WO2022032592A1

WO2022032592A1 - Interleukin-17d and cd93 as a new cytokine-receptor pair in the immune system

Info

Publication number: WO2022032592A1
Application number: PCT/CN2020/109014
Authority: WO
Inventors: Chen Dong; Jinling Huang; Xiaohu Wang
Original assignee: Tsinghua University
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2022-02-17

Abstract

Use of reagent in preparing medicament is provided, wherein the reagent is used for regulating IL-17D-CD93 axis, and the medicament is used for treating or prevention of inflammatory or immune diseases, including dysregulation of intestinal homeostasis.

Description

Interleukin-17D and CD93 as a new cytokine-receptor pair in the immune system

FIELD OF THE INVENTION

The present invention relates to the field of medicinal technology, in particular, to the treatment or prevention of intestinal homeostasis related diseases in a subject.

BACKGROUND OF THE INVENTION

Among the six structurally related members (IL-17A-F) in the IL-17 family, IL-17A and IL-17F, two most homologous cytokines produced by lymphocytes including Th17, ILC3 and γδ17 cells, signal through IL-17RA/IL-17RC and IL-17RA/IL-17RD complexes to promote the development of autoimmune diseases while playing important roles in host defense against bacterial and fungal infections. Other members in this family, including IL-17E, IL-17B, and IL-17C, are produced by epithelial cells and bind to receptor complexes on epithelial cells to regulate tissue inflammation, and also mediate differentiation and function of specific lymphocyte subsets.

While IL-17 family members emerge as important mediators in mucosal immunity, and serve as important drug targets in inflammatory disases, IL-17D is least studied in this family. Although it was initially shown to promote IL-6, IL-8, and GM-CSF production in endothelial cells, and recently, studies showed that overexpression of IL-17D in poorly immunogenic cancer cells mediate tumor rejection.

However, the physiological roles of IL-17D in the immune system are still largely unknown and its receptor has not been identified.

Innate lymphoid cells (ILCs) represent a family of heterogeneous lymphocytes that lack variable antigen receptors yet produce cytokines similar to CD4+ helper T cell subsets. Based on the expression of cytokines and transcription factors, ILCs are divided into three main groups: IFN-γ-producing ILC1s that depend on T-bet; IL-5-and IL-13-producing ILC2s that express Gata-3; IL-17-and/or IL-22-producing ILC3s that require RORγt. ILCs typically reside at mucosal tissues and promptly produce effector cytokines in response to environmental stimuli, thus playing critical roles in host defense against pathogens, regulation of adaptive immune responses, deveopment of lymphoid architectures, as well as tissue remodeling.

ILC3s, further subdividing into natural cytotoxicity receptor (NCR) -ILC3s, NCR+ ILC3s and LTi cells, were the first characterized ILC subset in tonsils in human and intestinal lamina propria in both mice and human. Previous studies have established the importance of ILC3 cells in intestinal homeostasis at the steady state and in models of intestinal inflammation; they produce IL-22 to trigger epithelial cells to produce antimicrobial peptides, such as RegIIIβ and RegIIIIγ, offering innate immunity to infection. Moreover, Il22-/-mice also exhibited accelerated tumorigenesis than wild-type mice in a colon cancer model.

ILC3 cells arise from hematopoietic progenitors in the bone marrow and ILC3 cells in peripheral tissues are functionally mature in expressing transcription factors RORγt and AhR, which allow their rapid production of IL-22 upon IL-23 stimulation. Thus, instead of de novo antigen-triggered effector differentiation in helper T cells, ILC3s act as “pre-primed” effector cells and respond rapidly to cytokine IL-23 to produce IL-22. Although the initial lineage development of ILC3 precursor cells in the bone marrow has been characterized, how they are regulated in the peripheral tissues for their effector function and IL-22 production in peripheral tissues remains poorly understood.

SUMMARY OF THE INVENTION

The following is only an overview of some aspects of the present invention, but is not limited thereto. All references of this specification are incorporated herein by reference in their entirety. When the disclosure of this specification is different with citations, the disclosure of this specification shall prevail.

In the research and development process, the inventors have characterized the role of IL-17D in the immune system and identified its receptor, CD93. They showed a role for this new cytokine-receptor pair in the selective regulation of ILC3s function during intestinal homeostasis. IL-17D-CD93 axis thus exert a protective role at the barrier surfaces against intestinal inflammation.

In the first aspect of present disclosure, there is provided use of reagent in preparing medicament, wherein the reagent is used for regulating IL-17D-CD93 axis, and the medicament is used for treating or prevention of inflammatory or immune diseases, including dysregulation of intestinal homeostasis related diseases. The inventors have found that IL-17D-CD93 axis exert a protective role during intestinal homeostasis, therefore, the medicament prepared with the reagent used for regulating IL-17D-CD93 axis can be used for regulation of intestinal homeostasis and treating or prevention of dysregulation of intestinal homeostasis related diseases effectively.

In some examples of present disclosure, the dysregulation of intestinal homeostasis related diseases comprise at least one of the following: microbiota dysbiosis, colitis, colon cancer, inflammatory bowel diseases (IBD) , pathogen infection. crohn’s disease and ulcerative colitis .

In some examples of present disclosure, the reagent is used for activating or upregulating IL-17D and/or CD93 or enhancing the binding of IL-17D and CD93, and the medicament is used for treating or prevention of microbiota dysbiosis, colitis, colon cancer, inflammatory bowel diseases or pathogen infection. In some examples of present disclosure, the reagent comprises at least one of the following: IL-17D, Nrf2 agonists, nucleic acid used for overexpression of IL-17D and/or CD93. For example, nucleic acid used for overexpression of IL-17D and/or CD93 has a nucleotide sequence shown as any one of SEQ ID NO: 1 and 2.

The sequence for over expression of IL-17D:

The sequence for over expression of CD93:

In some examples of present disclosure, the reagent is used for suppressing or downregulating IL-17D and/or CD93 or preventing the binding of IL-17D and CD93, and the medicament is used for treating or prevention of crohn’s disease and ulcerative colitis. In some examples of present disclosure, the reagent comprises at least one of the following: nucleic acid used for downregulating IL-17D and/or CD93, MMRN2 siRNA and MMRN2 inhibitor, wherein the MMRN2 siRNA or MMRN2 inhibitor can be used for downregulating CD93. And for example, nucleic acid used for downregulating IL-17D and/or CD93 has a nucleotide sequence shown as any one of SEQ ID NO: 3～5.

If using CRIPER-cas 9 to downregulate CD93, gRNA sequence can be as following:

CD93-gRNA: GCCCCAATGGGCTGTATAGC (SEQ ID NO: 3) .

If using shRNA to downregulate CD93, sequence can be as following:

If using shRNA to downregulate IL-17D, sequence can be as following:

Actually, ILC3 function as a double-edged sword in gut. ILC3 are responsible for intestinal homeostasis through moderate generation of IL-22 in the physiological state. However, ILC3 also contribute to the progression and aggravation of IBD (such as crohn’s disease and ulcerative colitis) while IL-22 is overexpressed by dysregulation of ILC3 in the pathological state.

