Αβ immunotherapy, non-steroidal anti-inflammatory drugs, drugs enhancing Αβ clearance, drugs inhibiting Αβ aggregation, drugs reducing Tau phosphorylation, drugs inhibiting Tau aggregation, passive anti-Tau immunotherapy in the form of monoclonal anti-Tau antibodies, active anti-Tau immunotherapy, and APOE-related treatment approaches.

All these compounds or active agents can be used alone or in combination.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from the disease or medical condition. The treatment may be administered to a subject having the medical disorder or who ultimately may acquire the disorder.. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of the disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.

A fourth aspect of the invention relates to a therapeutic composition comprising compound that reduces the presence or activity of neutrophils in blood and/or in brain according to the invention for use in the treatment of Alzheimer's disease in a patient with a rapid progression and/or a severe disease as described above.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. - -

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intrathecal or subcutaneous administration and the like.

Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

HC AD MCI AD dementia

(ii=22) (11=16) (n=26)

Age (mean) [min max] 71.6 [60-88] 71.0 [53-84] 66.6 [52-83]

Female Sex (n) [%] 16 [72.7] 8 [50.0] 18 [69.2]

MMSE (mean)[miii-max] 28.7 [27-30] 26.2 [12-30]° 17.3 [5-27]°*

C DR CDR = 0 CDR = 0.5 CDR>0.5

Carrier of APOE ε4 (n) {%] 2 [9.1] 10 [62.5]° 14 [53.8]°

(ll)C-PiB PET scan (tneaii)[miu-

1.53 [1.13-3.23] 2.65 [1.26-3.95]° 3.05 [1.51-4.69]° inax]

^1SF DPA 714 (mean)[min-max] 1.29 [1.02-1.82] 1.42 [1.13-1.87] 1.32 [1.03-2.03]

Cholinesterase inhibitors (u) [%] - 14 [87.5] 26 [100]

Memantlne (n) [%] __ 2 [12.5] 3 [11.5] - -

Table 3. Characteristics of patients with Alzheimer's disease and healthy subjects participating in the study. AD-MCI = prodromal Alzheimer's disease group; AD-D = Alzheimer's disease dementia group; CDR, Clinical Dementia Rating; MMSE, Mini-Mental State Examination; PiB, Pittsburgh compound B; n, number. °p<0.05 compared to healthy controls (HC). *p<0.05 compared to AD MCI. Statistical comparison of female sex, carrier of APOE ε4, subjects with CRP> 3 μg/mL and type of treatment was performed with the Chi2 test.

Figure 1. Circulating PMNs from AD patients are highly activated and produced increased levels of ROS.

(A) Surface expression of CD62L, CD 16b, CD l ib, and CDl lc on resting PMN was studied on whole blood samples maintained at 4°C stained with specific mAbs. Results are expressed as mean fluorescence intensity (MFI). (B, C) ROS production by PMNs was studied by using DHE oxidation. (B) ROS production by un-stimulated PMNs was studied after treatment of whole-blood samples for 50 minutes with PBS; results are expressed as MFI. (C) ROS production by stimulated PMNs was measured after pretreatment of whole-blood samples for 45 minutes with PBS, Pani3CSK₄ (TLR1/2 agonist, 1 μg/mL), or LPS (TLR4 agonist, 10 ng/ml), or TNF-a (TNF, 5 ng/ml) and incubation for 5 minutes with fMLP (10^"6M); results are expressed as MFI. (D) Quantification of circulating NETs was performed by MPO-DNA complex ELISA; the mean optical density was measured by capture ELISA and results are expressed as ng/μΐ DNA.

All the measurement were performed in healthy controls (HC, n=22) and AD patients (n=42). Values are means ± SEM. Statistical significance as determined by the nonparametric Mann-Whitney test is indicated. ^*Significantly different from HC p<0.05, **p<0.01, ***p<0.001. The two-way ANOVA test was used to determine whether differences observed were significant between the different conditions used for ROS production measurement (treatment with PBS or the various stimuli) for each group (controls and AD patients). For each parameter, samples stimulated with the different agonists were significantly different from sample incubated with PBS p< 0.001 (not shown).

Figure 2. Defective survival, migration and phagocytosis of PMNs from AD patients.

(A, B, C) PMN death was measured by staining with annexin V and 7-AAD (A, B) Spontaneous PMN death was measured immediately after sampling (TOh) and after 20h incubation at 37°C (T20h) by staining with annexin V and 7-AAD. Results are expressed as the percentages of apoptotic (A) and necrotic (B) PMNs. (C) Prolongation of PMN survival was - - analyzed by incubating whole-blood samples with PBS, or TLR1/2 agonist, or TLR4 agonist for 20 hours at 37°C. Results are expressed as the percentage inhibition of PMN apoptosis [1 - (% of total annexin V⁺/7-AAD^"PMN in TLR agonist-treated sample/% of total annexin V⁺/7- AAD PMN in PBS-treated sample)] X 100. (D) CD62L and (E) CD l ib expression on PMN surface were measured after incubation of whole-blood samples for 45 minutes with PBS, or TLR1/2 agonist, or TLR4 agonist, or TNF-a; results are expressed as MFI. (F) PMN migration was measured in Transwell plates; the lower wells were treated with PBS-BSA (1%) or IL-8 (25 ng/ml) or fMLP (10^~7 M) for 3h at 37°C. The results are expressed as the migration rate.

(G) Phagocytosis of PMNs was measured after incubation of whole blood samples with fluorescent opsonized E. coli bacteria for 10 minutes in a water bath at 37°C. Results are expressed as the PMN phagocytic index.

All measurements were performed in HC (n=22) and AD patients (n=42). Values are means ± SEM. Statistical significance as determined by the nonparametric Mann- Whitney test is indicated. "Significantly different from controls p<0.01 , * * *p<0.001. The two-way ANOVA test was used to determine whether differences were significant between the different conditions

(treatment with PBS or the various stimuli) for each group (controls and AD patients). For each parameter, samples stimulated with the different agonists were significantly different from sample incubated with PBS p< 0.001 (not shown).

Figure 3. PMNs from the AD prodromal group and the AD dementia group exhibit differential pro-inflammatory properties in relation with alterations of circulating neutrophils homeostasis.

ROS production by unstimulated (A) and stimulated PMNs (B) was studied by HE oxidation; results are expressed in MFI. (C) Quantification of circulating NETs was performed by MPO-DNA complex ELISA; results are expressed as ng/μΐ DNA. (D) Whole-blood samples were incubated for 45 minutes at 4°C with Pe-Cy7-anti-human CXCR4, PE-anti-human CD l ib, and APC-anti-human CD62L (BD Biosciences). Results are expressed as the percentages of the CXCR4^brigh7CD62L^dim senescent PMN subset. (E) Whole-blood samples were incubated for 45 minutes at 4°C with FITC-anti-human CD 16, PE-anti-human CD 11c, Pe-Cy7-anti-human CD l ib, and APC-anti-human CD62L (BD). Results are expressed as the percentages of the CD16^bright/CD62L^dim immunosuppressive PMN subset. (F) Ratio between the senescent and the immunosuppressive subsets. (G) Correlation between the percentage of the CXCR4^bright/CD62L^dim PMN subset and ROS production by TLR4-agonist-primed PMNs.

(H) Correlation between the percentage of the CD16^bright/CD62L^dim PMN subset and ROS production by TLR4-agonist-primed PMNs. - -

All measurements were made in controls (n=22), AD prodromal group (n=16) and AD dementia group (n=26). Values are means ± SEM. Statistical significance as determined by the nonparametric Mann- Whitney test is indicated. ^* Significantly different from controls p<0.05, **p<0.01, ***p<0.001.

Figure 4. Characterization of the activation state, chemotaxis, phagocytosis and apoptosis of PMNs from the AD prodromal group and the AD dementia group.

Surface expression of (A) CD62L, CD 16b, CD l ib, and CDl lc on resting PMN was studied on whole blood samples maintained at 4°C stained with specific mAbs. Results are expressed as mean fluorescence intensity (MFI). (B, C) PMN death was measured by staining with annexin V and 7-AAD. (B) Spontaneous PMN death was measured immediately after sampling (TOh) and after 20h incubation at 37°C (T20h); results are expressed as the percentages of apoptotic PMNs Results are expressed as the percentages of apoptotic PMNs. (C) Prolongation of PMN survival was measured by incubating whole-blood samples with PBS, or TLR1/2 agonist, or TLR4 agonist for 20 hours at 37°C. Results are expressed as the percentage inhibition of PMN apoptosis [1 - (% of total annexin V⁺/7-AAD^" PMN in TLR agonist-treated sample/% of total annexin V⁺/7-AAD^" PMN in PBS-treated sample)] X 100. (D) PMN migration toward IL-8 and fMLP. The results are expressed as the migration rate. (E) Phagocytosis of PMNs was measured after incubation of whole blood samples with fluorescent opsonized E. coli bacteria for 10 minutes in a water bath at 37°C. Results are expressed as the PMN phagocytic index.

All measurements were made in controls (n=22), AD prodromal group (n=16) and AD dementia group (n=26). Values are means ± SEM. Statistical significance as determined by the nonparametric Mann- Whitney test is indicated. NS: not significant. ^*Significantly different from controls p<0.05, **p<0.01, ***p<0.001.

Figure 5. PMN hyperactivation state and imbalance of circulating senescent and immunosuppressive PMN subsets are associated with the rapidity of AD evolution.

(A) Surface expression of CD62L, CD 16b, CD l ib, and CDl lc on resting PMN was studied on whole blood samples maintained at 4°C stained with specific mAbs. Results are expressed as mean fluorescence intensity (MFI). ROS production by unstimulated (B) and stimulated PMNs (C) was studied by HE oxidation; results are expressed in MFI. (D) Correlation between AMMSE and the ROS production by un-stimulated PMNs. (E) Quantification of circulating NETs was performed by MPO-DNA complex ELISA; results are expressed as ng/μΐ DNA. (F) Percentages of the CXCR4^bright/CD62L^dim senescent PMN subset. (G) Percentages of the CD16^bright/CD62L^dim immunosuppressive PMN subset. (H) Ratio - - between the CXCR4^bright/CD62L^dim senescent and the CD 16^bright/CD62L^dim immunosuppressive subsets. Correlation between AMMSE and the percentage of the CXCR4^brigh7CD62L^dim senescent PMN subset (I), the percentage of the CD16^brigh7CD62L^dim immunosuppressive PMN subset (J), and the ratio between the CXCR4^brigh7CD62L^dim senescent and the CD 16^brigh7CD62L^dim immunosuppressive subsets (K).

All measurements were made in slow decliners (SD) AD patients (n=14) and fast decliners (FD) AD patients (n=13). Values are means ± SEM. Statistical significance as determined by the nonparametric Mann- Whitney test is indicated. ^*Significantly different p<0.05, **p<0.01.

Figure 6. PMN survival, migration and phagocytosis in fast decliners and slow decliners

AD patients.

(A) Spontaneous PMN death was measured immediately after sampling (TOh) and after 20h incubation at 37°C (T20h); results are expressed as the percentages of apoptotic PMNs. (B) Prolongation of PMN survival was measured by incubating whole-blood samples with PBS, or TLR1/2 agonist, or TLR4 agonist for 20 hours at 37°C. Results are expressed as the percentage inhibition of PMN apoptosis [1 - (% of total annexin V77-AAD^" PMN in TLR agonist-treated sample/% of total annexin V⁺/7-AAD^" PMN in PBS-treated sample)] X 100. (C) PMN migration toward IL-8 and fMLP; results are expressed as the migration rate. (D) PMN phagocytosis; results are expressed as the PMN phagocytic index.

