WO2009115807A1

WO2009115807A1 - Inhibitors of the aryl hydrocarbon receptor for modulating the immune response

Info

Publication number: WO2009115807A1
Application number: PCT/GB2009/000739
Authority: WO
Inventors: Brigitta Stockinger; Marc Veldhoen
Original assignee: Medical Research Council
Priority date: 2008-03-20
Filing date: 2009-03-19
Publication date: 2009-09-24
Also published as: GB0805312D0

Abstract

The present invention relates to methods of modulating the immune response in the field of autoimmune diseases via targeting of the aryl hydrocarbon receptor (AhR) with inhibitors such as flavones antisense or interfering RNA molecules or 2-methyl-2H-pyrazole-3-carboxylic acid- (2-methyl-4-o-tolylazophenyl) -amide.

Description

Inhibitors of the Aryl Hydrocarbon Receptor for Modulating the Immune Response

Field of the Invention

The present invention relates to methods of modulating the immune response in the field of autoimmune diseases.

Background to the Invention

Autoimmune Disease

Autoimmune diseases are multifactorial, depending on intrinsic factors such as genetics, hormones or age and environmental factors, including infections, diet, drugs and chemicals. The increasing prevalence of certain autoimmune diseases in highly industrialized countries is likely to be connected to such environmental factors.

Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) is a ligand dependent transcription factor that mediates a wide range of critical cellular events in response to halogenated aromatic hydrocarbons and nonhalogenated polycyclic aromatic hydrocarbons such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)¹. AhR expression is ubiquitous in vertebrate cells suggesting important and widespread roles, but the full extent of the physiological role of AhR is not understood yet ². Mice with a targeted mutation of the AhR gene provided unequivocal evidence that the AhR is crucial to TCDD-induced toxicity and furthermore suggest a function for AhR in liver growth and development ^3'5.

The role of AhR in the immune system is not well defined in the art, although numerous publications describe adverse effects of TCDD on immune responses ^15"18.

lnterleukin 22 (IL-22)

lnterleukin-22 (IL-22) is a cytokine produced by activated T cells and is related to interleukin-10 (IL-10). IL-22 signals through a receptor complex comprised of CRF2-4, also known as IL- 10R. beta., and a new member of the class Il cytokine receptor family, interleukin-22 receptor (IL-22R) (Xie et al., J. Biol. Chem. (2000) 275, 31335 31339). US 6,939,545 refers to the use of IL-22 inhibitors to treat inflammatory states such as arthritis. Disclosure of the Invention

Environmental factors are believed to be a major factor in the increased prevalence of autoimmune diseases. The present invention relates to the finding that AhR activation during induction of experimental autoimmune encephalomyelitis (EAE) causes accelerated onset and increased pathology in wildtype, but not AhR deficient mice. Our findings thus indicate that AhR activation may represent a cofactor in the development of autoimmune diseases.

We have also found that in the CD4 T cell lineage of mice AhR expression is restricted to the TH17 subset and is essential for the production of the TH17 cytokine IL-22. The expression of IL-22 thus appears to be one of the mediators by which AhR activation exerts its effects.

Accordingly in one aspect the invention provides a method of treating an autoimmune disease which method comprises administering to a subject in need of treatment an inhibitor of AhR activation.

In another aspect, the invention provides an inhibitor of AhR activation for use in a method of treating an autoimmune disease.

The invention also provides a method for the treatment of an IL-22 mediated condition, such as autoimmune disease or an inflammatory disorder in a subject, which method comprises administering to the subject an AhR inhibitor. The method may be used to treat a disorder is mediated by IL-22 production in the Th17 subset of cells. In another aspect, the invention provides a method of inhibiting IL-22 production in a Th17 cell, which method comprises contacting the cell with an AhR inhibitor. This method may be practiced in vivo or in vitro.

The invention also provides a method for screening a compound for use as an inhibitor of an autoimmune disease, which method comprises determining whether said compound is able to inhibit AhR activation.

These and further aspects of the invention are set out further below.

Description of the Drawings

Fig. 1 : AhR is selectively expressed in the Th17 subset a,b) FACS sorted naϊve CD4 T cells from B6 (black bars) or AhR deficient mice (white bars) were stimulated under THO, TH2, iTreg or TH17 conditions. Cells were harvested day 5 for qPCR. The figure shows mRNA levels, normalised to Hprt expression, b) mRNA levels of AhR, IL-17A.F and IL-22 in human CD4 T cells stimulated under Th17 conditions for 4 days, c: mRNA levels for IL-22 in CD4 T cells subsets from B6 (black bars) or AhR deficient mice (white bars) on day 5.