In the second aspect of present disclosure, there is provided use of IL-17D-CD93 axis in screening monoclonal antibodies or other proteins/peptides or small molecules that preventing the binding of IL-17D and CD93.

In the third aspect of present disclosure, there is provided a method of enhancing IL-22 production by ILC3 cells. According to the embodiments of present invention, the method comprises: 1) using a chemical or biological activator to enhance the activity of IL-17D and/or CD93; 2) using a chemical or biological regent to enhance the amount of IL-17D and/or CD93; 3) using a chemical or biological regent to enhancing the binding of IL-17D and CD93.

In the fourth aspect of present disclosure, there is provided a method of impairing IL-22 production by ILC3 cells. According to the embodiments of present invention, the method comprises: 1) using a chemical or biological inhibitor to suppress the activity of IL-17D and/or CD93; 2) using a chemical or biological regent to reduce the amount of IL-17D and/or CD93; 3) using a chemical or biological regent to prevent the binding of IL-17D and CD93.

In the fifth aspect of present disclosure, there is provided a method of treating or prevention of dysregulation of intestinal homeostasis related disease, wherein the dysregulation of intestinal homeostasis related disease is caused by insufficient secretion of IL-22 by ILC3 cells or low activity of ILC3. According to the embodiments of present invention, the method comprises: administrating a promoter for IL-17D expression and secretion by colonic epithelial cells; administrating an activator for enhancing the activity of IL-17D; administrating a promoter for CD93 expressing on ILC3 cells; administrating an activator for enhancing the activity of CD93; or administrating an enhancer for enhancing the binding of IL-17D and CD93.

In some examples of present disclosure, the dysregulation of intestinal homeostasis related diseases comprise at least one of the following: microbiota dysbiosis, colitis, colon cancer, inflammatory bowel diseases, pathogen infection.

In the sixth aspect of present disclosure, there is provided a method of treating or prevention of dysregulation of intestinal homeostasis related disease, wherein the dysregulation of intestinal homeostasis related disease is caused by excessive secretion of IL-22 by ILC3 cells or over activity of ILC3. According to the embodiments of present invention, the method comprises: administrating an inhibitor for IL-17D expressing and secretion by colonic epithelial cells; administrating an inhibitor for suppressing the activity of IL-17D; administrating an inhibitor for CD93 expressing on ILC3 cells; administrating an inhibitor for suppressing the activity CD93; or administrating an inhibitor for preventing the binding of IL-17D and CD93.

In some examples of present disclosure, the dysregulation of intestinal homeostasis related diseases comprise at least one of the following: crohn’s disease and ulcerative colitis.

In the seventh aspect of present disclosure, there is provided a method of screening medicament, wherein the medicament is used for treatment or prevention of dysregulation of homeostasis related diseases, wherein the dysregulation of intestinal homeostasis related disease is caused by insufficient secretion of IL-22 by ILC3 cells or low activity of ILC3. According to the embodiments of present invention, the method comprises: contacting a candidate medicament with colonic epithelial cells, the elevation of the expression of IL-17D or the upregulating of IL-17D activity or the enhancing of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.

In the eighth aspect of present disclosure, there is provided a method of screening medicament, wherein the medicament is used for treatment or prevention of the dysregulation of intestinal homeostasis related diseases, wherein the dysregulation of intestinal homeostasis related disease is caused by insufficient secretion of IL-22 by ILC3 cells or low activity of ILC3. According to the embodiments of present invention, the method comprises: contacting a candidate medicament with ILC3 cells, the elevation of the expression of CD93 or the upregulating of CD93 activity or the enhancing of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.

In the ninth aspect of present disclosure, there is provided a method of screening medicament, wherein the medicament is used for treatment or prevention of the dysregulation of intestinal homeostasis related diseases, wherein the dysregulation of intestinal homeostasis related disease is caused by excessive secretion of IL-22 by ILC3 cells or over activity of ILC3. According to the embodiments of present invention, the method comprises: contacting a candidate medicament with colonic epithelial cells, the downregulating of the expression of IL-17D or the IL-17D activity or the inhibition of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.

In the tenth aspect of present disclosure, there is provided a method of screening medicament, wherein the medicament is used for treatment or prevention of the dysregulation of intestinal homeostasis related diseases, wherein the dysregulation of intestinal homeostasis related disease is caused by excessive secretion of IL-22 by ILC3 cells or over activity of ILC3. According to the embodiments of present invention, the method comprises: contacting a candidate medicament with ILC3 cells, the downregulating of the expression of CD93 or the CD93 activity or the weakening of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.

The methods described above can screen medicament used for treatment or prevention of homeostasis related diseases effectively.

The foregoing merely summarizes certain aspects disclosed herein and is not intended to be limiting in nature. These aspects and other aspects and additional embodiments, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference the accompanying schemes and drawings, in which:

Fig. 1 shows IL-17D protects against DSS-induced colitis,

Wherein, (A-C) Real-time RT-PCR analysis of Il17d mRNA expression in various tissues of adult mice (A and B) and neonatal mice (C) (n=3) . (D) Quantitative RT-PCR analysis of the Il17d mRNA in the small intestine and colon of SPF mice and GF mice (n=3) . (E) Weight loss of wild-type control mice (n=7) and Il17d-/-mice (n=5) during DSS-induced colitis. (F) Colon length (n=7, WT; n=5, Il17d-/-) at day 9 of the colitis model. (G and H) H&E staining (G) and histological scores (H; n=7, WT; n=5, Il17d-/-) of the colons as in (F) . Scale bar represents 100 μm. (I) Real-time RT-PCR analysis of mRNA expression of pro-inflammatory cytokines in colons of WT and Il17d-/-mice at day 0 or day 9 during DSS-induced colitis (n=4) . Data are a representative of three independent experiments. Data are shown as mean ± s.e.m. *p value < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA (B, C) or Student’s t test (E, F, H, I) ;

Fig. 2 shows IL-17D in non-hematopoietic cells is required for protection against colitis,

(A) Quantitative RT-PCR analysis of the Il17d mRNA in intestine epithelial cells (IECs) and CD45+ leukocytes isolated from wild-type mice at

day

0, 4, 8 post colitis induction (n=3) . (B to E) The bone marrow chimeric mice of indicated genotypes were fed with 2.5%DSS water for 5 days, and body weight (B) , colon length (C) , and histopathology (D and E) were analyzed (n=4) . Scale bars represent 100 μm. Data are representative of two independent experiments. Results shown are mean ± SEM. *p< 0.05, **p < 0.01 by two-way ANOVA.;

Fig. 3 shows IL-17D administration partially rescued the disease symptoms of colitis in Il17d-/-mice,