All measurements were made in slow decliners AD patients (n=14) and fast decliners

(FD) AD patients (n=13). Values are means ± SEM. Statistical significance as determined by the nonparametric Mann- Whitney test is indicated. ^*Significantly different p<0.05, **p<0.01, ***p<0.001.

Figure 7. Cytokinic environment in AD patients.

(Al , A2, A3) Circulating levels of pro- and anti-inflammatory cytokines were measured from serum using Luminex assays. Correlation between MMSE and IL-8 circulating levels (B), and IL-17 circulating levels (C).

Figure 8. Circulating levels of MMP-9 and TIMP-1 in AD patients.

Circulating levels of MMP-9 (A) and TIMP-1 (B) were measured from serum using Luminex assays. (C) Ratio between MMP-9 and TIMP-1 concentrations. (D) Correlation - - between the ratio between MMP-9 and TIMP-1 concentrations and ROS production by TLR4 agonist-primed PMNs (MFI).

All measurements were made in controls (n=22), AD prodromal group (n=16) and AD dementia group (n=26). Values are means ± SEM. Statistical significance as determined by the nonparametric Mann- Whitney test is indicated. * Significantly different from controls p<0.05, **p<0.01.

EXAMPLE 1:

Material & Methods

Study design and patients

All participants were enrolled in the prospective IMABio3 study (PHRC-0054-N 2010), which aimed to assess the neuroinflammation in Alzheimer's disease. The study was approved by the Ethics Committee of the Salpetriere Hospital. All subjects provided written informed consent prior to participating. This research has been registered on http://clinical- trials.gov/under number NCTO 1775696. Exclusion criteria in the control group included acute or chronic inflammatory conditions, and corticoid or non-steroidal anti-inflammatory drugs.

Patients with Alzheimer's disease were included according to the following criteria: (i) progressive episodic memory impairment, characterized by a low free recall not normalized with semantic cueing {Sarazin, 2007 #204} {Dubois, 2010 #205}; (ii) absence of extrapyramidal signs; and (iii) CSF Alzheimer's disease profile, when available, defined as score 50.8, calculated with the formula amyloid- 42/[240 + (1.18 x T-tau)] {de Souza, 2011 #206} .

Controls were recruited according to the following criteria: (i) Mini-Mental State Examination (MMSE) score >27/30 and normal neuropsychological assessment; (ii) Clinical Dementia Rating (CDR) score = 0; (iii) no history of neurological or psychiatric disorders; and (iv) no memory complaint or cognitive deficit. We did not include subjects with (i) severe cortical or subcortical vascular lesions; (ii) history of autoimmune and inflammatory diseases or chronic migraines; or (iii) history of psychiatric disorders or drugs abuse.

64 subjects were included in the study: 42 patients with Alzheimer's disease (mean age, 68.2+9.1 years) and 22 controls (mean age, 71.6+7.5 years). CSF biomarkers measures were available for 36 patients with Alzheimer's disease. None major abnormalities of the absolute count of leucocyte subpopulations was detected in our cohort.

Patients with Alzheimer's disease were classified in two groups according to disease - - severity assessed by the CDR scale: 16 patients had a score of 0.5 representing AD-MCI with isolated episodic memory impairments and only moderate impact on the activities of daily living, and 26 patients had a score > 0.5 representing AD-D. An extensive review was conducted of each patient's chart (Table 3).After written informed consent had been obtained from the patients and the controls, whole blood was sampled, kept on ice, and transported immediately to the laboratory.

Determination of surface molecule expression on resting and stimulated PMNs

Heparin whole-blood samples (500μ1) were either kept on ice or incubated with PBS, Tumor Necrosis Factor (TNF)-a (5ng/ml), the following Toll Like Receptor (TLR) agonists: Pani3CSK4 (TLR1/2 agonist, 1 μg/mL, Invivogen, San Diego, CA) or Lipopolysaccharide (LPS) from E. coli serotype R515 (TLR4 agonist, lOng/mL, Alexis Biochemicals, San Diego, CA) for 45 minutes at 37°C. Samples were stained with PE-anti-human CD l ib (clone ICRF44, BD Biosciences, San Jose, CA), APC-anti-human CD62L (clone DREG-56, BD Biosciences), PE- anti-human CD 16b (clone CLB-gran 11.5, BD Biosciences), and PE-anti-human CD1 lc (clone 3.9, Sony Biotech, San Jose, CA) as previously reported {Campillo-Gimenez, 2014 #22} . In order to investigate the senescent CXR4^bright/CD62L^dim PMN subset, samples kept on ice were incubated for 45 minutes with PE-Cy7-anti-human CXCR4 (clone 12G5, Sony Biotech, San Jose, CA), PE-anti-human CD l ib (BD Biosciences), and APC-anti-human CD62L (BD Biosciences). In order to investigate the immunosuppressive CD16^bnght/CD62L^dim PMN subset, samples kept on ice were incubated for 45 minutes with FITC-anti-human CD 16 (clone 1D3, Beckman Coulter), PE-anti-human CDl lc (clone 3.9, Sony Biotech,), PE-Cy7-anti-human CD l ib (clone Bear 1, Beckman Coulter), and APC-anti-human CD62L (BD Biosciences). In some experiments, whole-blood samples were incubated were incubated in 24-well tissue culture plates, for 3 or 5 hours, at 37°C with PBS, Αβ42 amyloid peptide (20μΜ), IL-6 (1-100 ng/ml, R&D Systems), IL-8 (1-100 ng/ml, R&D Systems), TNFa (1-100 ng/ml, R&D Systems), IL-17 (1-lOOng/ml, R&D Systems) or IL-18 (1-lOOng/ml, R&D Systems) before staining. The blood was then lysed with BD FACS lysing and cells were then resuspended with Cell Fix IX (BD Biosciences).

Measurement of phagocytosis

The percentage of PMN phagocytosing opsonized Escherichia Coli (E. coli) as well as the mean fluorescence intensity (MFI) was measured in whole blood by using the Phagotest Kit (Glycotope Biotechnology). Whole blood samples (ΙΟΟμΙ) were incubated with opsonized FITC-conjugated E. coli bacteria (2xl0⁷ per 20μ1) for 10 minutes in a water bath at 37°C, the - - phagocytosis was then stopped in ice and erythrocytes are lysed before analysis by flow cytometry {Sauce, 2017 #152} .

Measurement of the oxidative burst of PMNs

Superoxide anion (O2 ^" ) production by PMNs was measured using a flow cytometry based assay derived from the hydroethidine (HE) oxidation technique, as previously described {Campillo-Gimenez, 2014 #22} . Heparin-whole blood samples (500μί) were loaded for 15 minutes with 1500ng/mL HE (Fluka, Buchs, Switzerland) at 37°C, and then incubated for 45 minutes with PBS or various stimuli, as described above. Samples were then treated with PBS or 10^"6M fMLP (Sigma Chemical Co., St Louis, MO) for 5 minutes. Erythrocytes were lysed before analysis by flow cytometry.

Measurement of PMN apoptosis/necrosis

PMN cell death in whole blood was quantified with annexin V and the impermeant nuclear dye 7-amino-actinomycin D (7-AAD). Whole-blood samples (500μί) were incubated in 24-well tissue culture plates, for 20 hours, at 37°C with PBS or various stimuli as described above. Samples were incubated with APC-anti-CD 15 antibodies (clone HI98, BD Biosciences), FITC-annexin V, and 7-AAD (BD Biosciences) as previously described {Campillo-Gimenez, 2014 #22} and analyzed by flow cytometry.

Flow cytometry analysis

Cells were acquired with a Gallios™ flow cytometer and analyzed with Kaluza software. To determine the expression of surface molecules on PMN, and ROS production by PMNs, forward and side scatter characteristics were used to identify the PMN population and to gate out other cells and debris, and 10,000 events were counted per sample. Results were expressed as Mean Fluorescence Intensity (MFI). To measure PMN phagocytosis, a "live" gate on leukocyte was set during data acquisition allowing excluding of bacteria. 10,000 - 15,000 leukocytes were collected per sample and the granulocyte cluster was gated in the software program in the scatter diagram (lin FSC vs lin SSC). The MFI of FITC correlates with the number of bacteria per individual leukocyte. Data were expressed as the PMN phagocytic index: % phagocytic PMN x MFI. To measure cell death in whole blood by flow cytometry, PMNs were identified as CD15^hlgh cells and 2>< 10⁵ events were counted per sample. Results were expressed as the percentage of apoptotic (Annexin V⁺/7-AAD^") and necrotic PMNs (AnnexinV77-AAD⁺).

Measurement of PMN chemotactic activity

Chemo taxis was measured in Transwell plates (Corning Costar) containing 3-μιη pore- size polyvinylpyrrolidone-free polycarbonate filters {Berthelot, 2012 #153} . The lower well of - - each chamber received βθθμί of fMLP (10^"7M), or IL-8 (25ng/ml) diluted in PBS plus 1% human serum albumin. Spontaneous migration was measured with PBS plus 1% human serum albumin. The upper well received ΙΟΟμΙ of whole blood from patients or controls diluted 1/10 in PBS. The chambers were incubated for 3h at 37°C. Samples were stained with APC-anti- CD 15 (BD Biosciences) for 15 min, and 450μ1 of lysis solution were then added. The total number of PMN added to the upper well and the number of PMN that migrated to the lower well were counted by flow cytometry using TruCount tubes (BD Biosciences).

Measurement of intra- vascular NETosis

NETs are composed of a DNA backbone decorated with granule proteins such as MPO, cit-H3 and elastase. NETs are also the major source of circulating cell free DNA. To identify NETs, we quantified serum NETs level by detecting MPO-DNA complexes in serum samples.

In order to prepare the DNA standard, NETs from an healthy donor were produced and isolated, as previously described {Barrientos, 2014 #154} . Briefly, freshly isolated PMNs were seeded in 12-well culture plates (1.5xl0⁶ cells/well) and stimulated with 5 mM calciulm ionophore A23187 for 3 h at 37 °C with 5% C02. The cells were carefully washed twice with 1 ml PBS and then treated for 20 min at 37 °C with 20 U/ml restriction enzyme Alul in HBSS to recover large soluble NET fragments. Supematants were collected and centrifuged at 300 g for 5 min at 4 ^C to remove contaminating cells and debris. NET preparations were then pooled, aliquoted, and stored at -80°C until use. DNA was quantified in NET samples by using PicoGreen (Molecular Probes), as previously described {Barrientos, 2014 #154} .

NETs associated MPO-DNA complexes were then quantified as previously described {Kessenbrock, 2009 #155} . In brief, 5μg/ml of mouse anti-human MPO antibody (ABD Serotec) was coated to 96-well microtiter plates. After blocking with 1% BSA, serum sample was added together with a peroxidase-labeled anti-DNA monoclonal antibody (component 2 of the Cell Death ELISA kit, Roche). After incubation, the peroxidase substrate was added according to the manufacturer's instructions. The optical absorbance was measured at 405 nm in an ELISA reader (Bio-Rad 550; Bio-Rad Laboratories, Tokyo, Japan). Samples were compared to the standard curve (from O. lng/μΕ to lOOng/μΕ) and the results expressed in ng/μΕ.