Fig. 2: AhR ligation promotes the TM 7 program a) FACS sorted naϊve CD4 T cells from B6 were stimulated under THO, TH1 , TH2, or TH17 conditions in the presence (black bars) or absence (white bars) of 10OnM FICZ. qPCR was performed on day 5 after initiation of culture. The figure shows mean mRNA levels, normalised to Hprt expression with standard deviations of 3-5 experiments. b,c) CD4 T cells from B6 top panels and AhR deficient mice (lower panels) were cultured under Th17 conditions in the presence or absence of 10OnM FICZ and stained on day 5 for expression of IL-17A vs IL-17F (b) and IL-17A vs IL-22 (c) after restimulation for 4h with PdBU/ionomycin. The figure shows representative dot plots-a summary of several experiments is shown as scattergram in Fig. 6b.

Fig. 3: Retroviral transduction of AhR restores IL-22 expression a,b) FACS sorted naϊve CD4 T cells from AhR deficient mice were cultured under TH 17 conditions (other conditions are shown in Supplement. Fig.3) and transduced with either vector control (RV-GFP) or AhR containing construct (RV-AhR -GFP) in the presence (lower panels) or absence (upper panels) of FICZ. IL-22 intracellular staining vs GFP expression a) and 1L-17A expression in gated GFP⁺ cells (b) was assessed 5 days later, c) qPCR for IL-17A, IL-22 and CYP1A1 in TH17 ceils from AhR deficient mice transduced with control retroviral vector (vertical hatched bar), control vector in the presence of 10OnM FICZ (horizontal hatched bar), AhR containing vector (black bar) or AhR containing vector in the presence of FICZ (white bar).

Fig. 4: EAE is enhanced by AhR ligation a) RT-PCR analysis for IL-17A, F, IL-22 and AhR from MOG/CFA of sorted CCR6CD4 T cells (black bars) or CCR6⁺ CD4 T cells (white bars) isolated from draining lymph nodes FACS sorted on day 7 after MOG/CFA immunization, b) mean absolute numbers of IL-17A positive cells in spinal cord 18 days after immunization of B6 mice with MOG/CFA (black bar) or MOG/CFA with FICZ (white bar) as well as MOG/CFA immunized AhR deficient (hatched bar) mice. Mean values and standard deviation of four mice per group are shown, c) B6 (squares) or AhR deficient mice (dots) were immunized with MOG/CFA in the absence (filled symbols) or presence (open symbols) of 200ng FICZ. The figure shows mean clinical score +/-SEM of 8-14 animals per group, d) Mean clinical scores of MOG/CFA immunized chimeras, AhR-B6 (dots), B6-Ahr (squares), e) table detailing incidence, mean day of onset and mean maximal scores for the mice in A, B. p values were determined by Mann Whitney non-directional test and are p= 0.0013 for mean day of onset in B6 vs B6+FICZ and p= 0.03 for B6 vs AhR. Fig. 5: AhR is not expressed in iTreg or nTreg and FICZ stimulation does not influence cytokines and transcription factors of other CD4 T cell subsets a) FACS sorted naive CD4 T cells from AhR deficient mice were stimulated under iTreg (TGFβ only), TH17 (lL-6+TGFβ), or in the presence of IL-6 alone. Furthermore, natural Treg (nTreg) were FACS sorted on the basis of CD25 expression. AhR expression was assessed in all 4 populations by qPCR. The figure shows mRNA levels, normalised to Hprt expression, b) Foxp3 expression in CD4 T cells from lymph node and spleen of B6 (black bar) or AhR deficient mice (white bar) was assessed by intracellular staining for Foxp3. Mean values with standard deviations from 3 mice per group are shown, c) qPCR of AhR expression in THO, TH1, TH2 and three individual TH17 samples generated as in d) vs liver expression of AhR d) FACS sorted naive CD4 T cells from B6 mice were cultured under THO, TH1 , TH2, iTreg, or TH17 conditions with (white bars) or without FICZ (black bars). Cells were harvested on day 5 and qPCR was performed. The figure shows mRNA levels, normalised to Hprt expression.

Fig. 6: Effect of Ahr ligation on Th17 program a) FACS sorted naive CD4 T cells were cultured under TH17 conditions in the absence or presence of indiated amounts of FICZ. qPCR for cytokines was performed 5 days later and mRNA levels normalised to Hprt expression is shown, b) Scattergram summary of experiments showing % positive cells for IL-17A, F and IL- 22 expression following stimulation under TH17 conditions in the absence or presence of 10OnM FICZ. c) cells treated as in a) and stimulated in the absence or presence of indicated amounts of βnaphtoflavone (Sigma). The figure shows % positive cells for IL-17A or IL-22 expression determined by intracellular staining on day 5.