(A) Relative mRNA expression of indicated genes in colons of WT and Il17d-/-mice at day 9 of colitis (n=4) . (B to D) The body weight change (B, n=4) and colon length (C; D, n=4) in indicated mice treated with or without IL-17D in the colitis model. (E) Representative colon HE staining (left) and histological score (right) of indicated mice are shown. Scar bars represent 100 μm. (F and G) Relative Il22 (F, n=4) , Reg3b and Reg3g mRNA expression (G, n=4) in the colon tissue of indicated mice. Data are representative of at least two independent experiments or pooled from three independent experiments (B) . Results are shown as mean ± SEM. *p< 0.05, **p<0.01, ****p < 0.0001 by Two-way ANOVA (B, D, H) or Student’s t test (A, E, G, I) ;

Fig. 4 shows IL-17D is essential for IL-22 production by ILC3,

Small intestinal lamina propria lymphocytes (LPLs) were isolated from 6-to 8-week-old wild-type and Il17d-/-mice. (A) Analysis of different populations of ILC3s (live CD45+CD3-CD90.2+ RORγt+ cells) by flow cytometry. Numbers indicate percentages of cells in each gate. (B) Absolute numbers of total ILC3s, Nkp46+ ILC3s, CD4+ ILC3s, CD4-Nkp46- (DN) ILC3s (n=6, WT; n=4, Il17d-/-) . (C) Expression of Ki-67 by ILC3s in SI LPLs of WT and Il17d-/-mice (n=4, WT; n=3, Il17d-/-) . (D and E) Representative cytokine staining for analysis of IL-22 production by ILC3s without (D) or with (E) IL-23 stimulation in vitro for 4 hrs. (F and G) Statistic analysis of the percentages (F) and absolute numbers (G) of IL-22-producing ILC3 subsets stimulated with IL-23 (n=5, WT; n=4, Il17d-/-) . (H) Heatmap of selected differentially expressed genes between ILC3s from wild-type and Il17d-/-mice. Data are a representative of three (A to G) independent experiments. Data are shown as mean ± s.e.m. *p < 0.05, **p < 0.01, ****p < 0.0001 (unpaired t test) ;

Fig. 5 shows Il17d-deficient mice show dysregulated fecal microbiota,

(A to D) Operational taxonomic unit (OUT) abundances (A) , Shannon-Wiener diversity index (Shannon index) (B) , observed species index (C) , and Principal coordinates analysis (PCoA) (D) of fecal isolated from WT (n=4) and Il17d-/- (n=4) mice on day 0 or day 9 of colitis by 16S rRNA sequencing. (E) The relative abundance of microbiota at phylum level in the fecal samples (n=4) as in (A) . (F) Heatmap analysis of the relative abundance of microbiota at family level in the fecal samples (n=4) as in (A) . Data are representative of two independent experiments. Results shown are mean ± SEM. *p< 0.05, **p < 0.01, ***p < 0.001 by Student’s t test;

Fig. 6 shows IL-17D binds to CD93,

(A) Cultured RAW 264.7 cells were cross-linked with 30 μg/ml human IgG or mouse IL-17D-human IgG1 Fc fusion protein, followed by Western blot analysis probed with HRP conjugated anti-human IgG. (B to E) Flow cytometry analysis of IL-17A or IL-17D binding to 293T cells transfected with empty, IL-17RA, or CD93 expression vector. 293T cells were subsequently incubated 30 min with His-tagged proteins (B) , human IgG fusion proteins (C) , biotin labelled proteins (D) , or commercial proteins bought from R&D systems (E) , followed by staining with anti-His (B) , anti-human IgG (C) , streptavidin (E) , anti-IL-17A or anti-IL-17D antibodies (E) . (F) Schematic diagram of CD93 extracellular domains with different deletions. CTLD, C-type lectin like domain; X, domain of unknown structural function; EGF-like, EGF-like repeats; Mucin, mucin-like domain; TM, transmembrane domain. (G) Flow cytometry analysis of biotin labelled IL-17D binding to 293T cells transfected as in F. (H) SPR affinity measurements of IL-17D to the immobilized extracellular domain of CD93. Representative sensorgrams are plotted as resonance units (RUs) versus time. Data are a representative of three (B-E, G) or two (H) independent experiments;

Fig. 7 shows CD93 plays a protective role in DSS-induced colitis and colitis-associated colon cancer (CAC) ,

(A) Flow cytometric staining of CD93 expression in ILC3s isolated from the small intestine lamina propia in WT mice stimulated with IL-23 (Gated on live CD45+ CD3-CD90.2+ RORγt+cells) . (B) Flow cytometry staining of IL-17D-hIg binding to ILC3s isolated from the SI LPL of WT and Cd93-/-mice. Left: CD93 staining; Middle: hIg binding control; Right: IL-17D-hIg binding. (C) Weight loss of WT (n=7) and Cd93-/-mice (n=5) in the colitis model. The colon length (n=7, WT; n=5, Cd93-/-) in DSS-induced colitis model (Day 8) . (D) WT mice (n=5) and Cd93-/-mice (n=4) were subjected to develop CAC model and euthanized on day 80. Representative images of colon tumors and tumor number were shown. (E and F) Relative mRNA expression of indicated genes in colons of WT and Cd93-/-mice (n=6-7) . (G and H) The body weight change (G) and colon length (H) on day 8 in indicated mice treated with or without IL-22 in the colitis model (n=4-5) . Data are a representative of three (A to C) or two (D, G, H) independent experiments, or pooled from two experiments (E and F) . Data are shown as mean ± s.e.m. *p < 0.05, **p < 0.01, ****p < 0.0001 by unpaired t test (C to F, H) , two-way ANOVA (G) ; and

Fig. 8 shows CD93 regulates ILC3 function,

(A) Mixed bone marrow chimera were generated by reconstituting CD45.1 lethally irradiatd recipients with WT (CD45.1CD45.2) and Cd93-/- (CD45.2) bone marrow. (B, D) Analysis of the frequency of wild-type or CD93-knockout ILC3s and IL-22-producing ILC3s among total ILCs in the colon (B) and small intestine (D) of mice (n=5) . (C, E) Analysis of the ratio of different intestine lymphocyte subsets (T, ILC1, ILC2, ILC3, IL-22+ ILC3) from WT to CD93-deficient donor. Data are a representative of two independent experiments. Data are shown as mean ± s.e.m. **p < 0.01 by paired t-test (B, C) .

MOBODIMENT

Definition

Unless otherwise indicated, otherwise the following terms and phrases as used herein will have the following meanings:

As used herein, the term “IL-17D-CD93 axis” refers to IL-17D in the intestine identifies its receptor, CD93. IL-17D-CD93 axis is in the selective regulation of ILC3s function during intestinal homeostasis.