Measurement of soluble pro- and anti-inflammatory mediators

Soluble cytokines (interleukin (IL)- 1 β, IL-6, TNF-a, G-CSF, IL-17, IL-18, IL-22, IL- 23, IL-10, TGF-β), chemokines (IL-8, MCP-1), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metallo-proteinases (TIMP)-l were detected from plasma using Luminex - - assays (Luminex Performance Assays , R&D systems, Abingdon, UK); EDTA-whole-blood samples were centrifuged for 15 minutes at lOOOg within 30 minutes of collection. Assays were performed on crystored samples and diluted according to manufacturer's instructions (R&D systems).

Statistical analysis

Data are reported as means±SEM. The Mann- Whitney U test or two-way AN OVA tests with a Tukey posttest were used to determine whether differences were significant between groups, and the Spearman correlation to examine the associations between T-cell activation, neutrophil activation, and cytokine production. We used the Shapiro-Wilk normality test to ensure the normality of distributions in compared groups when using a parametric test. The significance level was set at p<0.05. All analyses were performed with GraphPad Prism 5.

Results

Patients' characteristics

Table 3 summarizes patients' clinical and AD-related characteristics. The baseline

MMSE of AD patients was 21 (range : 5-30) points. The group featured a slight imbalance with more females. As expected, there were significant differences across the diagnostic groups (MCI-AD and AD) for amyloid burden and cognitive deficits (MMSE and CDR). The prevalence of APOE e4 carriers was higher among patients with Alzheimer's disease. The level of C reactive protein levels was below 3 μg/ml in 54 subjects (18 controls and 36 patients) and did not exceed 10 μg/ml for both controls and patients.

Circulating PMNs from AD patients are highly activated and produce increased levels of ROS

PMN activation is associated with the modulation of surface molecules, specifically, a decrease in L-selectin (CD62L) or FCyRIIIB (CD 16b) and an increase in 2-integrins (CD l ib/CD 18, CDl lc/CD18) through either stimulus-induced shedding [Moldovan, 1999; Venturi, 2003] or translocation from intracellular granules [Sengelov, 1993]. As Fig 1 A shows, we observed lower expression of CD62L and of CD 16b, associated with higher CD l ib and CDl lc expression, on resting PMNs from AD patients than from HC. Because PMNs are reported to trigger microbicidal mechanisms on activation [Borregaard, 2010] , we measured, in unstimulated whole-blood samples (sample incubated with PBS+PBS), ROS production and found that it was significantly higher in AD patients than in HC (Fig IB). These results reflect the basal hyperactivation of circulating PMNs from AD patients. - -

We then analyzed whether the increased constitutive ROS production by PMNs from AD patients was associated with an increased capacity of PMNs to produce ROS in response to various stimuli. We have previously reported that, in whole blood, a single stimulus gives rise to minimal ROS production by PMN [Elbim, 1993; Elbim, 1994]. We therefore studied PMN oxidative burst in response to bacterial formyl peptides (fMLP) after priming with TNF- α or TLR agonists. ROS production by control PMNs (sample pre-incubated with PBS and stimulated with fMLP) was significantly higher in AD patients than in HC (Fig 1C). After pretreatment of whole blood with TNF-a or TLR agonists followed by fMLP stimulation, ROS production by PMNs from HC and AD patients was significantly increased compared to sample pre-incubated with PBS alone (Fig 1C). For the different stimulating conditions, ROS production was significantly higher in AD patients than in HC (Fig 1C) demonstrating the hypereactivity of PMNs to produce ROS in response to various stimuli.

ROS production by PMNs is a prerequisite for NETosis [Fuchs, 2007] . During NETosis, PMN release neutrophil extracellular traps (NETs) that can entangle pathogens but also contribute to various inflammatory diseases. We thus measured the levels of circulating intravascular NETs which were defined as complexes of nucleosomes and MPO. We found increased levels of circulating NETosis-derived products remnants in AD patients compared to HC (Fig ID).

Altogether, PMN hyperreactivity in terms of ROS combined with the increase in intravascular netosis in AD patients may play a role in establishing chronic inflammation in AD patients.

Defective survival, migration and phagocytosis of PMNs from AD patients

As PMNs can undergo activation-induced apoptosis [Aleman, 2005; 11 si eh. 2007], we measured the percentage of PMNs undergoing spontaneous apoptosis, immediately after sampling (TOh) as well as after 20h incubation (T20h). We found increased percentages of apoptotic (Fig B) as well as necrotic (Fig 2B) PMNs in AD patients compared with HC. In addition, the capacity of TLR1/2 and TLR4 agonists to increase PMN survival [Gabelloni 2013] was significantly decreased in AD patients than in HC (Fig 2C).

Increased PMN apoptosis in AD patients may result in the decrease in some functional capacities of PMNs to respond to various stimuli [Dransfield, 1995]. One of the main steps in PMN migration from the bloodstream to an inflammatory site is the modulation of adhesion molecule expression on both PMN and endothelial ceils. Stimulation of whole-blood samples with TNF-a as well as TLR agonists induced a normal maximal capacity of PMNs from AD - - patients to shed L-selectin (Fig 2D) and to translocate CD 1 l b (Fig 2E). PMN are attracted by a variety of molecules, including formyl-methionyl-leucyl-phenyl-alaninc (fMLP), a bacterial peptide, and chemokines such as IL-8, which are released from sites of inflammation or injury. As Fig 2F shows, PMN chemota is towards IL-8 and fMLP was significantly reduced in AD patients compared with HC (Fig 2F). We also studied the capacity of PMN to phagocytose opsonized bacteria. As previously reported [Davvdova 2003], we observed that the phagocytic index was significantly lower in AD patients reflecting an intrinsic defect in the capacity of PMN phagocytosis (Fig 2G), which might be related at least in part to the decreased CD 16b (FcyPJII) expression at PMN surface [Rivas-Fuentes, 2010].

Altogether, decreased PMN survival in response to inflammatory mediators associated with decreased migration and phagocytosis may be involved in the enhanced susceptibility to bacterial and fungal infections reported in AD patients [Bu, 2015; Alonso, 2014] leading to a vicious cycle further amplifying the inflammatory response. In addition, as PMN death by necrosis induces the release of harmful intracellular contents [Gabelloni, 2013], the increased percentage of circulating necrotic PMNs detected in AD patients might participate in promoting proinflammatory responses in AD patients.

PMNs from the AD-D and AD-MCI patients exhibit differential pro-inflammatory properties in relation with alterations of circulating neutrophils homeostasis

We then investigated PMN phenotype and functions separately in the prodromal AD group (AD-MCI) and in patients with dementia (AD-D). ROS production by PMNs and the level of circulating intravascular NETs were increased in AD-D patients compared to HC ; in contrast, no significant difference was observed between MCI- AD patients and HC (Fig. 3A, B and C). In addition, primed PMNs from AD-D patients exhibit higher ROS production in response to formyl peptides compared to PMNs from AD-MCI patients (Fig. 3B). Altered expression of L-selectin, CD1 lb and CD1 lc was only detectable in AD-D patients (Fig. 4A).

PMNs undergo phenotypic and functional changes from the time they are released into blood (fresh neutrophils) to the time they disappear from the circulation (aged neutrophils) in the absence of inflammation. Aged or senescent CXCR4^hlghPMNs are characterized by lower expression of CD62L, increased expression of β2 integrins, as well as increased production of ROS and susceptibility of NET formation compared to the total circulating pool [Zhang, 2015] . Conversely, an immunosuppressive PMN subset CD16^bright/CD62L^dim, showing decreased adhesion properties and ROS production, has been reported during acute inflammation [Pillay, 2012] and healthy ageing [Sauce, 2017]. Differential phenotypic and functional features of PMNs from the prodromal AD group and from the AD dementia group might thus be related - - to an imbalance between these two PMN subsets. We observed an expansion of the senescent CXCR4^high/CD62L^l0W PMN subset in the two groups of AD patients; the percentage of the senescent PMN subset was higher in AD-D than in AD-MCI patients (Fig 3D). Of note, we observed an increased percentage of the CD16^bright/CD62L^dim immunosuppressive subset in AD-MCI patients compared with HC and AD-D patients; in addition, the percentage of this subset was significantly decreased in AD-D patients compared to HC (Fig 3E). Accordingly, the ratio between the senescent and the immunosuppressive subsets was greater in AD-D patients compared to AD-MCI patients (Fig 3F). Moreover, ROS production by TLR4-primed PMNs from patients correlated with the percentages of the senescent CXCR4^high/CD62L^low subset (Fig 3G) as well as with the percentage of the immunosuppressive PMN subset (Fig 3H). Similar results were observed after TLR1/2 or TNF priming and fMLP stimulation (not shown). Altogether, these results suggest than the expansion of the immunosuppressive PMN subpopulation in the AD-MCI group can be involved, at least in part, in the normal ROS production and normal NETs formation despite the increase in the senescent subset. In the latter stage of the disease, the decrease of the immunosuppressive subpopulation associated with an increase in the senescent subpopulation leads to greater PMN oxidative burst and intravascular NETosis.

Finally, in accordance with the fact that senescence precedes cell death [Adrover, 2016], we observed an increased percentage of apoptotic PMNs (Fig. 4B) associated with a decreased PMN survival in response to inflammatory mediators in the AD dementia group compared to the AD prodromal group (Fig 4C). On the same line, CD 16b expression (Fig. 4A), PMN migration (Fig. 4D) and PMN phagocytosis (Fig. 4E) were significantly lower in the AD-D patients compared to the AD-MCI patients.

PMN hyperactivation state and alterations of circulating neutrophils homeostasis are greater in fast decliners than in slow decliners AD patients

We then investigated whether the PMN abnormalities observed in AD patients might be associated with the rapidity of disease progression. 27 subjects were followed-up over 2 years, with a standardized protocol including a CDR. AD patients were dichotomized into slow decliners (SD) or fast decliners (FD) based on the progression of the CDR score at the last visit. n= 14 patients with Alzheimer's disease remained stable (SD), showing unchanged CDR score (n = 9 prodromal Alzheimer's disease and n = 5 Alzheimer's disease dementia at baseline), whereas n=13 patients with Alzheimer's disease declined (FD), with an increase of 0.5 or more of the CDR score (n = 8 prodromal Alzheimer's disease and n = 5 Alzheimer's disease dementia at baseline). All phenotypic and functional characterizations of PMNs have been carried out at - - year one. Basal PMN activation was greater in FD than in SD as shown by lower expression of CD62L and CD 16, and increased expression of CD l ib and CD 11c (Fig 5 A), associated with higher levels of constitutive ROS production (Fig 5B) as well higher ROS production by primed-PMNs (Fig 5C). These phenotypic and functional properties of PMNs from FD patients were associated with an increased percentage in the senescent hyperactive PMN subset (Fig 5F) and a decreased percentage in the immunosuppressive subset (Fig 5G) resulting in an increased ratio between the senescent and immunosuppressive PMN subset (Fig 5H). Of note, we observed a positive correlation between the deltaMMSE (MMSE score at recruitment - MMSE score after the two-year period of follow-up) and the level of ROS production by unstimulated (Fig 5D) as well as the percentage of the senescent PMN subset (Fig 51), the percentage of the immunosuppressive PMN subset (Fig 5J) and the ratio between the senescent and immunosuppressive PMN subsets (Fig 5K). The correlations were adjusted to age and CDR score. Altogether, these results strongly suggest that combined altered homeostasis of circulating PMNs combined with PMN hyperactivation is associated with the rapidity of Alzheimer's disease progression.