Fig. 7: Retroviral transduction of AhR in non-Th17 subsets does not induce IL-22 FACS sorted naϊve CD4 T cells were cultured under THO, TH1 , TH2, iTreg conditions or with IL-6 and retrovirally transduced with RV-AHR-GFP. The figure shows IL-22 vs IL17A staining in gated GFP⁺CeIIs. A representative example of GFP gating is shown-levels of GFP expression were similar in all transduced subsets.

Fig. 8: FICZ but not IL-23 induces maximal IL-22 production. B6 mice were immunized with MOG/CFA (open bars) or MOG/CFA and FICZ (black bars) and AhR deficient mice were immunized with MOG/CFA (hatched bars). Seven days later 5x10⁵ cells from peripheral LN were stimulated with 50μg MOG peptide in the presence or absence of 25ng/ml IL-23 for 48h. Intracellular staining was performed after 2h stimulation with PdBU/ionomycin/brefeldin A. The figure shows % positive CD4 T cells for IL-17A or IL-22.

Fig. 9: SEQ ID NO:1 (AhR gene). Fig. 10: SEQ ID NO:2 (AhR protein sequence).

Detailed Description of the Invention

By "treating", it is meant both therapeutic treatment of ongoing disease intended to cure the disease, or to provide relief from the symptoms of the disease, as well as prophylactic treatment to prevent disease in a subject at risk of developing an autoimmune disease or an immune pathology. In the case of treatment of an autoimmune disease, the subject may be an individual recovering from an autoimmune disease, or an individual who has been exposed to a risk factor, such as an environmental pollutant (e.g. a dioxin), for the disease. In the case of the treatment or prophylaxis of an immune pathology, the invention may find particular application in the setting of retained antigenic stimulation, such as found in chronic infection of the lung³⁹.

By "autoimmune disease" it is meant any condition which involves an overactive immune response of the body against substances and tissues normally present in the body. Specific autoimmune diseases include, autoimmune hematological disorders (including e.g. hemolytic anemia, aplastic anemia, pure red cell anemia and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, sclerodoma, Wegener granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic sprue, (autoimmune) inflammatory bowel disease (including e.g. ulcerative colitis and Crohn's disease), endocrine ophthalmopathy, Graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial lung fibrosis, COPD, psoriatic arthritis, glomerulonephritis (with and without nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minimal change nephropathy) and juvenile dermatomyositis.

Autoimmune diseases of particular interest include multiple sclerosis and Psoriasis. Diseases due to immune pathology in chronic infection include COPD and asthma.

By "administering" it is meant providing to a subject via any suitable route of administration the inhibitor of AhR activation.. Thus such inhibitors may be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

For solid compositions, conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound as defined above may be formulated as suppositories using, for example, polyalkylene glycols, acetylated triglycerides and the like, as the carrier. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see "Remington: The Science and Practice of Pharmacy", 20th Edition, 2000, pub. Lippincott, Williams & Wilkins. The composition or formulation to be administered will, in any event, contain a quantity of the active compound(s) in an amount effective to alleviate the symptoms of the subject being treated.

Dosage forms or compositions containing active ingredient in the range of 0.25 to 95% with the balance made up from non-toxic carrier may be prepared.

For oral administration, a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, sodium crosscarmellose, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium, carbonate, and the like. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like. Such compositions may contain 1%-95% active ingredient, more preferably 2-50%, most preferably 5-8%.

Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, triethanolamine sodium acetate, etc.

Parenteral administration may also employ the implantation of a slow-release or sustained- release system, such that a constant level of dosage is maintained.

The percentage of active compound contained in such parental compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject. However, percentages of active ingredient of 0.1% to 10% in solution are employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages. Preferably, the composition will comprise 0.2-2% of the active agent in solution.

For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment or gel containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

By "a subject" it is intended a mammalian subject, including but not limited to a human subject. Non-human mammalian subjects may be treated in accordance with the invention. This includes rodents such as mice, rats or other rodents used as animal models of disease.