As used herein, the term "treat" , "treating" or "treatment" of any disease or disorder refers to all processes wherein there may be a slowing, interrupting, arresting, controlling, or stopping of the progression of the disease or disorder, but does not necessarily indicate a total elimination of all the disorder symptoms, as well as the prophylactic therapy of the mentioned conditions, particularly in a patient who is predisposed to such disease or disorder. In some embodiments, "treat" , "treating" or "treatment" refers to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof) . In other embodiments, "treat" , "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In other embodiments, "treat" , "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom) , physiologically, (e.g., stabilization of a physical parameter) , or both. In other embodiment, "treat" , "treating" or "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder. As used herein, the term "therapeutically effective amount" or "therapeutically effective dosage" refers to the amount of the regent of the invention which is capable of eliciting biological or medical response (Such as alleviating symptoms, slowing or delaying the development of the disease, or preventing diseases, etc. ) of an individual. In one non-limiting embodiment, the term "therapeutically effective amount" refers to, when the regent of the present invention is administered to a subject. In other embodiment, the term "therapeutically effective amount" refers to, when administering the cell, or organ, or non-cellular biological material, or medium, an effective amount of the regent of the present invention, which can be at least used for regulating IL-17D-CD93 axis.

Regents or pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising the reagent used for regulating IL-17D-CD93 axis. According to the specific examples of the present invention, the pharmaceutical composition can further comprise pharmaceutically acceptable excipient, carrier, adjuvant, solvent and a combination thereof. The present invention provides a method of treating, preventing or ameliorating a disease or disorder, comprising administrating a safe and effective amount of a combination of drugs containing regents and one or more therapeutic active agents. Among them, the combination of drugs comprises one or more additional drugs for treatment of dysregulation of intestinal homeostasis related disease. Other drugs for treatment autoimmune diseases are not limited to: Aminosalicylates (5-ASA) , Antibiotics, Biologics, Corticosteroids, Immunomodulators, Small molecules.

The amount of the regent of the pharmaceutical composition disclosed herein refers to an amount which can be effectively regulating IL-17D-CD93 axis. The dosage of active ingredient in the compositions of this invention may be varied, however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The active ingredient may be administered to patients (animals or human) in need of such treatment in dosage that will provide optimal pharmaceutical efficacy. The selected dosage upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. The dosage will vary from patient to patient depending upon the nature and severity of disease, the patient's weight, special diet then being followed by a patient, concurrent medication, and other factors which those skilled in the art will recognize.

It will also be appreciated that certain of the regents of the present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative or a prodrug thereof. A pharmaceutically acceptable derivative includes pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative which upon administration to a patient in need thereof is capable of providing, directly or indirectly, a regent as otherwise described herein, or a metabolite or residue thereof.

When the pharmaceutical compositions of the present invention also contain one or more other active ingredients, in addition to a regent of the present invention, the weight ratio of the regent of the present invention to the second active ingredient may be varied and depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a regent of the present invention is combined with another agent, the weight ratio of the regent of the present invention to the other agent will generally range from about 1000: 1 to about 1: 1000, such as about 200: 1 to 1: 200. Combinations of a regent of the present invention and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.

"Pharmaceutically acceptable excipient" as used herein means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled, such that interactions which would substantially reduce the efficacy of the regent of the invention when administered to a patient and would result in pharmaceutically unacceptable compositions are avoided. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically acceptable.

Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting the regent of the present invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance. Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweetners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents.

The skilled artisan will appreciate that certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation. Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically acceptable excipients in appropriate amounts for use in the invention. In addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company) , The Handbook of Pharmaceutical Additives (Gower Publishing Limited) , and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press) .

In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams &Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, the contents of each of which is incorporated by reference herein, are disclosed various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the regents of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component (s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention.

The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company) .

Therefore, another aspect of the present invention is related to a method for preparing a pharmaceutical composition. The pharmaceutical composition contains the regent disclosed herein and pharmaceutically acceptable excipient, carrier, adjuvant, vehicle or a combination thereof, the method comprises mixing various ingredients. The pharmaceutical composition containing the regent disclosed herein can be prepared at for example environment temperature and under barometric pressure.

The pharmaceutical compositions provided herein may be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.

The pharmaceutical compositions provided herein may be administered parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral administration, as used herein, include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

The pharmaceutical compositions provided herein may be formulated for single or multiple dosage administration. The single dosage formulations are packaged in an ampoule, a vial, or a syringe. The multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.

In one embodiment, the pharmaceutical compositions are provided as ready-to-use sterile solution. In another embodiment, the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders and hypodermic tablets, to be reconstituted with a vehicle prior to use. In yet another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile suspensions. In yet another embodiment, the pharmaceutical compositions are provided as sterile dry insoluble products to be reconstituted with a vehicle prior to use. In still another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile emulsions.

The regents of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agents. The regents of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition .

The following examples are provided so that the invention might be more fully understood. However, it should be understood that these embodiments merely provide a method of practicing the present invention, and the present invention is not limited to these embodiments.

How group 3 innate lymphoid cells (ILC3) , important in mucosal immunity and barrier maintenance, are regulated in peripheral tissues is unclear. Here we show that interleukin 17D (IL-17D) , expressed by colonic epithelial cells, regulates ILC3 function. Il17d deficiency impaired IL-22 production by ILC3 cells, which resulted in microbiota dysbiosis, worsened severity in acute colitis and increased development of colon cancer. Via protein purification, IL-17D was found to interact with CD93, a glycoprotein expressed on ILC3 cells. Mice lacking Cd93 also showed aggravated colonic inflammation in experimental colitis and CD93 expression in ILC3 cells was required for their function. Thus, our work has demonstrated an IL-17D-CD93 axis important in tissue-specific regulation of ILC3 cell function required for intestinal homeostasis.

The related methods are described as follows:

Mice

All animal experiments conducted at Tsinghua University were approved by the Institutional Animal Care and Use Committee. Mice used in this study were housed under specific-pathogen-free (SPF) condition and on the C57BL/6 background. C57BL/6 and GF mice were obtained from animal facility of Tsinghua University. Il17d-/-mice was purchased from MMRRC facility (032380-UCD-SPERM) as cryo-preserved spermatozoa. Cd93-/-mice were generated at Tsinghua University as described below.

Generation of Cd93-/-mice

The pcDNA3.1-Cas9 plasmid was digested with XbaI to synthesis Cas9 mRNA using mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies) . The pUC19-sgRNA plasmid encoding gRNA sequence was in vitro transcribed using MEGAshortscript T7 Transcription Kit (Life Technologies) . Cas9 mRNA and gRNA were subsequently purified with MEGAclearTM Transctiption Clean-Up Kit (Life Technologies) and resuspended in RNase-free water. The mixture of Cas9 mRNA and gRNA was injected into 0.5-day zygotes of C57BL/6 mice. CRISPR/Cas9 knockout founder mice were crossed with WT mice to generate F1 generation mice.