In contrast, no difference was observed between the two groups concerning the level of intravascular NETs (Fig 5E) as well as spontaneous apoptosis (Fig 6A), survival (Fig 6B), migration capacity (Fig. 6C) and phagocytosis (Fig 6D).

ROS production by PMNs from AD patients is associated with increased MMP-9 activity

Matrix metalloproteinase (MMP)-9 belong to a family of zinc containing endopeptidases that o degrade components of the extracellular matrix which is important for normal blood-brain barrier function [Yong, 2001]. Although circulating levels of MMP-9 were significantly lower in the two groups of AD patients than in HC, we found a decrease in serum levels of tissue inhibitors to the metalloproteinases (TIMP)-l (which inhibits preferentially MMP-9) [Brew, 2000] in AD-D patients compared to HC. This decrease leads the MMP- 9/TIMP-l ratio to increase. Of note, the MMP-9/TIMP-1 ratio correlated with the level of ROS produced by stimulated PMNs (Fig 8).

Pro-inflammatory environment in AD patients.

As PMN activity is highly regulated by pro-inflammatory mediators such as cytokines

[Elbim, 1993; Elbim, 1994; Gabelloni, 2013], we investigated the cytokine environment in AD patients. Concerning pro-inflammatory cytokines, serum IL-Ιβ, IL-6, TNFa and IL-18 levels were significantly higher in the two groups of AD patients than in HC (Fig 7A1, A2 and A3); serum levels of IL-8 and IL-17 were significantly higher in the dementia AD group than in HC - -

(Fig 7A1, A2 and A3) and positively correlated with the MMSE score (Fig 7B and C). Serum level of IL-22, a bifunctional cytokine with both proinflammatory and protective functions, was significantly lower in the dementia AD group. Serum level of IL-10, an anti-inflammatory cytokine, was significantly decreased in the AD dementia group compared to HC and the prodromal AD group. Levels of other pro- (IL-23, G-CSF, MCP-1) and anti-inflammatory (TGFP) cytokines were similar in the two groups of AD patients and HC (Fig 7A1 , A2 and A3). No difference was observed for the different cytokine levels measured between both slow and fast decliner AD patients (not shown).

We then looked for potential association between markers of PMN hypereactivity and levels of pro-inflammatory cytokines. Increased CD l ib expression induced by suboptimal stimulation (PBS-stimulated samples) positively correlated with the IL-6 (data not shown) and IL-8 circulating levels (data not shown) suggesting a possible in vivo preactivation of PMNs by these cytokines. In addition, we observed a positive correlation between serum levels of IL- 8 plasma and the level of ROS production by fMLP-stimulated PMNs (data not shown) suggesting an in vivo priming of PMNs from AD patients by this cytokine. Furthermore, IL- 18 circulating level positively correlated with the percentage of the immunosuppressive PMN subset (data not shown) while IL-6 and IL-8 (circulating levels positively correlated with the percentage of the senescent PMN subset (data not shown) as well as with ratio between senescent and immunosuppressive PMN subsets (data not shown). We thus investigated using in vitro studies the effect of these cytokines on the percentage of the two PMN subsets. Incubation of whole samples from young healthy subjects with IL-6 and IL-8 for 5 hours induced a significant increase in the ratio between the senescent and the immunosuppressive PMN subset while IL-18 significant increased the percentage of the immunosuppressive PMN subset (data not shown).

EXAMPLE 2:

Material & Methods

Study design

All participants were enrolled in the prospective longitudinal IMABio3 study (PHRC- 0054-N 2010), which aims to assess neuroinflammation in Alzheimer's disease. The Ethics Committee of Salpetriere Hospital approved the study, and all subjects provided written informed consent before participating. This research is registered at http://clinicaltrials.gov/ as number NCTO 1775696. Exclusion criteria in the control and patient groups included acute or - - chronic inflammatory conditions and treatment with corticosteroid or non-steroidal antiinflammatory drugs.

Forty-two patients with Alzheimer's disease met the following inclusion criteria: (i) progressive episodic memory impairment, characterized by low free recall not normalized by semantic cueing; (ii) absence of extrapyramidal signs; and (iii) a cerebrospinal fluid (CSF) profile of Alzheimer's disease, when available, defined as a score < 0.8, calculated with the formula amyloid-P42/[240 + (1.18 x T-tau)] . Twenty-two control subjects were recruited if they met the following criteria: (i) Mini-Mental State Examination (MMSE) score >27/30 and normal neuropsychological assessment; (ii) Clinical Dementia Rating (CDR) score = 0; (iii) no history of neurological or psychiatric disorders; and (iv) no memory complaint or cognitive deficit. We did not include subjects with (i) severe cortical or subcortical vascular lesions; (ii) history of autoimmune and inflammatory diseases or chronic migraines; or (iii) history of psychiatric disorders or drug abuse. CSF biomarker measurements were available for thirty-six patients with Alzheimer's disease. No major abnormality of the absolute numbers of leukocyte subpopulations was detected in our cohort.

Patients with Alzheimer's disease were classified in two groups according to disease severity, assessed by the CDR scale on the day blood samples were taken: sixteen patients had a score of 0.5, indicative of prodromal Alzheimer's disease with isolated episodic memory impairments and only moderate impact on the activities of daily living, and twenty-six patients had a score > 0.5, considered to demonstrate Alzheimer's disease dementia. An extensive review was conducted of each patient's chart (Table 3). The two groups of patients were similar for age, prevalence of APOE ε4 carriers, duration and type of treatment, and levels of C reactive protein. Patients with Alzheimer's disease were also dichotomized into slow decliners (SD) or fast decliners (FD), based on the progression of the CDR score between the visit at baseline (assessment of neutrophil markers) and 1 year later (20). There were sixteen SD patients, who remained stable with no change in their CDR score (n = 6 ADMCI and n = 10 AD-D at baseline), and eleven patients who declined (FD), with an increase of at least 0.5 of the CDR score (n = 4 AD-MCI and n = 7 AD-D at baseline). The percentages of AD-MCI patients and AD-D patients did not differ significantly between the SD and FD groups (p>0.99).

All participants underwent the same procedure including a complete clinical and neuropsychological assessment, 3 T brain MRI, and (11) C-Pittsburgh compound B (PIB) and F-DPA-714 PET imaging, as described previously. Controls included in our study were defined by a negative PIB-PET. Patients with unavailable CSF biomarkers had a positive PIB-PET. - -

In order to check the part of recurrent infections occurring in patients with Alzheimer's disease, due to their way of living (i.e. poor ability to care for themselves), in the occurrence of neutrophil abnormalities, we analyzed in parallel two other groups of patients: i. patients without dementia and suffering from bronchiectasis who have elevated frequency of lung infections (pneumonia and bronchial infections) [n=l l, aged 64-87 (mean age, 70.7 years)] ; i.i. patients with dementia of other types, i.e. patients with corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) or frontotemporal dementia (FTD) [n=l l, aged 63-99 years (mean age, 74.2 years); MMSE : 13-28 (mean MMSE, 20.95)]. The differential diagnosis between non-Alzheimer's disease dementia and Alzheimer's disease was based on a non AD CSF biomarker profile defined by an Innotest Amylo^'id Tau Index > 1.2 (index IATI: ratio Αβΐ- 42/(240 + 1,18 x tau).

Patients and controls were included at a minimal distance of two months from an acute infectious episode. Whole blood of controls and patients was sampled, kept on ice, and transported to the laboratory immediately.

Determination of surface molecule expression on resting neutrophils

Surface expression of CD62L, CD16b, CDl lb, CDl lc, CD83 and CD152 on resting neutrophils was studied on whole-blood samples maintained at 4°C and stained with specific mAbs (22). To investigate the senescent CXCR4high/CD62Llow neutrophil subset, samples kept on ice were incubated for 45 minutes with PE-Cy7-anti-human CXCR4 (clone 12G5, Sony Biotech, San Jose, CA), PE-anti-human CDl lb (clone ICRF44, BD Biosciences), and APC anti-human CD62L (BD Biosciences). The immunosuppressive CD16bright/CD62Ldim neutrophil subset was studied after samples on ice were incubated for 45 minutes with FITC anti-human CD16 (clone 1D3, Beckman Coulter, Brea, CA), PE-anti-human CD1 lc (clone 3.9, Sony Biotech), PE-Cy7-anti-human CDl lb (clone Bear 1, Beckman Coulter), and APC anti- human CD62L (BD Biosciences). Samples were analyzed by flow cytometry.

Measurement of whole-blood neutrophil phagocytosis, oxidative burst and cell death The percentage of neutrophils phagocytosing opsonized FITC-conjugated Escherichia Coli (E. coli) and mean fluorescence intensity (MFI) were both measured in whole blood with the Phagotest Kit (BD Biosciences). Superoxide anion (02-°) production by neutrophils was measured with a flow cytometry-based assay derived from the hydroethidine (HE) oxidation technique. Neutrophil cell death was quantified with annexin V and the impermeant nuclear dye 7-amino-actinomycin D (7-AAD). Samples were then analyzed by flow cytometry.

Measurement of neutrophil chemotactic activity - -

Chemotaxis was assessed in Transwell plates (Corning Costar) containing 3-μηι pore polyvinylpyrrolidone-free polycarbonate filters. The lower well of each chamber was filled with 600 μΐ. of fMLP (10-7M) or of IL-8 (25 ng/niL) diluted in PBS plus 1% human serum albumin. Spontaneous migration was evaluated with PBS containing 1 % human serum albumin. The upper well was filled with 100 μΐ, of whole blood from patients or controls diluted 1/10 in PBS. The chambers were incubated for 3 hours at 37°C and the migration index was evaluated as previously described.

Measurement of intravascular NETosis

NETs are composed of a DNA backbone peppered with granule proteins, including myeloperoxidase (MPO), cit-H3, and elastase. To prepare the DNA standard, NETs from a healthy donor were produced and isolated as previously described. Briefly, freshly isolated neutrophils were seeded in 12-well culture plates (1.5x 106 cells/well) and stimulated with 5 mM calcium ionophore A23187 for 3 hours at 37 °C with 5% C02. The cells were carefully washed twice with 1 mL PBS and then treated for 20 minutes at 37 °C with 20 U/mL restriction enzyme Alul in HBSS to recover large soluble NET fragments. Supernatants were collected and centrifuged at 300 g for 5 minutes at 4 °C to remove contaminating cells and debris. NET preparations were then stored at -80°C until use. DNA was quantified in NET samples with PicoGreen (Molecular Probes, Waltham, MA). NET-associated MPO-DNA complexes were then quantified as previously described. In brief, 96-well microtiter plates were coated with 5 μg/mL of mouse anti-human MPO antibody (ABD Serotec). After blocking with 1% BSA, serum samples were added together with a peroxidase-labeled anti-DNA monoclonal antibody (component 2 of the Cell Death ELISA kit, Roche) to the wells. After incubation, the peroxidase substrate (ABTS) was added according to the manufacturer's instructions. Optical absorbance was measured at 405 nm in an ELISA reader (Bio-Rad 550; Bio-Rad Laboratories, Tokyo, Japan). Samples were compared to the standard curve (from 0.1 ng/μΕ to 100 ng/μΕ), and the results expressed in ng/μΕ.