By "an inhibitor of AhR activation" it is meant any substance which is capable of inhibiting the normal biological response pathway that is activated by biding of an aryl hydrocarbon (e.g. dioxin) to the aryl hydrocarbon receptor. The activation of the AhR biological response pathway may be determined in a sample of T_H17 subset of cells (e.g. murine or human cells) which have a functional AhR by determining whether a substance, e.g. a dioxin or other aryl compound such as FICZ) mediates the up- regulation of IL-22 expression. Substances which act as inhibitors of AhR will be able to compete against the up-regulating substance in such an assay.

Inhibitors of AhR include nucleic acid inhibitors such as anti-sense RNA and RNAi molecules, as well as small molecules. These are discussed further below.

The inhibitor may also be an antisense compound, particularly an oligonucleotide, for use in modulating the function of nucleic acid molecules encoding AhR or IL-22, ultimately modulating the amount of the proteins produced. This is accomplished by providing oligonucleotides which specifically hybridize with nucleic acids, preferably mRNA, encoding AhR or IL-22.

An antisense oligonucleotide which targets a portion of the mRNA of AhR or IL-22 may be used. It may be necessary to determine a site or sites within the nucleic acid sequence for the antisense interaction to occur such that modulation of gene expression will result. This may be done by routine experimentation known to persons of skill in the art as such.

The mRNA sequence of AhR may be determined by reference to databases such as the

Genbank database, the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences. The human AhR gene is accession no. Genbank AK307495.

Antisense oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to non-coding or coding parts of the mRNA. Thus for example the oligonucleotide may be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5' cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5'- or 3'-untranslated region. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide to give the desired modulation.

The antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 bases in length, e.g. from about 8 to about 30 bases, such as from about 15 to about 30 bases. Also suitable as an inhibitor is a double-stranded RNA (dsRNA) molecule which is capable of inducing RNA interference. Such molecules have been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products.

These double stranded RNA molecules target regions similar to those targeted by antisense oligocleotides and have similar effects. These double stranded RNA molecules are generally 19-23, e.g. 19-21 base pairs in length, but may range between 8 and 50 bases. The production of siRNA molecules is known as such in the art and it will be appreciated that any desired siRNA targeted to AhR or IL-22 may be synthesized by conventional oligonucleotide synthesis techniques. Generally, the dsRNA may comprise two separate annealed strands, e.g. each of about 19-23, such as about 21 , bases in length in which one or two of the terminal 3' nucleotides overhang, or to a single stem loop, often referred to as a short hairpin RNA (shRNA) in which the stem comprises about 19-23 (though this may be from 8-50 as above) base pairs.

Antisense and interfering RNAs may include one or more modified e.g. non-naturally occurring intemucleoside linkages. Oligonucleotides having modified intemucleoside linkages include internucleoside linkages that retain a phosphorus atom and intemucleoside linkages that do not have a phosphorus atom. Examples of modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphoro-dithioates, phosphotriesters, aminoalkyiphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.

Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

A small molecule is typically an organic compound having a molecular weight of from about 200 Da to 2000 Da in size, which can be formulated in a pharmaceutically acceptable manner for deliver to its target. Small molecules include but are not limited to compounds of the peptide, steroid, benzodiazepine, prostaglandin and opiate classes.

Small molecules of particular interest include members of the flavone class, e.g. dietary flavonoids such as flavone, apigenin and naringenin (see US 20040077080 A1), as well as flavonoid compounds of the formula:

5'

in which the 5' position is hydrogen or iodo, the 4' position is selected from hydrogen, iodo, azido, nitro, a group -NCS, cyano, amino or a group -NHCOCH₃; and the 3' position is hydroxy or lower alkoxy having from 1 to 3 carbon atoms, which may be saturated or unsaturated. Compounds of this class are described in Henry et al⁴⁰. Preferred flavone compounds of this class include those with a 3'-methoxy group and a 4'-substituent having one or more terminal atoms of high electron density (-N₃, -NO₂, or -NCS). Particular compounds include 3'-methoxy-4'-nitroflavone.

A further compound which is an AhR antagonist is CH-223191 , 2-methyl~2H-pyrazole-3- carboxylic acid-(2-methyl-4-o-tolylazophenyl)~amide, of formula:

This compound is commercially available (Calbiochem® Cat. No. 182705) and further described in Kim et al⁴¹.

In another aspect, the invention provides a method for determining a polymorphism in the AHR and linking the polymorphism to a risk factor for an autoimmune disease or an inflammatory disorder in a subject.

There are a number of known polymorphisms in the coding sequence of AHR. The wild-type AHR gene, which is shown in Figure 9 as SEQ ID NO:1 (Genbank Accession no. AK307495). The gene contains an open reading frame, residues 71 to 2617 of SEQ ID NO:1 , the translation of which is shown as SEQ ID NO:2.