Mouse model

Age-matched male mice were given drinking water containing 2.5%DSS for 5 days, and then followed by regular water for 3-4 days. The DSS water was replaced daily. For the IL-17D rescue experiment, mice were injected i.p. with 0.5 μg recombinant mouse IL-17D (R&D) or IL-22 (R&D) everyday starting from Day 3. Body weight was monitored throughout the experiments. To induce colon cancer model, WT and Il17d-/-mice were injected intraperitoneally with AOM (10mg/kg) . 5 days later, 2.5%DSS was added to the drinking water for 5 consecutive days, followed by regular water for 14 days. This cycle was repeated three times. Mice were sacrificed for analysis on day 80 of the experiments. Mice were orally gavaged with 2×109 CFU C. rodentium (ATCC 51459) and weighed daily after infection. Bacterial load was assessed by plating fecal homogenates overnight on MacConkey.

LPMCs Isolation and Flow Cytometry Analysis

The intestine tissues were harvested from indicated mice, longitudinally opened and cut into small pieces. The LPMCs were isolated as described previously. Briefly, the tissue pieces were incubated in pre-digestion media (RPMI 1640 containing 20mM HEPES, 5mM EDTA, 1%penicillin and streptomycin, 1mM DTT) for 30min at 37 ℃. After washing, the pieces were incubated in digestion buffer (RPMI 1640 supplemented with 1%penicillin and streptomycin, 20mM HEPES, 0.5mg/ml Collagenase D, 1mg/ml Dispase, 0.5mg/ml DNase) for 30min at 37 ℃. After filtering, leukocytes were purified on Percoll density-gradient separation. Purified LPMCs were washed and stimulated with 5ng/ml IL-23 or 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of GolgiPlug (BD) for 4 hr prior to staining for flow cytometry analysis by LSR Fortessa (BD) . Flurochrome-conjugated antibodies against CD45 (clone 30-F11) , CD3 (clone 500A2) , CD90.2 (clone 53-2.1) , Nkp46 (clone 29A1.4) , CD4 (clone RM4-5) , RORγt (clone Q31-378) , IL-22 (clone IL22JOP) , CD45.1 (clone A20) , CD45.2 (clone 104) , CD93 (AA4.1) , TCRγδ (clone GL3) , IFN-γ (clone XMG1.2) , IL-17A (clone eBio17B7) , Ki67 (BD, catalog no. 556026) were purchased from eBioscience or BD Biosciences. Cell surface staining was performed by incubating cells with antibodies for 30 min at 4 ℃ after blocking with Fc antibody. RORγt, Ki67, IL-22, IFN-γ, IL-17A staining was carried out using intracellular transcription factor kit (eBioscience) or cytokine staining kit (BD Biosciences) .

Histology

Colons from indicated mice were dissected and immediately fixed in 10%formalin. Paraffin-embedded sections were staining with H&E. Histological scores of colon tissue were assessed with scoring system of Wirtz et al.

RNA Isolation and Gene Expression Analysis

Total RNA was extracted from indicated colon tissues with TRIzol reagent (Invitrogen) and subjected to reverse-transcription with M-MLV system (Invitrogen) according to the manufacturer’s protocol. The cDNA samples were amplified by real-time PCR using Hieff ^TM qPCR

Green Master Mix with specific primers. Rpl13a or Gapdh was chosen as the internal control for the quantitative PCR analysis. Il17d: 5’ -TGAGTGCATAATTGTAGTGCTCAG-3’ and 5’ -ACTCAGGGACAGGGCACA-3’ ; Il22: 5’ -CATGCAGGAGGTGGTACCTT-3’ and 5’ -CAGACGCAAGCATTTCTCAG-3’ ; Reg3b: 5’ -CTCTCCTGCCTGATGCTCTT-3’ and 5’ -GTAGGAGCCATAAGCCTGGG-3’ ; Reg3g: 5’ -TCAGGTGCAAGGTGAAGTTG-3’ and 5’ -GGCCACTGTTACCACTGCTT-3’ ; Il6: 5’-GATGGATGCTACCAAACTGGAT-3’ and 5’ -CCAGGTAGCTATGGTACTCCAGA-3’ ; Il1b: 5’-TGTAATGAAAGACGGCACACC-3’ and 5’ -TCTTCTTTGGGTATTGCTTGG-3’ .

Microbiota sequencing

Fresh mouse fecal samples were collected and microbial DNA was isolated from stools of indicated mice by using QiAamp DNA Stool Mini Kit (QIAGEN) according to manufacturer’s instructions. DNA concentration was checked with Nanodrop (Thermo Scientific) . The V4 regionsof the bacteria 16S rRNA gene were amplified by PCR using primers 515F-806R. The 16S rDNA amplicons were sequenced on an Illumina (HiSeq 2500) platform by Beijing Genomics Institute.

Generation of BM Chimera

Bone marrow cells from WT and Il17d-/-donor mice were collected and flushed with 1x PBS. Cell suspension containing 3×10 ⁶ donor BM cells was intravenously injected into lethally irradiated WT and Il17d-/-recipient mice (1050 rads) . To generate mixed bone marrow chimeras, bone marrow cells were isolated from CD45.1/2 Cd93+/+ and CD45.2 Cd93-/-mice. After depleted red blood cells, the remaining cells were resupended in PBS for injection at a ratio of 1: 1 (WT: CD93 KO) , 3×10 ⁶ cell mixture was injected intravenously into lethally irradiated CD45.1 recipients (2x550 rads, at an interval of 2h) . Experiments were performed 6-7 weeks after reconstitution.

Expression of IL-17D fusion protein and binding assay

The expression vectors of IL-17D-hIg fusion protein or his-tagged IL-17D were first generated by cloning the Il17d gene sequence (Genbank accession number NM_145837.3) into pVRC vector and transfected into 293F cells. The secreted IL-17D-hIg and IL-17D-his protein were purified with a protein A column and

Metal Affinity Resins, respectively. For the binding assay, 293T cells were transfected with indicated vectors, The IL-17D fusion proteins or recombinant proteins bought from R&D System were added to the transfected 293T cells for 30 min, followed by staining with indicated antibodies conjugated with proper flurochrome.

Cross-linking with IL-17D-hIg Protein and IP-MS

RAW264.7 cells plated on 15 cm dishes were incubated with 30 μg/ml IL-17D-hIg fusion protein or control human IgG for 30 min. Cells were cross-linked by 0.1%formaldehyde and then stopped with 0.2M Glycine, and subsequently lysed with 2 ml of buffer containing 50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10%glycerol, 0.5%NP-40, and 1× protease inhibitor cocktail. The cell lysates were precipitated with 250 μl of Protein A beads, washed with NETN buffer (20 mM Tris-HCl pH 7.8, 100 mM NaCl, 1 mM EDTA, 0.5%NP-40) , and eluted with 50 μl of non-5 reducing SDS-PAGE loading buffer. The samples were then subjected to 8%SDS-PAGE followed by western blot analysis probed with HRP conjugated anti-human IgG or coomassie blue staining. The indicated gels bands were cut and subjected to mass spectrometry by Centre for Protein Technology of Tsinghua University.