Flow cytometry analysis

Cells were analyzed with a GalliosTM flow cytometer and the data with Kaluza software (Beckman Coulter). To determine neutrophil expression of surface molecules and ROS production, we used forward and side scatter characteristics to identify the neutrophil population and to gate out other cells and debris; 10,000 events were analyzed per sample. To measure neutrophil phagocytosis, a "live" leukocyte gate was set during data acquisition to exclude bacteria, and 10,000-15,000 leukocytes were collected per sample, with the granulocyte cluster gated in the software program in the scatter diagram (lin FSC vs lin SSC). To measure - - cell death in whole blood by flow cytometry, neutrophils were identified as CD15high cells and 2x 105 events were analyzed per sample.

Measurement of soluble pro- and anti-inflammatory mediators

Soluble cytokines, chemokines, MMP-9, and TIMP-1 were detected from serum with Luminex assays (Luminex Performance AssaysTM, R&D Systems). Whole-blood samples were centrifuged for 15 minutes at 1000 g within 30 minutes of collection. Assays were performed on cryostored samples frozen at -80°C and diluted according to manufacturer's instructions (R&D Systems).

Isolation of neutrophils

Neutrophils were isolated in LPS-free conditions by Dextran sedimentation and Ficoll-

Hypaque centrifugation of freshly drawn blood; neutrophils were further purified by negative selection with pan anti-human HLA class II-coated magnetic beads (Miltenyi Biotec, CA) to deplete B lymphocytes, activated T lymphocytes and monocytes, as previously described. Less than 1% of cells were positive by non specific esterase staining, and flow cytometry showed the absence of CD45+/CD14high, CD45+/CD3+ or CD45+/CD19+ cells; this showed that the neutrophils were highly purified, without contaminating monocytes.

Cytokine production by neutrophils

Isolated neutrophils (5xl06/ml) were cultured for 24 h at 37°C with 5% C02 in 24-well tissue culture plates (Costar) in RPMI 1640 medium (Sigma- Aldrich) in the presence of PBS Tumor necrosis factor (TNF)-a, or the following Toll-Like Receptor (TLR) agonists: Pam3CSK4 (TLR1/2 agonist, 10 μg/mL), or LPS (TLR4 agonist, 100 ng/mL). Supernatants were stored at -80°C for no longer than 15 days before assay. IL-8, IL-6, IL-Ιβ, TNFa, and IL- 17 were detected simultaneously in supernatants with Luminex assays.

Isolation of RNA

RNA was isolated using Trizol-chloroform, precipitated in isopropanol, and cleaned using the RNeasy mini-kit (Qiagenkit, Hilden, Germany) including a DNase digestion step as previously described. RNA was quantified using a NanoDrop (ND-1000, ThermoFisher Scientific, Waltham, MA) and checked for quality using the Agilent Bioanalyser. Total RNA was enriched for mRNA using poly-A selection.

Library generation and sequencing

Libraries were prepared with 500 nanograms of total RNA from each individual using the TruSeq Stranded Total RNA (Illumina, San Diego, CA) according to the manufacturer's instructions. Libraries were analyzed on a Tapestation (Agilent Technologies, Les Ulis, France) - - and then sequenced on a NextSeq500 (Illumina) as 75-bp paired-end reads with a sequencing depth of 30 million reads.

Read Mapping and Gene Annotation

Sequenced reads passing the quality controls using FastQC and Trimmomatic were aligned to the human reference genome hgl9 using Galaxy (https://usegalaxy.org/). Gene quantification and normalization were performed using FeatureCounts and EdgeR and transcripts that had no sequence reads across all samples were discarded from further analysis. A minimum CPM threshold of expression of 20 was applied.

Bioinformatic analysis of neutrophil transcriptome

All manipulations and statistical analysis were implemented using the R freeware. For each sample, raw data consisting of the CPM were log2 -transformed and normalized through trimmed mean of M-values (TMM) under the R freeware (http://www.r-project.org). Statistical analysis was performed on these normalized data under the R freeware. Independent samples were compared by two-tailed unpaired Student's t -test filtered at P < 0.05. These values were used to select the 100 most differently expressed genes in order to generate a graphical representation of the expression profile in form of heat map depending both on expression levels and differential expression. Functional profiling of the 100 more differentially expressed genes was performed using gProfiler.

Statistical analysis

The freely available statistical software Rstudio 1.0.143 (http://www.rstudio.com/) was used to conduct the statistical analysis. All tests were two-tailed, with a significance level of a = 0.05. When a parametric test was used, normality of distribution was tested with the Shapiro- Wilk test. Differences between groups were assessed with the chi-square test or ANOVA followed by the Tukey post-hoc test, as appropriate. ANOVA, adjusted for age, was used to compare neutrophil markers between the groups of patients and controls (with age as a covariate), and ANOVA with confounding factors (age, and initial MMSE score) as covariates, to compare them between fast and slow decliners. Bonferroni correction was used for multiple comparisons. Correlations were performed by linear partial correlation analysis, with adjustment for covariates.

Results

Circulating neutrophils from the Alzheimer disease dementia group are highly activated and produce higher levels of ROS and intravascular NETs than those from healthy subjects - -

We investigated neutrophil phenotype and functions separately in the prodromal Alzheimer's disease group (AD-MCI) and the Alzheimer's disease dementia group (AD-D). We observed lower expression of CD62L and CD 16b, together with higher CD1 lb and CD1 lc expression, on resting neutrophils from AD-D patients than from healthy controls (HC) (Data not shown) , indicating basal hyperactivation of circulating neutrophils from patients. Neutrophils from AD-MCI patients expressed normal levels of L-selectin, CD1 lb, and CD1 lc, but lower levels of CD 16b than those from HC. Basal CD 16b expression at the cell surface was modulated more strongly in AD-D than AD-MCI patients (Data not shown).

Upon activation, neutrophils are reported to trigger microbicidal mechanisms, such as ROS production and release of NETs. We observed significantly higher ROS production in unstimulated neutrophils from AD-D patients than HC (Data not shown). In contrast, ROS production did not differ significantly between MCI- AD patients and HC. In addition, ROS production by non-primed neutrophils (sample preincubated with PBS and stimulated with the bacterial formyl peptide, fMLP) was significantly higher in AD-D patients than in either HC or AD-MCI patients (Data not shown). Moreover, under the different priming conditions, ROS production was significantly higher in AD-D patients than in the HC and AD-MCI groups (Data not shown), thereby demonstrating the neutrophil hyperreactivity in the AD-D group. In contrast, ROS production by primed neutrophils did not differ significantly between MCI-AD patients and HC. Finally, we found higher levels of circulating NETs in AD-D patients than in HC. In contrast, again, no significant difference was observed between AD-MCI patients and HC (Data not shown).

Neutrophils from patients without dementia but with an increased susceptibility to infections, as well as neutrophils from patients with dementia of other types, exhibit normal expression of adhesion molecules, normal ROS production and normal levels of circulating NETs.

Impaired survival, migration, and phagocytic activity of neutrophils from patients with Alzheimer's disease

Because neutrophil activation can induce apoptosis, we measured the percentage of neutrophils undergoing spontaneous apoptosis. Both groups of patients had higher percentages of apoptotic neutrophils (Data not shown) than HC did. We also observed greater percentages of necrotic neutrophils in AD-D patients than in HC (Data not shown). Moreover, the capacity of TLR1/2 and TLR4 agonists to increase survival of neutrophils was significantly lower in patients with Alzheimer's diseases (Data not shown). Importantly, these differences with HC were also greater in AD-D than AD-MCI patients. - -

Increased neutrophil apoptosis in patients may impair some functional capacities of these cells to respond to various stimuli. Neutrophil chemotaxis towards IL-8 or fMLP was significantly lower in AD-D patients than in either HC or AD-MCI patients (Data not shown). Similar results was observed in supplemental experiments performed on isolated neutrophils (Data not shown) ruling out the influence of dendritic cells and monocytes derived cytokines and chemokines on chemotaxis of whole-blood neutrophils. As previously reported, we observed a significantly lower phagocytic index in patients with Alzheimer's disease (Fig Data not shown), perhaps related, at least, in part to the decreased CD 16b (FcyRIII) expression on neutrophil surfaces (Data not shown). Neutrophil phagocytic activity was also significantly lower in the samples from AD-D than AD-MCI patients.

Neutrophils from patients without dementia but with an increased susceptibility to infections, as well as neutrophils from patients with dementia of other types, exhibit normal survival, migration, and phagocytic activity.

Homeostasis of the circulating neutrophils differed for neutrophils from the prodromal group and from the dementia groups

Evidence over the last decade has demonstrated unexpected phenotypic heterogeneity and functional versatility in the neutrophil population. Differential phenotypic and functional features of neutrophils from AD-MCI and AD-D patients might be related to an imbalance between neutrophils subsets, i.e. the overly active subset of senescent CXCR4high neutrophils and the immunosuppressive CD 16bright/CD62Ldim subset of neutrophils exhibiting decreased proinflammatory properties. We observed an expansion of the senescent CXCR4high/CD62Llow subset of neutrophils in both groups of patients, and the percentage of neutrophils in the senescent subset was higher in AD-D than in AD-MCI patients (Data not shown).

The percentage of neutrophils in the CD 16bright/ CD62Ldim immunosuppressive subset was higher in AD-MCI patients than in HC, but significantly lower in AD-D patients than in HC (Data not shown). Accordingly, the ratio between the senescent and immunosuppressive subsets was higher in AD-D than in AD-MCI patients (Data not shown).

Neutrophils from patients without dementia but with an increased susceptibility to infections, as well as neutrophils from patients with dementia of other types, exhibit normal homeostasis.

Gene expression profile in neutrophils from patients with Alzheimer's disease

Phenotypic changes associated with neutrophil activation involve both molecular rearrangements to change the activity and/or sub-cellular localisation of pre-existing molecules. - -

Activation also includes increased ability of neutrophils to synthesize de novo proteins such as cytokines and chemokines, although neutrophils possess 10-20 times less RNA than other leukocytes. Transcriptome sequencing (RNA-Seq) was carried out on mRNA purified from freshly isolated neutrophils from healthy controls and patients with Alzheimer's disease. Analysis of RNA isolated from unstimulated neutrophils revealed expression (CPM >20) of 12289 known genes among a total of 22675 genes analyzed.