A number of variants of the coding sequence are known, including SNP: rs2066853 (rs numbers as defined on the http://snpper.chip.org/ database) in which amino acid 554 Arginine is changed to Lysine and SNP: rs4986826 in which amino acid 570 Valine is changed to

Isoleucine. The frequencies of these two polymorphisms is known to differ between Japanese and Caucasian populations. The latter variant, as well as a further change at codon 517 (P517S), have been shown to have an effect on AhR responses³⁶. A further variant, M786V, is also known. A further polymorphism, Genbank accession no. D16354 is caused by a frameshift leading to substitutions at positions 807 and 808 of SEQ ID NO:2 and truncation of the remaining protein (L807F:N808K:Δ809-848). It has been reported that the truncation results in a two-fold increase in affinity for dioxin⁴².

Thus in one embodiment the invention provides: determining the presence of one or more alleles of AHR in individuals from a population; and correlating the presence of an allele with the incidence of an autoimmune disease or an immune pathology in said population.

The one or more (e.g. one, two, three, four or five) alleles may be one of the above-mentioned alleles, i.e. R554K, V570I, P517S, M786V, or L807F:N808K:Δ809-848. The allele may further be an allele of the 5' untranslated sequence or 5' regulatory sequences of the gene. For example, a comparison of the AhR Genbank entries AB384754, AK307495, BC069390, BC070080, D16354 and L19872 show that a number of such 5' nucleotides are polymorphic.

Where a particular polymorphism is found to be associated in a population with an increased risk of an autoimmune disease or an immune pathology the invention further provides a method of predicting the risk of an autoimmune disease or an immune pathology in an individual free of disease by determining whether or not a sample of nucleic acid comprising the AhR gene obtained from the individual contains that polymorphism. The invention also provides a method of predicting the response of a patient with an autoimmune disease or an immune pathology to a treatment, by determining whether or not a sample of nucleic acid comprising the AhR gene obtained from the individual contains that polymorphism. The nucleic acid may be a DNA sample or RNA₁ wherein in the case of RNA the sample is obtained from a tissue that expresses the AhR gene.

Kits comprising nucleic acid primers for determination of an AhR variant of interest, together with instructions for their use in the above methods, form a further aspect of the invention.

The invention also provides a method for screening a compound for use as an inhibitor of an autoimmune disease, which method comprises determining whether said compound is able to inhibit AhR activation. Such a method may be practiced in a variety of ways. In its most general aspect, the method will comprise bringing an AhR into contact with a substance under conditions in which in the absence of the substance the AhR is able to bind a known AhR ligand (e.g. dioxin) and trigger one or more biological responses. In a preferred embodiment, the method is practiced on a sample of cells comprising T_H17 cells (e.g. murine or human cells). In this aspect, the biological response determined may be the production of IL-22.

Suitable compounds for use in the screening method of the invention include small molecules as defined above.

Autoimmune diseases are multifactorial, depending on intrinsic factors such as genetics, hormones or age and environmental factors, including infections, diet, drugs and chemicals. The increasing prevalence of certain autoimmune diseases in highly industrialized countries is likely to be connected to such environmental factors. Our data linking a transcription factor responsive to environmental factors to the TH 17 program provide a linkage between such factors and the role of AhR in human autoimmune diseases.

The effects of TCDD on the thymus¹⁹' ²⁰ are in contrast to the specific mechanistic effects we describe here, but it is worth remembering that RORyt, the lineage defining transcription factor for TH17 differentiation has distinct functions in the thymus as well as in lymphoid inducer cells²¹ which are unrelated to its function in the TH17 pathway. Our data show that the AhR, in addition to driving the expression of IL-22, enhances TH17 development and the expression levels of IL-17A/F and consequently increases autoimmune pathology. Blockade of IL-17A with an auto-vaccine as well as neutralizing antibody can completely prevent the development of EAE ^{22i Z3} emphasizing the central role of IL-17A in the pathogenesis of this autoimmune disease.