Surface Plasmon Resonance (SPR)

CD93-ECD-Fc (R&D, catalog no. 1696-CD-050) was immobilized to a CM5 sensorchip (GE Healthcare) to a level of ～750 response units (RUs) using a Biacore T200 (GE Healthcare) and a running buffer composed of 10 mM HEPES pH 7.2, 150 mM NaCl and 0.05%Tween 20. Serial dilutions of mIL-17D (R&D, catalog no. 2274-ML-025/CF) was flowed through with a concentration ranging from 150 to 9.375 nM. The resulting data were fit to a 1: 1 binding model using Biacore Evaluation Software (GE Healthcare) .

RNA-seq

CD45 ^lowCD3-CD90.2 ^high ILC3s were sorted from small intestine LPLs of indicated mice. Total RNAs were isolated with TRIzol reagent (Invitrogen) and the RNA-seq library was built by the Beijing Genomics Institute. Sequence reads were obtained by BGISEQ-500 and the clean reads were mapped to mouse genome. Gene expression was indicated by RPKM (Reads Per Kilobases per Million reads) , and genes with at least 1.5 fold changes were used to select as differentially expressed genes.

Statistical Analysis

The data were analyzed by Graph Prism 6.0 software and presented as mean ± SEM. A two-tailed paired or unpaired student’s t test was used for statistic analysis. For multiple group comparisons, statistical analysis was performed with two-way ANOVA. p values less than 0.05 were considered statistically significant.

Results

Il17d expression in epithelial cells is required to control colonic inflammation

To understand the physiological function of IL-17D, we first assessed its mRNA expression in multiple mouse tissues by quantitative RT-PCR. High levels of Il17d expression was observed in the colon as well as lung, but not in thymus or bone mrrow of adult mice (Fig. 1A-B) , while small intestine and colon of newborn mice also expressed substantial levels of Il17d (Fig. 1C) . Il17d expression in intestine exhibited no difference between SPF and germ-free animals (Fig. 1D) , suggesting that it was not dependent on microbiota. Interestingly, the expression of Il17d was higher in colon than in small intestine (Fig. 1B-D) .

Il17d-/-mice did not exhibit any significant difference in their T cells, dendritic cells or macrophages from thymus, spleen, small intestine and colon compared to WT mice, and appeared to have normal mesenteric lymph node and Peyer’s patches (not shown) . To characterize the function of IL-17D in colon inflammation, we induced acute colitis by treating wild-type (WT) and Il17d-/-mice with 2.5%dextran sulfate sodium (DSS) for 5 days. In WT mice, DSS administration led to a reduction in body weight, which was subsequently recovered at around day 7 (Fig. 1E) . However, Il17d-deficient animals exhibited further decrease in body weights between days 6 and 9 (Fig. 1E) , with increased colon shortening than WT mice when they were sacrificed on day 9 (Fig. 1F) . Moreover, there was stronger inflammation and destruction of intestinal epithelium structure in Il17d-/-mice (Fig. 1G-H) . Next, we performed qPCR to analyze the expression of cytokine mRNA in colon tissue after DSS administration and found that ablation of Il17d resulted in significantly increased expression in pro-inflammatory cytokines IL-6 and IL-1β (Fig. 1I) . These data indicate that IL-17D plays an important protective role in DSS colitis model.

Given the importance of IL-17D in colonic inflammation, we next explored its cellular source by isolating CD45+ and CD45-cells from colonic tissues of normal and DSS-treated WT mice. Il17d mRNA was highly expressed by the CD45-fraction, most of which were colon epithelial cells (Fig. 2A) , similar to other IL-17 family members.

In order to confirm that IL-17D expression in non-hematopietic cells was important in DSS colitis, we generated bone marrow chimeras with Il17d deficiency in hematopoietic and/or non-hematopoietic compartments. Mice with Il17d deficiency in non-immune cells displayed worsened colitis after DSS treatment (Fig. 2B-E) , suggesting that colon epithelial cells (CECs) is the main functional source of IL-17D in DSS-induced colitis.

IL-17D is required for IL-22 production by ILC3s

We then investigated the possible target cells of IL-17D. IL-17A-and IFN-γ-expressing T cells are important regulators in inflammatory diseases. We thus examined them in the intestinal lamina propria leukocytes (LPLs) , but found that the percentages of IFN-γ+, IL-17A+ and IL-22+cells among CD4+ T and γδ T populations were similar in WT and Il17d-deficient mice during colitis, suggesting that the worsened colitis in Il17d-/-15 mice was unlikely to be related to pathogenic T cells.

Previous studies have established that IL-22 and its downstream effector proteins, such as RegIIIβ and RegIIIγ, are critical for tissue repair and immune homeostasis in intestine. Thus, we examined the mRNA expression of Il22, Reg3b and Reg3g. Compared with colons of WT mice, Il17d-/-20 mice showed a significant reduction in Il22, Reg3b and Reg3g expression during DSS-induced colitis (Fig. 3A) , indicating that IL-17D is critical for IL-22 production in the intestine. Moreover, administration of recombinant IL-17D i.p. reduced the severity of DSS-induced colitis in Il17d-deficient mice, including significant improvement in body weights, colon lengths and intestinal epithelium organization (Fig. 3B-E) , and most importantly, in Il22 and Reg3b/g gene expression (Fig. 3F-G) . Notably, IL-22 treatment improved severe colitis in DSS-treated Il17d-/-mice (Fig. 3H-I) . These data suggest that IL-22 may be an important factor downstream of IL-17D signaling to protect mice against DSS-induce colitis.

RORγt+ILC3 cells, an important source of IL-22, are critical mediators in host defense against pathogens and in tissue repair, especially at the intestinal barrier. Mice lacking IL-22 or RORγt + ILC3s exhibited increased susceptibility to DSS-induced colitis similar to mice deficient in Il17d. We thus focused on ILC3 cells and tested if they could be the target cells for IL-17D. An IL-17D-hIg fusion protein was generated and was found to specifically bind to ILC3 but not T cells or CD3-CD90.2-cells in small intestine LPLs, suggesting that IL-17D might regulate ILC3s in the gut. Thus, we analyzed ILC3 cells in LPLs from small intestine and colon. When compared to control mice, the frequencies, absolute numbers, subsets and proliferation status of ILC3 cells were not changed in Il17d-/-mice (Fig. 4A-C) . However, the percentages of ILC3 cells constitutively expressing IL-22 were reduced in mice deficient in Il17d than in wild-type mice in small intestine and colon (Fig. 4D) . Furthermore, when we assessed IL-23-induced IL-22 production from ILC3 subsets that differentially expressed Nkp46 and CD4, significant differences between WT and Il17d-/-groups of animals were observed in all subsets in small intestine (Fig. 4E-G) ; Il17d-/-mice also showed decreased IL-22 expression in their ILC3 cells in colon. Consistently in DSS-treated mice (Fig. 1G) , expression of IL-22-downstream genes Reg3b and Reg3g was significantly decreased in Il17d-/-mice as compared with control wild-type mice at steady state. Therefore, these results indicate that IL-17D is necessary for ILC3 function in the gut. However, the addition of IL-17D to IL-23-or IL-1β stimulated ILC3s did not alter the proliferation of ILC3s, nor the percentages of IL-22-or GM-CSF-producing cells.