Hierarchical cluster analysis of genes expressed (CPM>20) in neutrophils from healthy controls and AD-D patients was performed and an expanded heat map of the top 100 genes differentially expressed between healthy controls and AD-D was realized (Data not shown). Neutrophils from patients with Alzheimer's disease showed different profiles as compared to controls. Interestingly, AD-MCI patients clustered in between controls and AD-D patients. Neutrophils from patients with Alzheimer's disease showed upregulation of transcripts with functions mostly associated with inflammatory and cell-mediated immune responses as well as organization of cytoplasm and cytoskeleton. Of note, actin cytoskeleton is required for vesicle trafficking to the plasma membrane and granule exocytosis allowing the NADPH oxidase cytosolic components to translocate to the membrane and to form the catalytically active enzyme complex. Functions associated with cell senescence and death, various signaling pathways, nucleic acid metabolism, lipid and protein metabolism, regulators of protein synthesis or stability including ubiquitination machinery and chaperones were similarly upregulated (Data not shown). Neutrophils from AD-D patients showed significant upregulation (p<0.05) of 18 transcripts including inflammatory and host defense genes (TAP1, CD83, CTLA4, TMIGD2, S100B, EDAR, MDS2) (Data not shown). However, we did not observe any significant upregulation of pro-inflammatory cytokines and chemokines transcripts, a classical signature of in vitro activated neutrophils. In accordance, neutrophil production of the classical proinflammatory cytokines IL-1, IL-6, IL-7, IL-8 and TNFa, measured at the protein level by Luminex (Data not shown) did not differ between patients with Alzheimer's disease and controls, in line with previous data. The up-regulation of two host- defense genes, CD83 and CTLA4 shown by RNAseq (Data not shown) was associated with increased expression of these two molecules at the surface of neutrophils from AD-D patients (Data no shown). CD83 encodes an Ig supergene family member, best known as a marker for mature dendritic cells, but has also been identified as a differentially regulated gene in neutrophil-transcription profiles. CD83 surface antigen has been reported previously in neutrophils cultured in vitro with cytokines and bacterial peptides or during acute bacterial infection and chronic inflammatory disease in vivo. Increased expression of CD83 transcript - - was also associated with up-regulation of TAP 1, involved in the proteolytic steps of MHC-I antigen processing by neutrophils suggesting that neutrophils from AD-D patients can adapt a function as antigen presenting cell. CTLA4 encodes Cytotoxic T-Lymphocyte Associated Protein 4 (CD 152) which transmits an inhibitory signal to T cells and is also involved in neutrophil regulation. Importantly, in AD-D patients we observed significant increased expression of four genes (SIK1 , CRIP2, GRASP, and AK5) involved in regulation of signalling pathways. SIK1, a serine/threonine kinase, that belongs to the AMP activated protein kinase family, and CRIP2, that belongs to the cysteine-rich intestine protein family 1 act as transcriptional repressors of NF-KB-mediated transcription induced by neutrophil in response to pro-inflammatory mediators such as TLR agonists. Upregulation of this negative regulatory circuit might thus be involved at least in part in the decreased capacity of activated neutrophils to produce a variety of proteins, such as cytokines and chemokines, as well as in the decreased TLR-dependent survival of neutrophils. AK5 is an adenylate kinase that catalyzes the reversible transfer of the γ-phosphate group from a phosphate donor (normally ATP) to AMP, and generate the intracellular second messenger cAMP. Interestingly, cAMP significantly inhibits ROS production by neutrophils in young subjects but increases ROS level in the 50-80 years old individuals. In the same line, GRASP promotes activation of ADP-ribosylation factor-6 (Arf6), a low molecular weight GTPase that regulates key aspects of endocytic recycling pathways and that has been involved in neutrophil degranulation and oxidative burst. Furthermore, upregulation of functions associated with ubiquitination machinery (DTX1, PSMB8) in AD-D patients could result in increased neutrophil death by apoptosis as well as by NETosis in these patients. Finally, we observed an increased expression of PERI gene (p=0.06) (Data not shown). PERI encodes for period circadian protein homo log 1 protein, a key regulator of internal biological clock in neutrophils, which has been involved in the mobilization of young neutrophils from the bone marrow and their ageing in the peripheral circulation.

Fast decliner patients have greater neutrophil hvperactivation and greater impairments in homeostasis than slow decliner do

We then investigated whether the neutrophil abnormalities observed in patients with Alzheimer's disease might be associated with the rate of disease progression, studying twenty- seven subjects who were followed up over 1 year with a standardized protocol including a CDR score. Patients were dichotomized into slow decliners or fast decliners as described in Materials and Methods. Basal neutrophil activation at baseline was greater in fast decliner than in slow decliner patients, as shown by their lower expression of CD62L and CD 16 (Data not shown), although their expression of CD1 lb and CD1 lc remained similar. Constitutive ROS production - -

(Data not shown) and ROS production by primed-neutrophils (Data not shown) were both higher in fast decliner than in slow decliner patients, as was the level of intravascular NETs (Data not shown). These phenotypic and functional properties of neutrophils from fast decliner patients were associated with a higher percentage of neutrophils in the senescent hyperactive subset (Data not shown) and a lower percentage in the immunosuppressive subset (Data not shown) and resulted in a higher ratio between these subsets in the fast decliner group (Data not shown), as well as a higher rate of spontaneous apoptosis (Data not shown). In contrast, neutrophil survival did not differ between these two groups, nor did migration capacity or phagocytosis (Data not shown). This analysis of the prognostic value of our neutrophil markers also considered the variations of both cognitive (MMSE) and functional (Clinical Dementia Rating Sum of Boxes, CDRSOB) indicators, which are complementary dimensions. Of note, we observed a negative correlation between the AMMSE with both CD62L expression (Data not shown) and the percentage of immunosuppressive neutrophils (Data not shown), as well as a positive correlation between the AMMSE and each of the following: the level of ROS production by unstimulated neutrophils, the percentage of apoptotic and senescent neutrophils (Data not shown), and the ratio between the senescent and immunosuppressive subsets (Data not shown). These markers were similarly correlated with the ACDRSOB (Data not shown).

Of note, neutrophil markers were similarly associated with the overall disease progression profile over a two-year follow-up period, determined by retrospectively taking into account the clinical scores one year before baseline. We observed that both the neutrophil activation parameters and the percentage of the different neutrophil subpopulations were correlated with AMMSE and ACDR-SOB (Data not shown).

ROS production by peripheral neutrophils from patients with Alzheimer's disease correlates with brain amyloid burden

We then investigated correlations between neutrophil abnormalities and pathological features in patients, i.e., brain amyloid deposits and microglial activation, as measured by PET imaging, and Αβ and Tau CSF biomarkers. C-PiB binding was used to quantify brain amyloid burden, and F-DPA-714 binding as a marker of microglial activation, with low affinity binders excluded from the analysis. We found that PiB binding was positively correlated with ROS production by stimulated neutrophils (Data not shown) as well as with the percentage of senescent neutrophils (Data not shown) and the senescent/immunosuppressive subset ratio (Data not shown). In addition, the latter two were negatively correlated with F-DPA-714 binding (Data not shown). The percentage of senescent neutrophils was positively correlated with CSF phospho-Tau levels (Data not shown). - -

ROS appear to be the initial trigger for the activation of matrix metalloproteinase (MMP)-9, which belongs to a family of zinc-containing endopeptidases. MMP-9 degrades components of the extracellular matrix, which is important for normal blood-brain barrier (BBB) function. Active MMP-9 forms 1 : 1 complexes with tissue inhibitor of metalloproteinases-1 (TIMP-1), which preferentially inhibits it, and the balance between them reflects the net proteolytic activity in several physiological processes, The MMP-9 and TIMP- 1 levels were significantly lower in both groups of patients than in HC (Data not shown). This led to a higher MMP-9/TIMP-1 ratio, which was moreover significantly different in ADD patients than in HC (Data not shown). The MMP-9/TIMP-1 ratio also correlated with the level of ROS produced by unstimulated and fMLP-stimulated neutrophils (Data not shown).

Proinflammatory environment in patients with Alzheimer's disease

Pro-inflammatory cytokines have been reported to be overproduced by PBMCs from patients with Alzheimer's disease compared to healthy controls. As neutrophil activity is highly regulated by proinflammatory mediators such as cytokines, we thus investigated the peripheral cytokine environment in patients. Serum levels of the proinflammatory cytokines Interleukin (IL)-ip, IL-6, TNFa, and IL-18 were significantly higher in both groups of patients than in HC (Data not shown). Serum levels of IL-8 and IL-17 were also significantly higher in the AD-D patients than in HC (Data not shown) and negatively correlated with the MMSE score (Data not shown). The serum level of IL-22, a bifunctional cytokine with both proinflammatory and protective functions, was significantly lower in the AD-D group than in HC. The serum level of IL-10, an anti-inflammatory cytokine, was significantly lower in the AD-D group than in either HC or AD-MCI patients (Data not shown).

We then looked for potential correlations between markers of neutrophil hyperreactivity and levels of proinflammatory cytokines. CDl lb expression on resting neutrophils was positively correlated with serum IL-6 level (Data not shown), as was higher CDl lb expression induced by suboptimal stimulation (that is, PBS-stimulated samples) with circulating levels of IL-6 and IL-8 (Data not shown). These findings suggest that these cytokines might possibly preactivate neutrophils in vivo. Similarly, the positive correlation between serum levels of IL- 8 and the level of ROS production by fMLP-stimulated neutrophils (Data not shown) indicates that this cytokine might prime neutrophils from patients in vivo. Furthermore, the circulating levels of IL-6 and IL-8 were positively correlated with the percentage of senescent neutrophils (Data not shown). We therefore used in vitro studies to investigate the effect of proinflammatory cytokines on the percentage of neutrophils in the senescent and immunosuppressive subsets. Incubation of whole samples from young healthy subjects with IL- - -

6, IL-8, TNFa, and IL-17 for 5 hours induced a significant increase in the percentage of senescent neutrophils (Data not shown), whereas TNFa and IL-17 significantly decreased the percentage of immunosuppressive neutrophils (Data not shown). REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Adrover, A.M. 2016. Answer to the Letter to the Editor of L. P. Ardigo et al. concerning "Validity and reliability of photographic measures to evaluate waistline asymmetry in idiopathic scoliosis" by Matamalas A, Bago J, D Agata E, Pellise F (2016) Eur Spine J; 2016 Mar 14 [Epub ahead of print]. Eur Spine J 25:3019-3020.

Agrawal, S.M., L. Lau, and V.W. Yong. 2008. MMPs in the central nervous system: where the good guys go bad. Semin Cell Dev Biol 19:42-51.

Aleman, M., S.S. de la Barrera, P.L. Schierloh, L. Alves, N. Yokobori, M. Baldini, E.

Abbate, and M.C. Sasiain. 2005. In tuberculous pleural effusions, activated neutrophils undergo apoptosis and acquire a dendritic cell-like phenotype. J Infect Dis 192:399-409.

Alonso, Pv., D. Pisa, A.I. Marina, E. Morato, A. Rabano, and L. Carrasco. 2014. Fungal infection in patients with Alzheimer's disease. J Alzheimers Dis 41 :301-311.

Barrientos, L., A. Bignon, C. Gueguen, L. de Chaisemartin, R. Gorges, C. Sandre, L.

Mascarell, K. Balabanian, S. Kerdine-Romer, M. Pallardy, V. Marin-Esteban, and S. Chollet- Martin. 2014. Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells. J Immunol 193:5689-5698.

Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Pillars Article: Flow Cytometric Studies of Oxidative Product Formation by Neutrophils: A Graded Response to Membrane Stimulation. J. Immunol. 1983. 130: 1910-1917. J Immunol. 2016 Aug l;197(3):683-90.

Bateman, R.J., C. Xiong, T.L. Benzinger, A.M. Fagan, A. Goate, N.C. Fox, D.S. Marcus, N.J. Cairns, X. Xie, T.M. Blazey, D.M. Holtzman, A. Santacruz, V. Buckles, A. Oliver, K. Moulder, P.S. Aisen, B. Ghetti, W.E. Klunk, E. McDade, R.N. Martins, C.L. Masters, R. Mayeux, J.M. Ringman, M.N. Rossor, P.R. Schofield, R.A. Sperling, S. Salloway, J.C. Morris, and N. Dominantly Inherited Alzheimer. 2012. Clinical and biomarker changes in dominantly inherited Alzheimer's disease. N Engl J Med 367:795-804. - -

Berthelot, F., L. Fattoum, S. Casulli, J. Gozlan, V. Marechal, and C. Elbim. 2012. The effect of HMGB1, a damage-associated molecular pattern molecule, on polymorphonuclear neutrophil migration depends on its concentration. J Innate Immun 4:41-58.

Bianca, V.D., S. Dusi, E. Bianchini, I. Dal Pra, and F. Rossi. 1999. beta-amyloid activates the 0-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease. J Biol Chem 274:15493-15499.