It has been suggested that IL-22 may contribute to autoimmune pathology since, like IL-17, it disrupts blood-brain barrier tight junctions²⁴. We have observed that IL-22 deficient mice do not seem to have altered susceptibility to EAE induction, suggesting that this cytokine may be dispensable for the development of pathology. Thus, the enhancement of IL-17 production by AhR ligation may be more crucial than the induction of IL-22 in determining disease severity. There is literature (reviewed in ²⁵) describing interactions of AhR with other key regulatory proteins including for instance NF-kB²⁶ , which plays a role in the induction of EAE²⁷. The core nucleotide sequence to which the nuclear AHR complex binds, also termed the 'xenobiotic responsive element' ^2δ occurs frequently in the mammalian genome ²⁹and is also represented in IL-17A.F, IL-22 and ROR vt. While basal expression of AhR and IL-22 is readily detectable in TW 7 T cells in the apparent absence of a ligand, it is now clear that there are numerous endogenous agents that can activate AhR, such as prostaglandins, bilirubin at high concentration, modified low-density lipoprotein (LDL) and various modifications of tryptophan whose UV irradiated photoconversion into the high affinity ligand FICZ is only one example (reviewed in ³⁰). IL-22 expression is reported to be dependent on IL-23 ⁸' ⁹, but at least in vitro there seems to be little effect of IL-23 as AhR ligation in the absence of any source of IL-23 was sufficient to drive IL-22 expression.

Inbred mouse strains can be classified into high and low responder strains on the basis of inducibility of AhR ^{31> 32}. The C57BI/6 strain expresses the high responder AhR^b allele, whereas DBA/2 mice express the low responder AhR^d allele. Interestingly, DBA/2 mice were reported to be resistant to EAE induction³³ and highly susceptible to infection with Candida albicans³⁴ , both important features of TH17 responses. In our studies, we have compared TH17 differentiation from naive T cells of DBA/2 and B6 mice in the absence or presence of FICZ and found that 1L-17A, F and IL-22 production in DBA/2 is attenuated, as well not being enhanced by FICZ. Human AhR appears to have impaired binding affinity as human cells require approximately 10- fold higher concentrations of TCDD in vitro than rodent cells to respond with enzyme induction ³⁵. Nevertheless some individuals have AhR receptors with binding affinities close to that of high responder B6 mice³⁶.

The invention is further illustrated by the following examples. Examples

Overview of Examples

We show here that in the CD4 T cell lineage of mice AhR expression is restricted to the TH17 subset and essential for the production of the TH17 cytokine IL-22. AhR is also expressed in human TH17 cells, indicating the close similarity of the mouse and human Th17 program. Activation of AhR by a high affinity ligand during TH17 development markedly increases the proportion of TH17 T cells and their production of cytokines. CD4 T cells from AhR deficient mice can develop TH17 responses, but fail to produce IL-22 and do not show enhanced TH17 development when confronted with AhR ligand. AhR activation during induction of experimental autoimmune encephalomyelitis (EAE) causes accelerated onset and increased pathology in wildtype, but not AhR deficient mice. AhR activation may therefore represent a cofactor in the development of autoimmune diseases.

Example 1 -AhR is selectively expressed in the Th17 subset

During gene array analysis of CD4 effector T cell subsets we noticed that the TH17 CD4 T cell subset, in addition to the lineage defining transcription factor RORyt⁶, expresses AhR. Quantitative PCR analysis of CD4 T cell subsets polarized to TH1 , TH2, iTreg or TH17 effector profile established that the lineage defining transcription factors Tbet, GATA-3, RORyt and Foxp3 respectively are expressed in a comparable manner in effector T cell subsets of wildtype and AhR deficient B6 mice, as are their marker cytokines IFNy, IL-4, IL-17A and IL-17F (Fig.1a, Fig.5a, b). AhR was only found in the TH17 subset with levels of expression similar to liver (Fig.5c), whereas it is absent in Th17 CD4 T cells from AhR deficient mice. Importantly, AhR expression is also found in human TH17 cells, which suggests close similarity in the TH17 program of humans and mice (Fig.1b).

While CD4 T cells cells from AhR deficient mice can differentiate into TH17 cells, they lacked, however, the expression of IL-22 (Fig.1c). IL-22, originally defined as an hepatocyte stimulating factor ⁷ is co-expressed with IL-17 by TH17 T cells and its expression is thought to be enhanced by dendritic cell-derived IL-23 ^{8i 9}.The biological functions of IL-22 are not fully understood; on the one hand IL-22 appears to be pro-inflammatory, inducing dermal inflammation and proinflammatory gene expression in the skin ^{8ι 10}, on the other hand IL-22 delivery ameliorates T cell mediated liver injury ¹¹. In T cell mediated hepatitis IL-22 is a survival factor for hepatocytes via activation of STAT3¹². AhR resides in the cytoplasm complexed with hsp90 until binding of ligand triggers conformational changes resulting in an exchange of hsp90 for the nuclear translocation component ARNT₁ reviewed in¹. ARNT was found expressed in all CD4 T cell subsets. Ligation of AhR by 6-formylindolo[3,2-b] carbazole (FICZ), a tryptophan derived photoproduct that is thought to be an endogenous ligand with high affinity for the AhR receptor¹³ upregulates genes encoding xenobiotic metabolizing cytochrome P450 enzymes such as CYP1A1¹⁴.