To better understand the mechanisms by which IL-17D regulates ILC3 cells, we conducted RNA sequencing in CD45 ^loCD3-CD90 ^hi LPLs from small intestine of WT and Il17d-deficient mice at steady states, among which 96%were RORγt+ 5 ILC3 cells, consistent with the literature. Substantial transcriptional changes in ILC3s were observed as a result of Il17ddeficiency; of note, Th17 and Th1/Th2 differentiation pathways were among the top pathways regulated by IL-17D. Moreover, combined with previously reported gene expression profiling of intestine innate lymphoid cells , our analysis revealed that ILC3-associated genes, including Cd93, Ahr, Foxs1, Il17f, Il1r2 and Il22, were down-regulated, whereas some genes associated with ILC1 or ILC2 cells, including klrd1, Ifng, Gata3, Il4, Il17rb and Ccr8, were up-regulated in ILC3 cells from Il17d-deficient mice (Fig. 4H) . These results were independently confirmed by real-time RT-PCR. Thus, IL-17D is required for reinforcing ILC3 transcriptional programs in the gut.

Intestinal inflammation is associated with cancer development. We thus tested the role of IL-17D in a colon cancer model. WT and Il17d-deficient mice were subject to AOM-DSS-induced tumorigenesis. Consistent with their defects in ILC3 function, Il17d-/-mice showed increased tumor loads as compared to WT mice. Interestingly, we observed decreased percentages of ILC3s, but comparable Th17 cells, in colon of Il17d-/-mice in this model, supporting a selective regulatory role of IL-17D on ILC3 cells in the gut. Since IL-22-producing ILC3s are essential for host defense against C. rodentium infection, we also investigated the function of IL-17D in this model, results showed that Il17d-deficient mice were more susceptible to C. rodentium infection than WT mice, reflected by decreased weight recovery and increased bacterial burden in the feces. Moreover, IL-22 secretion from colon ILC3s was also reduced in Il17d-/-mice, indicating defective IL-22 production might account for the susceptibility to this pathogen.

IL-22-producing ILC3 cells are critical for the maintenance of microbiota homeostasis, and microbiota dysbiosis has been reported to contribute to the pathogenesis of colitis. We thus performed 16S rRNA sequencing in feces from WT and Il17d-deficient mice. In both steady state and DSS-induced colitis, fecal from Il17d-deficient mice showed a remarkable decrease of bacterial diversity (Fig. 5A-C) and changes in bacterial composition (Fig. 5D) , when compared to WT mice, consistent with reports on mice deficient in IL-22. Verrucomicrobia and Proteobacteria, two bacteria strains increased after DSS treatment shown in a previous study, were the most significantly upregulated phyla in Il17d-deficient mice at steady state and during colitis, respectively (Fig. 5E) . Enterobacteriaceae, which are susceptible to the antimicrobial Verrucomicrobiaceae and Enterobacteriaceae, belonging to the Verrucomicrobia and Proteobacteria phyla separately, were the most increased families in Il17d-deficient mice at steady state and colitis, respectively (Fig. 5F) . Therefore, our data indicate that Il17d deficiency results in microbiota dysbiosis, consistent with defective ILC3 function in these mice proteins and can be suppressed by IL-22, mediate DSS-induced colitis.

IL-17D binds to CD93

To further understand the molecular mechanisms underlying IL-17D function in ILC3 cells, we pursued the identification of IL-17D receptor. First, we used our IL-17D-hIg fusion protein and tested its binding with 293T cells transfected with various IL-17R family members. However, we did not observe any binding of IL-17D-hIg to the homodimers or heterodimers of IL-17 receptors, suggesting that IL-17D may not bind a typical member of the IL-17R family. After CD93 protects against colon inflammation through IL-22 expression searching in cell lines, we found that IL-17D-hIg bound to RAW264.7 cells, similar as IL-17A-hIg but not PD-L1-hIg (Fig. S6B) . The RAW264.7 cells were incubated with IL-17D-hIg or control protein (hIg) , followed by cross-linking with formaldehyde. The potential interacting proteins were precipitated by protein A beads. Western blotting with HRP-conjugated anti-human 5 IgG revealed two bands of ～100 kDa (IL-17D-hIg protein) and ～250 kDa in IL-17D-hIg but not control hIgG lane (Fig. 6A) . Reasoning that ～250 kDa band might be the complex of IL-17D-hIg protein with its partner, we subjected it to mass spectrometry analysis. Among about 260 proteins identified, we focused on 21 transmembrane proteins, and expressed their cDNA in 293T cells to examine the binding with IL-17D. We found that IL-17D specifically bound to 293T cells expressing CD93, but not to cells transfected with IL-17RA, which could be recognized by IL-17A (Fig. 6B-E) . Most importantly, we validated the binding of CD93 by IL-17D protein using IL-17D recombinant proteins with hIgG and His tags, detected with anti-His (Fig. 6B) , anti-human IgG (Fig. 6C) or via biotin-streptavidin system (Fig. 6D) , and using proteins generated by our lab or from R&D Systems (Fig. 6E) . CD93 consistently interacted with IL-17D in all these experiments when expressed in 293T cells.

CD93, also known as C1qRp, is a cell-surface glycoprotein of 645 amino acids with its extracellular region composed of a C-type lectin-like domain (CTLD) , five epidermal growth factor (EGF) -like repeats and a mucin-like domain. CD93 was originally proposed as a surface receptor involved in C1q-mediated phagocytosis. However, contradictory results were reported subsequently showing no interaction between CD93 with C1q. Asking which part of CD93 was required for the binding to IL-17D, we generated several CD93 deletional mutants (Fig. 6F) , and transfected in 293T cells to test the binding of IL-17D. Extracellular CTLD domain and the unknown structure between CTLD and EGF-like repeats of CD93 were necessary for binding by IL-17D (Fig. 6G) . Furthermore, we confirmed the binding of IL-17D to CD93 by surface plasmon resonance (Kd values: 62nM) (Fig. 6H) . Hence, our data for the first time identify CD93 as a putative interacting protein for IL-17D.

CD93 protects against colon inflammation through IL-22 expression

To further understand the roles of CD93 in mucosal immunity, we examined the expression of CD93 by flow cytometry. In isolated small intestine LPLs, ILC3 cells, but not Th17 cells, displayed high levels of CD93 expression (Fig. 7A) , consistent to its mRNA expression derived from the publically available RNA-seq data. Of note, most of CD93 expression was observed on IL-22+ cells (Fig. 7A) . Moreover, CD93 was highly expressed by RORγ+ILCs from SI, but not from skin or lung (Fig. S7B) . When gating on CD45+LPLs, CD93 were mainly expressed by CD3-and CD90.2+ population, and among CD3-CD90.2+ cells, CD93 was selectively expressed by three subsets of RORγ+ ILC3s including CCR6+ CD4-ILC3, CCR6+ CD4+ ILC3 and CCR6-ILC3. Gating on CD45+ CD93+ revealed that 88%of these cells were RORγt-expressing cells. Previous single-cell RNA-sequencing analysis also revealed ILC3s, but ILC1s or ILC2s, were enriched for expression of Cd93 transcript, consistent with an earlier report suggesting that CD93 is specifically expressed by ILC3 cells. In order to confirm that CD93 is a physiological receptor for IL-17D, we generated a mouse strain deficient in Cd93. Cd93 deletion diminished surface staining of CD93, as well as the binding of IL-17D, on ILC3 cells (Fig. 7B) . Therefore, our findings indicate that IL-17D interacts directly with ILC3 cells in a CD93-dependent manner. CD93 appears to be a physiological receptor for IL-17D.