Borregaard, N. 2010. Neutrophils, from marrow to microbes. Immunity 33:657-670. Bowman, G.L., J.A. Kaye, M. Moore, D. Waichunas, N.E. Carlson, and J.F. Quinn. 2007. Blood-brain barrier impairment in Alzheimer disease: stability and functional significance. Neurology 68: 1809-1814.

Brew, K., D. Dinakarpandian, and H. Nagase. 2000. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 1477:267-283.

Bu, X.L., X.Q. Yao, S.S. Jiao, F. Zeng, Y.H. Liu, Y. Xiang, C.R. Liang, Q.H. Wang, X. Wang, H.Y. Cao, X. Yi, B. Deng, C.H. Liu, J. Xu, L.L. Zhang, C.Y. Gao, Z.Q. Xu, M. Zhang, L. Wang, X.L. Tan, X. Xu, H.D. Zhou, and Y.J. Wang. 2015. A study on the association between infectious burden and Alzheimer's disease. Eur J Neurol 22: 1519-1525.

Campillo-Gimenez, L., S. Casulli, Y. Dudoit, S. Seang, G. Carcelain, S. Lambert-Niclot, V. Appay, B. Autran, R. Tubiana, and C. Elbim. 2014. Neutrophils in antiretroviral therapy- controlled HIV demonstrate hyperactivation associated with a specific IL-17/IL-22 environment. J Allergy Clin Immunol 134: 1142-1152 el 145.

Cattaneo, A., N. Cattane, S. Galluzzi, S. Provasi, N. Lopizzo, C. Festari, C. Ferrari, U.P. Guerra, B. Paghera, C. Muscio, A. Bianchetti, G.D. Volta, M. Turla, M.S. Cotelli, M. Gennuso, A. Prelle, O. Zanetti, G. Lussignoli, D. Mirabile, D. Bellandi, S. Gentile, G. Belotti, D. Villani, T. Harach, T. Bolmont, A. Padovani, M. Boccardi, G.B. Frisoni, and I.-F. Group. 2017. Association of brain amyloidosis with pro-inflammatory gut bacterial taxa and peripheral inflammation markers in cognitively impaired elderly. Neurobiol Aging 49:60-68.

Chen, J.M., G.X. Jiang, Q.W. Li, Z.M. Zhou, and Q. Cheng. 2014. Increased serum levels of interleukin-18, -23 and -17 in Chinese patients with Alzheimer's disease. Dement Geriatr Cogn Disord 38:321-329.

Chiu, M.J., S.Y. Yang, T.F. Chen, J.J. Chieh, T.Z. Huang, P.K. Yip, H.C. Yang, T.W. Cheng, Y.F. Chen, M.S. Hua, and H.E. Horng. 2012. New assay for old markers-plasma beta amyloid of mild cognitive impairment and Alzheimer's disease. Curr Alzheimer Res 9: 1142- 1148. - -

Chiu, M.J., S.Y. Yang, H.E. Horng, C.C. Yang, T.F. Chen, J.J. Chieh, H.H. Chen, T.C. Chen, C.S. Ho, S.F. Chang, H.C. Liu, C.Y. Hong, and H.C. Yang. 2013. Combined plasma biomarkers for diagnosing mild cognition impairment and Alzheimer's disease. ACS Chem Neurosci 4:1530-1536.

Cunningham, C, and E. Hennessy. 2015. Co-morbidity and systemic inflammation as drivers of cognitive decline: new experimental models adopting a broader paradigm in dementia research. Alzheimers Res Ther 7:33.

Dansokho, C, D. Ait Ahmed, S. Aid, C. Toly-Ndour, T. Chaigneau, V. Calle, N. Cagnard, M. Holzenberger, E. Piaggio, P. Aucouturier, and G. Dorothee. 2016. Regulatory T cells delay disease progression in Alzheimer-like pathology. Brain 139: 1237-1251.

Davydova, T.V., V.G. Fomina, N.I. Voskresenskaya, and O.A. Doronina. 2003. Phagocytic activity and state of bactericidal systems in polymorphonuclear leukocytes from patients with Alzheimer's disease. Bull Exp Biol Med 136:355-357.

de Souza, L.C., F. Corlier, M.O. Habert, O. Uspenskaya, R. Maroy, F. Lamari, M. Chupin, S. Lehericy, O. Colliot, V. Hahn-Barma, D. Samri, B. Dubois, M. Bottlaender, and M. Sarazin. 2011. Similar amyloid-beta burden in posterior cortical atrophy and Alzheimer's disease. Brain 134:2036-2043.

Dransfield, I., S.C. Stocks, and C. Haslett. 1995. Regulation of cell adhesion molecule expression and function associated with neutrophil apoptosis. Blood 85:3264-3273.

Dubois, B., H.H. Feldman, C. Jacova, J.L. Cummings, S.T. Dekosky, P. Barberger-

Gateau, A. Delacourte, G. Frisoni, N.C. Fox, D. Galasko, S. Gauthier, H. Hampel, G.A. Jicha, K. Meguro, J. O'Brien, F. Pasquier, P. Robert, M. Rossor, S. Salloway, M. Sarazin, L.C. de Souza, Y. Stern, P.J. Visser, and P. Scheltens. 2010. Revising the definition of Alzheimer's disease: a new lexicon. Lancet Neurol 9: 1118-1127.

Elbim, C, S. Bailly, S. Chollet-Martin, J. Hakim, and M.A. Gougerot-Pocidalo. 1994.

Differential priming effects of proinflammatory cytokines on human neutrophil oxidative burst in response to bacterial N-formyl peptides. Infect Immun 62:2195-2201.

Elbim, C, S. Chollet-Martin, S. Bailly, J. Hakim, and M.A. Gougerot-Pocidalo. 1993. Priming of polymorphonuclear neutrophils by tumor necrosis factor alpha in whole blood: identification of two polymorphonuclear neutrophil subpopulations in response to formyl- peptides. Blood 82:633-640.

Francois S, E Benna J, Dang PM, Pedruzzi E, Gougerot-Pocidalo MA, Elbim C. Inhibition of neutrophil apoptosis by TLR agonists in whole blood: involvement of the - - phosphoinositide 3 -kinase/ Akt and NF-kappaB signaling pathways, leading to increased levels of Mcl-1, Al, and phosphorylated Bad. J Immunol. 2005 Mar 15;174(6):3633-42.

Fuchs, T.A., U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn, Y. Weinrauch, V. Brinkmann, and A. Zychlinsky. 2007. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 176:231-241.

Gabelloni, M.L., A.S. Trevani, J. Sabatte, and J. Geffner. 2013. Mechanisms regulating neutrophil survival and cell death. Semin Immunopathol 35:423-437.

Gidday, J.M., Y.G. Gasche, J.C. Copin, A.R. Shah, R.S. Perez, S.D. Shapiro, P.H. Chan, and T.S. Park. 2005. Leukocyte-derived matrix metalloproteinase-9 mediates blood-brain barrier breakdown and is proinflammatory after transient focal cerebral ischemia. Am J Physiol Heart Circ Physiol 289:H558-568.

Hamelin, L., J. Lagarde, G. Dorothee, C. Leroy, M. Labit, R.A. Comley, L.C. de Souza, H. Corne, L. Dauphinot, M. Bertoux, B. Dubois, P. Gervais, O. Colliot, M.C. Potier, M. Bottlaender, M. Sarazin, and I.t. Clinical. 2016. Early and protective microglial activation in Alzheimer's disease: a prospective study using 18F-DPA-714 PET imaging. Brain 139: 1252- 1264.

Holmes, C, C. Cunningham, E. Zotova, D. Culliford, and V.H. Perry. 2011. Proinflammatory cytokines, sickness behavior, and Alzheimer disease. Neurology 77:212-218.

Holmes, C, C. Cunningham, E. Zotova, J. Woolford, C. Dean, S. Kerr, D. Culliford, and V.H. Perry. 2009. Systemic inflammation and disease progression in Alzheimer disease. Neurology 73:768-774.

Hsieh, S.C., H.S. Yu, S.H. Cheng, K.J. Li, M.C. Lu, C.H. Wu, C.Y. Tsai, and C.L. Yu. 2007. Anti-myeloperoxidase antibodies enhance phagocytosis, IL-8 production, and glucose uptake of polymorphonuclear neutrophils rather than anti-proteinase 3 antibodies leading to activation-induced cell death of the neutrophils. Clin Rheumatol 26:216-224.

Huang, C.W., S.J. Wang, S.J. Wu, C.C. Yang, M.W. Huang, C.H. Lin, and I.H. Cheng. 2013. Potential blood biomarker for disease severity in the Taiwanese population with Alzheimer's disease. Am J Alzheimers Dis Other Demen 28:75-83.

Kamp, V.M., J. Pillay, J.W. Lammers, P. Pickkers, L.H. Ulfman, and L. Koenderman. 2012. Human suppressive neutrophils CD16bright/CD62Ldim exhibit decreased adhesion. J Leukoc Biol 92:1011-1020.

Kessenbrock, K., M. Krumbholz, U. Schonermarck, W. Back, W.L. Gross, Z. Werb, H.J. Grone, V. Brinkmann, and D.E. Jenne. 2009. Netting neutrophils in autoimmune small- vessel vasculitis. Nat Med 15:623-625. - -

Kitazawa, M., S. Oddo, T.R. Yamasaki, K.N. Green, and F.M. LaFerla. 2005. Lipopolysaccharide-induced inflammation exacerbates tau pathology by a cyclin-dependent kinase 5-mediated pathway in a transgenic model of Alzheimer's disease. J Neurosci 25:8843- 8853.

Lambeth, J.D. 2004. NOX enzymes and the biology of reactive oxygen. Nat Rev

Immunol 4: 181-189.

Larbi, A., G. Pawelec, J.M. Witkowski, H.M. Schipper, E. Derhovanessian, D. Goldeck, and T. Fulop. 2009. Dramatic shifts in circulating CD4 but not CD8 T cell subsets in mild Alzheimer's disease. J Alzheimers Dis 17:91-103.

Lehner, C, R. Gehwolf, H. Tempfer, I. Krizbai, B. Hennig, H.C. Bauer, and H. Bauer.

2011. Oxidative stress and blood-brain barrier dysfunction under particular consideration of matrix metalloproteinases. Antioxid Redox Signal 15: 1305-1323.

Lewkowicz, P., N. Lewkowicz, A. Sasiak, and H. Tchorzewski. 2006. Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J Immunol 177:7155-7163.

Licastro, F., M.C. Morini, L.J. Davis, P. Malpassi, D. Cucinotta, R. Parente, C. Melotti, and G. Savorani. 1994. Increased chemiluminescence response of neutrophils from the peripheral blood of patients with senile dementia of the Alzheimer's type. J Neuroimmunol 51 :21-26.

Moldovan, I., J. Galon, I. Maridonneau-Parini, S. Roman Roman, C. Mathiot, W.H.

Fridman, and C. Sautes-Fridman. 1999. Regulation of production of soluble Fc gamma receptors type III in normal and pathological conditions. Immunol Lett 68: 125-134.

Motta, M., R. Imbesi, M. Di Rosa, F. Stivala, and L. Malaguarnera. 2007. Altered plasma cytokine levels in Alzheimer's disease: correlation with the disease progression. Immunol Lett 114:46-51.

Nathan, C. 2006. Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 6: 173-182.