Example 2 - AhR ligation promoters the TH 17 program

In order to test whether exposure of T cells to FICZ influences differentiation of naive CD4 T cells to effector cells, we added FICZ during the in vitro differentiation of CD4 effector T cell subsets. The addition of FICZ did not did not induce AhR or its downstream target CYP1 A1 in THO, TH1, TH2 or iTreg (Fig.2a) and did not alter the expression of IFNγ, IL-4, IL-21 nor the lineage defining transcription factors in these subsets (Fig.δd). However, the presence of a high affinity AhR ligand during TH17 inducing conditions led to strong upregulation of IL-17A, IL-17 F and particularly of IL-22 mRNA expression (Fig.2a and Fig.δa). Expression of IFNy and TNFα in TH 17 cells was not influenced by AhR ligation.

Comparison of Th17 differentiation in naive CD4 T cells from wildtype B6 and AhR deficient mice by intracellular staining showed that exposure of B6 CD4 T cells to FICZ under TH17 conditions strongly enhanced IL-17A/F production (Fig.2b) and also increased the proportion and staining intensity of cells producing IL-22 (Fig.2c). A similar response was seen with another AhR ligand, β-Naphthoflavone (Fig.βc). In contrast, IL-17A/F production was attenuated in TH17 from AhR deficient mice and no IL-22 was detectable whether FICZ was present or not (Fig.2c).

Example 3 - Retroviral transduction of AhR restores IL-22 expression

In order to test whether AhR expression on its own is essential and sufficient to drive IL22 expression, we performed retroviral transduction of sorted naive AhR deficient CD4 T cells with an AhR-GFP construct or a GFP vector control construct. Transduction under neutral, Th1 , Th2 or iTreg conditions did not reconstitute IL-22 expression even in the presence of FICZ (Fig.7). However, under TH17 conditions reconstitution of AhR expression by retroviral transduction induced expression of IL-22 (Fig.3a top right panel) and increased the proportion of IL-17 producing cells (Fig.3b top right panel, gated on the GFP⁺ population). Exposure to FICZ resulted in a substantial increase in IL-22 (Fig.3a bottom right panel) as well as the enhanced expression of IL-17A (Fig.3b bottom right panel, gated on the GFP⁺ population). RT-PCR analysis of cultured AhR transduced Th17 cells confirmed the increase in IL-17 expression, the enhancement of IL-22 as well as the induction of the AhR target CYP1A1 upon exposure to FICZ (Fig.3c). Thus, AhR expression is only functional in CD4 T cells that have differentiated to the TH17 lineage.

Example 4 - EAE is enhanced by AhR ligation

TH 17 T cells play a prominent role in the pathology of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), which is induced by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 and CFA. CCR6⁺ CD4 T cells from immunized B6 mice, which are enriched in TH17 cells, express AhR, IL-17A, F, IL-22 and RORγt, whereas the CCR6 negative CD4 T cell fraction lacked expression of AhR and TH17 markers, confirming that physiological differentiation in vivo recapitulates in vitro differentiation (Fig.4a). Analysis of spinal cord at the height of the EAE response on day 18 showed increased numbers of TH17 cells in B6 mice treated with MOG/CFA and FICZ and reduced numbers of TH17 cells in AhR deficient mice (Fig.4b).

AhR deficient mice developed EAE with delayed kinetics in line with the attenuated TH17 differentiation seen in vitro (Fig.4c). Despite the delayed onset of EAE most AhR deficient mice succumbed to disease eventually (Fig.4e). Stimulation of AhR by inclusion of FICZ in the antigen emulsion accelerated the onset and increased the severity of EAE in B6 mice, but, as expected, did not influence the onset or severity of EAE in AhR deficient mice (Fig.4c left). In order to assess the influence of AhR deficiency in haematopoietic or non-haematopoietic cells, we also induced EAE in chimeras constructed either by injection of AhR deficient bone marrow into irradiated wildtype mice (AhR-B6) or by injection of B6 wildtype bone marrow into irradiated AhR deficient mice (B6-AhR). AhR-B6 chimeras showed attenuated EAE like AhR deficient mice, whereas B6-AhR chimeras developed EAE with kinetics and severity similar to wildtype mice (Fig.4d).

The IL-17 and IL-22 responses to stimulation, in the presence or absence of IL-23, of peripheral lymph node (LN) cells in mice immunised with MOG and FICZ was examined and it was observed that the response in cells was not significantly different in either case.

Materials and Methods

The examples set out above were performed in accordance with standard laboratory techniques. For the convenience of the reader, the following discussion of the methods used is provided; routine variations or additions to such methods may be used in reproducing the examples.

Mice

C57BI/6 (B6), DBA/2 and AhR deficient mice on a B6 background (B6 BRA AhRKO) ³ originally obtained from the Jackson Laboratory via Dr. A. Smith at Leicester University were bred in the SPF facility at NIMR. All animal experiments were done according to institutional guidelines and Home Office regulations.

In vitro T cell differentiation and cytokine staining

FACS sorted naive T cells (CD4⁺, CD25^", CD44^|OW) were cultured in 24 well plates as described in detail³⁷ under THO (αlFNy + αlL-4 + αTGFβ), TH1 (IL-12), TH2 (IL4) , iTreg (TGFβ) or TH17 (IL-6 + TGFβ+ 1L-1 β) conditions. Neutralizing antibodies were used at a concentration of 10μg/ml, clone R46A2 anti IFNy, clone 11 B11 anti-IL-4 and clone 1D11 anti-TGFβ. The Ahr ligand 6-formylindolo[3,2-b] carbazole (BioMol) was added in some experiments at the start of culture. For measurements of intracellular cytokines T cells were restimulated with 500ng/ml Phorbol dibutyrate (PdBU), 500ng/ml ionomycin in the presence of brefeldin A for 4h on day 5 after initiation of cultures. IL-17A antibody was obtained from eBioSciences, IL-17F antibody from R&D. The anti-IL-22 antibody (MH22B2) was generated in IL-22 KO Balb/c mice immunized with recombinant human IL-22 crosslinked to ovalbumin in the presence of glutaraldehyde and cross-reacts with human and murine IL-22.

Human T cell culture

Human PBMC were cultured in plates coated with anti-CD3 (1μg/ml) and anti-CD28 (1μg/ml) in the presence of 10ng/ml IL-1 and 40ng/ml IL-6. qPCR analysis for Th17 markers and AhR expression was performed on day 4.

Real time PCR The expression of mRNA for transcription factors and cytokines in CD4 T cell subsets was analysed 4-5 days after T cell activation using specific primers from Applied Biosystems and expression was normalized to the housekeeping gene HPRT.

Retroviral transduction

AhR was cloned into vector plRES2-EGFP (Clontech) generating a bicistronic mRNA encoding AhR and, separated by an IRES element, EGFP. Viruses were generated by simultaneous

CaCI2-mediated transient transfections of 293T cells with three plasmids providing vector, gag- pol, and env functions. FACS sorted naive CD4 T cells were plated in antibody coated wells as described above in the absence of cytokines on day 0. On days 1 and 2 fresh retrovirus supernatant was added and the cells were spun for 1h at 1200 rpm. Thereafter the cells were cultured in the presence or absence of 10OnM AhR ligand FICZ for another 4 days and then assayed by RT-PCR and intracellular staining.

EAE induction EAE was induced and scored as described previously³⁸. Some mice received 200ng FICZ in the antigen emulsion. CD4 T cells from draining lymph nodes of MOG immunized mice were FACS sorted on the basis of CCR6 expression and processed for RT-PCR analysis as described above. Spinal cord was isolated on day 18 after EAE induction for determination of T H17 cell numbers.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described invention will be apparent to those of skill in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

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Claims

1. An inhibitor of AhR activation for use in a method of treating an autoimmune disease in a subject.

2. An inhibitor for use according to claim 1 wherein said inhibitor is a fiavone or 2-Methyl- 2H-pyrazole-3-carboxyiic acid-(2-methyl-4-o-tolylazophenyl)-amide.

3. An inhibitor for use according to claim 1 wherein said inhibitor is an antisense or interfering RNA molecule.

4. An inhibitor for use according to any one of the preceding claims wherein said autoimmune disease is multiple sclerosis.

5. An inhibitor for use according to any one of claims 1 to 3 wherein said autoimmune disease is multiple sclerosis.

6. A method for screening a compound for use as an inhibitor of an autoimmune disease, which method comprises determining whether said compound is able to inhibit AhR activation.

7. A method for the treatment of an IL-22 mediated autoimmune disease or an inflammatory disorder in a subject, which method comprises administering to the subject an AhR inhibitor.

8. The method of claim 8 wherein said disorder is mediated by IL-22 production in the Th 17 subset of cells.