Nauseef, W.M., and N. Borregaard. 2014. Neutrophils at work. Nat Immunol 15:602-

611.

Nemeth, T., and A. Mocsai. 2012. The role of neutrophils in autoimmune diseases.

Immunol Lett 143:9-19.

Ouyang, W., and P. Valdez. 2008. IL-22 in mucosal immunity. Mucosal Immunol 1 :335-338. - -

Pallister, I., R. Bhatia, G. Katpalli, D. Allison, C. Parker, and N. Topley. 2006. Alteration of polymorphonuclear neutrophil surface receptor expression and migratory activity after isolation: comparison of whole blood and isolated PMN preparations from normal and postfracture trauma patients. J Trauma 60:844-850.

Perry, V.H., and C. Holmes. 2014. Microglial priming in neurodegenerative disease.

Nat Rev Neurol 10:217-224.

Pillay, J., V.M. Kamp, E. van Hoffen, T. Visser, T. Tak, J.W. Lammers, L.H. Ulfman, L.P. Leenen, P. Pickkers, and L. Koenderman. 2012. A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1. J Clin Invest 122:327-336.

Pun, P.B., J. Lu, and S. Moochhala. 2009. Involvement of ROS in BBB dysfunction.

Free Radic Res 43:348-364.

Ramanan, V.K., S.L. Risacher, K. Nho, S. Kim, L. Shen, B.C. McDonald, K.K. Yoder, G.D. Hutchins, J.D. West, E.F. Tallman, S. Gao, T.M. Foroud, M.R. Farlow, P.L. De Jager, D.A. Bennett, P.S. Aisen, R.C. Petersen, C.R. Jack, Jr., A.W. Toga, R.C. Green, W.J. Jagust, M.W. Weiner, A.J. Saykin, and I. Alzheimer's Disease Neuroimaging. 2015. GWAS of longitudinal amyloid accumulation on 18F-florbetapir PET in Alzheimer's disease implicates microglial activation gene IL1RAP. Brain 138:3076-3088.

Rivas-Fuentes, S., E. Garcia-Garcia, G. Nieto-Castaneda, and C. Rosales. 2010. Fcgamma receptors exhibit different phagocytosis potential in human neutrophils. Cell Immunol 263: 114-121.

Sarazin, M., C. Berr, J. De Rotrou, C. Fabrigoule, F. Pasquier, S. Legrain, B. Michel, M. Puel, M. Volteau, J. Touchon, M. Verny, and B. Dubois. 2007. Amnestic syndrome of the medial temporal type identifies prodromal AD: a longitudinal study. Neurology 69: 1859-1867.

Sauce, D., Y. Dong, L. Campillo-Gimenez, S. Casulli, C. Bayard, B. Autran, J. Boddaert, V. Appay, and C. Elbim. 2017. Reduced Oxidative Burst by Primed Neutrophils in the Elderly Individuals Is Associated With Increased Levels of the CD16bright/CD62Ldim Immunosuppressive Subset. J Gerontol A Biol Sci Med Sci 72:163-172.

Scali, C, C. Prosperi, L. Bracco, C. Piccini, R. Baronti, A. Ginestroni, S. Sorbi, G. Pepeu, and F. Casamenti. 2002. Neutrophils CD l ib and fibroblasts PGE(2) are elevated in Alzheimer's disease. Neurobiol Aging 23:523-530.

Scapini, P., and M.A. Cassatella. 2014. Social networking of human neutrophils within the immune system. Blood 124:710-719. - -

Sengelov, H., L. Kjeldsen, M.S. Diamond, T.A. Springer, and N. Borregaard. 1993. Subcellular localization and dynamics of Mac- 1 (alpha m beta 2) in human neutrophils. J Clin Invest 92: 1467-1476.

Shaffer, J.L., J.R. Petrella, F.C. Sheldon, K.R. Choudhury, V.D. Calhoun, R.E. Coleman, P.M. Doraiswamy, and I. Alzheimer's Disease Neuroimaging. 2013. Predicting cognitive decline in subjects at risk for Alzheimer disease by using combined cerebrospinal fluid, MR imaging, and PET biomarkers. Radiology 266:583-591.

Shoemark, D.K., and S.J. Allen. 2015. The microbiome and disease: reviewing the links between the oral microbiome, aging, and Alzheimer's disease. J Alzheimers Dis 43:725-738.

Solomon PR, Hirschoff A, Kelly B, Relin M, Brush M, DeVeaux RD, Pendlebury WW.

A 7 minute neurocognitive screening battery highly sensitive to Alzheimer's disease. Arch Neurol. 1998 Mar;55(3):349-55.

Song, C, M. Vandewoude, W. Stevens, L. De Clerck, M. Van der Planken, A. Whelan, H. Anisman, A. Dossche, and M. Maes. 1999. Alterations in immune functions during normal aging and Alzheimer's disease. Psychiatry Res 85:71-80.

Tecchio, C, A. Micheletti, and M.A. Cassatella. 2014. Neutrophil-derived cytokines: facts beyond expression. Front Immunol 5:508.

Ullen, A., E. Singewald, V. Konya, G. Fauler, H. Reicher, C. Nusshold, A. Hammer, D. Kratky, A. Heinemann, P. Holzer, E. Malle, and W. Sattler. 2013. Myeloperoxidase-derived oxidants induce blood-brain barrier dysfunction in vitro and in vivo. PLoS One 8:e64034. van de Haar, H.J., S. Burgmans, P.A. Hofman, F.R. Verhey, J.F. Jansen, and W.H. Backes. 2015. Blood-brain barrier impairment in dementia: current and future in vivo assessments. Neurosci Biobehav Rev 49:71-81.

Venturi, G.M., L. Tu, T. Kadono, A.I. Khan, Y. Fujimoto, P. Oshel, C.B. Bock, A.S. Miller, R.M. Albrecht, P. Kubes, D.A. Steeber, and T.F. Tedder. 2003. Leukocyte migration is regulated by L-selectin endoproteolytic release. Immunity 19:713-724.

Vitte, J., B.F. Michel, P. Bongrand, and J.L. Gastaut. 2004. Oxidative stress level in circulating neutrophils is linked to neurodegenerative diseases. J Clin Immunol 24:683-692.

Yong, V.W., C. Power, P. Forsyth, and D.R. Edwards. 2001. Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci 2:502-511.

Zenaro, E., E. Pietronigro, V. Delia Bianca, G. Piacentino, L. Marongiu, S. Budui, E. Turano, B. Rossi, S. Angiari, S. Dusi, A. Montresor, T. Carlucci, S. Nani, G. Tosadori, L. Calciano, D. Catalucci, G. Berton, B. Bonetti, and G. Constantin. 2015. Neutrophils promote - -

Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin. Nat Med 21 :880- 886.

Zenewicz, L.A., X. Yin, G. Wang, E. Elinav, L. Hao, L. Zhao, and R.A. Flavell. 2013. IL-22 deficiency alters colonic microbiota to be transmissible and colitogenic. J Immunol 190:5306-5312.

Zhang, D., G. Chen, D. Manwani, A. Mortha, C. Xu, J.J. Faith, R.D. Burk, Y. Kunisaki, J.E. Jang, C. Scheiermann, M. Merad, and P.S. Frenette. 2015. Neutrophil ageing is regulated by the microbiome. Nature 525:528-532.

Zuliani, G., G. Guerra, M. Ranzini, L. Rossi, M.R. Munari, A. Zurlo, A. Ble, S. Volpato, A.R. Atti, and R. Fellin. 2007. High interleukin-6 plasma levels are associated with functional impairment in older patients with vascular dementia. Int J Geriatr Psychiatry 22:305-311.

Claims

CLAIMS:

A method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the neutrophils are hyperactivated as compared to the predetermined reference or predicting a slow progression when the neutrophils are not hyperactivated as compared to the predetermined reference.

A method for predicting the rapidity of progression of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive neutrophil subsets ii) comparing the ratio between the senescent and the immunosuppressive neutrophil subsets at step i) with its predetermined reference and iii) predicting that the patient will have a rapid progression when the ratio between the senescent and the immunosuppressive neutrophil subsets is higher as compared to the predetermined reference or predicting a slow progression when the ratio between the senescent and the immunosuppressive neutrophil subsets did not differ from its predetermined reference.

A method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the expression level determined at step i) is higher as compared to the predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower as compared to the predetermined reference value.

A method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive neutrophil subsets ii) comparing the ratio between the senescent and the immunosuppressive neutrophil subsets at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the ratio determined at step i) is higher as compared to the predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the ratio determined at step i) is lower as compared to the predetermined reference value.

5. A method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the level of circulating intravascular Neutrophil Extravascular Traps (NETs) ii) comparing the level of circulating intravascular NETs at step i) with its predetermined reference and iii) determining that the patient has a severe Alzheimer's disease when the level determined at step i) is higher as compared to the predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the level determined at step i) is lower as compared to the predetermined reference value.

6. A method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the expression level of MMP-9 and the expression level of TIMP-1 ii) comparing the ratio determined at step i) with its predetermined reference value and iii) determining that the patient has a severe Alzheimer's disease when the ratio determined at step i) is higher as compared to the predetermined reference value or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower as compared to the predetermined reference value.

7. A method for predicting the severity of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the expression level of at least one proteins selected in the group consisting in IL-Ιβ, IL-6, TNF-a, IL-18, IL-8, IL17, IL- 22 and IL10 ii) comparing the expression level determined at step i) with their predetermined reference values and iii) determining that the patient has a severe Alzheimer's disease when the expression level determined at step i) is higher than their predetermined reference values for at least one of the proteins IL-Ιβ, IL-6, TNF-a, IL- 8 and IL17 or when the expression level determined at step i) is lower than their predetermined reference values for at least one of the proteins IL-18, IL-22 and IL10 or determining that the patient has not a severe Alzheimer's disease when the expression level determined at step i) is lower than their predetermined reference values for at least one of the proteins IL-Ιβ, IL-6, TNF-a, IL-8 and IL17 or when the expression level determined at step i) is higher than their predetermined reference values for at least one of the proteins IL-18 IL-22 and IL10.

8. A method for monitoring the efficacy of a treatment of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the activation state of neutrophils ii) comparing the activation state of the neutrophils at step i) with its predetermined reference and iii) predicting that the treatment is effective when the neutrophils are not hyperactivated than its predetermined reference or predicting that the treatment is not effective when the neutrophils are hyperactivated than its predetermined reference. 9. A method for monitoring the efficacy of a treatment of Alzheimer's disease in a patient comprising i) determining in a sample obtained from the patient the ratio between the senescent and the immunosuppressive PMN subsets ii) comparing the ratio between the senescent and the immunosuppressive PMN subsets at step i) with its predetermined reference and iii) predicting that the treatment is not effective when the ratio between the senescent and the immunosuppressive PMN subsets is higher than its predetermined reference or predicting that the treatment is effective when the ratio between the senescent and the immunosuppressive PMN subsets did not differ from its predetermined reference.

10. A method according to claims 1 to 9 wherein the neutrophils are the polymorphonuclear neutrophils (PMN).

11. A compound that reduces the presence or the activity of neutrophils in blood and/or in brain for use in the treatment of Alzheimer's disease in a patient with a rapid progression and/or a severe disease according to claim 1 to 10.

12. A therapeutic composition comprising compound that reduces the presence or activity of neutrophils in blood and/or in brain according to the invention use in the treatment of Alzheimer's disease in a patient with a rapid progression and/or a severe disease according to claims 1 to 10.