Next, we investigated the functional role of CD93 in DSS-induced colitis. Consistent with Il17d deficient mice, mice with Cd93 deficiency showed worsened colitis, including a significant decrease in body weight and shortened colon length (Fig. 7C) . In AOM-DSS-induced tumor model, these mice also exhibited increased tumor numbers (Fig. 7D) . Since IL-17D regulates IL-22 production by ILC3 cells, we examined mRNA expression of Il22, Reg3b, and Reg3g in total colon tissues of WT mice as well as Cd93-deficient mice at steady state. We found that the expression of these genes was significantly decreased in Cd93-/-mice than in wild-type mice (Fig. 7E-F) . Interestingly, i.p. admisitration of IL-22 partially rescued phenotype of Cd93-/-5 mice (Fig. 7G-H) , indicative of IL-22 as a functional factor downstream CD93.

To test if CD93 is intrinsically required for ILC3 cell function, we generated mixed bone marrow chimeras by reconstituting lethally irradiated CD45.1 mice with a 1: 1 mixture of WT (CD45.1/CD45.2) and Cd93-/- (CD45.2) bone marrow cells (Figure 8A) . The chimaeric mice had comparable percentages of wild-type and Cd93-deficient T, ILC1 or ILC2 cells (Fig. 8C, E) . Consistent with this, there was no ILC1/2 defect in Cd93-deficient mice when compared with WT mice. However, Cd93-deficient ILC cells in both small intestine and colon of chimeric mice showed reduced RORγ+ ILC3 cells than wild-type ILCs (Fig. 8B-D) . Even more strinkingly, Cd93-deficient ILC cells in both small intestine and colon of the chimeric mice showed defective IL-22 production than wild-type cells (Fig. 8B-D) , though with similar proliferation in small intestine. Taken together, mice with Cd93 deletion phenocopied Il17d knockout mice, indicating that CD93 is a functional receptor for IL-17D.

Reference throughout this specification to "an embodiment" , "some embodiments" , "one embodiment" , "another example" , "an example" , "a specific example" or "some examples" means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as "in some embodiments, " "in one embodiment" , "in an embodiment" , "in another example, "in an example, " "in a specific example, " or "in some examples, " in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

Use of reagent in preparing medicament, wherein the reagent is used for regulating IL-17D-CD93 axis, and the medicament is used for treating or prevention of inflammatory or immune diseases.
Use of claim 1, wherein the inflammatory or immune diseases including dysregulation of intestinal homeostasis related diseases.
Use of claim 2, wherein the dysregulation of intestinal homeostasis related diseases comprise at least one of the following:

microbiota dysbiosis, colitis , colon cancer, inflammatory bowel diseases, pathogen infection.crohn’s disease and ulcerative colitis .
Use of claim 2 or 3, wherein the reagent is used for activating or upregulating IL-17D and/or CD93 or enhancing the binding of IL-17D and CD93, and the medicament is used for treating or prevention of microbiota dysbiosis, colitis , colon cancer, inflammatory bowel diseases or pathogen infection .
Use of claim 2 or 3, wherein the reagent is used for suppressing or downregulating IL-17D and/or CD93 or preventing the binding of IL-17D and CD93, and the medicament is used for treating or prevention of crohn’s disease and ulcerative colitis.
Use of claim 4, wherein the reagent comprises at least one of the following: IL-17D, Nrf2 agonists, nucleic acid used for overexpression of IL-17D and/or CD93.
Use of claim 6, wherein the Nrf2 agonists comprises tert-butylhydroquinone.
Use of claim 5, wherein the reagent comprises at least one of the following: nucleic acid used for downregulating IL-17D and/or CD93, MMRN2 siRNA and MMRN2 inhibitor.
Use of IL-17D-CD93 axis in screening monoclonal antibodies or other proteins/petides or small molecules that preventing the binding of IL-17D and CD93.
A method of enhancing IL-22 production by ILC3 cells, comprising:

1) using a chemical or biological activator to enhance the activity of IL-17D and/or CD93;

2) using a chemical or biological regent to enhance the amount of IL-17D and/or CD93;

3) using a chemical or biological regent to enhancing the binding of IL-17D and CD93.
A method of impairing IL-22 production by ILC3 cells, comprising:

1) using a chemical or biological inhibitor to suppress the activity of IL-17D and/or CD93;

2) using a chemical or biological regent to reduce the amount of IL-17D and/or CD93;

3) using a chemical or biological regent to prevent the binding of IL-17D and CD93.
A method of treating or prevention of dysregulation of intestinal homeostasis related disease, wherein the dysregulation of intestinal homeostasis related disease is caused by insufficient secretion of IL-22 by ILC3 cells or low activity of ILC3, comprising:

administrating a promoter for IL-17D expressing and secretion by colonic epithelial cells;

administrating an activator for enhancing the activity of IL-17D;

administrating a promoter for CD93 expressing on ILC3 cells;

administrating an activator for enhancing the activity of CD93; or

administrating an enhancer for enhancing the binding of IL-17D and CD93.
Method of claim 12, wherein the intestinal homeostasis related diseases comprise at least one of the following:

microbiota dysbiosis, colitis, colon cancer, inflammatory bowel diseases, pathogen infection.
A method of treating or prevention of dysregulation of intestinal homeostasis related disease, wherein the dysregulation of intestinal homeostasis related disease is caused by excessive secretion of IL-22 by ILC3 cells or over activity of ILC3, comprising:

administrating an inhibitor for IL-17D expressing and secretion by colonic epithelial cells;

administrating an inhibitor for suppressing the activity of IL-17D;

administrating an inhibitor for CD93 expressing on ILC3 cells;

administrating an inhibitor for suppressing the activity CD93; or

administrating an inhibitor for preventing the binding of IL-17D and CD93.
Method of claim 14, wherein the dysregulation of intestinal homeostasis related diseases comprise at least one of the following:

crohn’s disease and ulcerative colitis.
A method of screening medicament, wherein the medicament is used for treatment or prevention of dysregulation of intestinal homeostasis related diseases, wherein the dysregulation of intestinal homeostasis related disease is caused by insufficient secretion of IL-22 by ILC3 cells or low activity of ILC3, comprising:

contacting a candidate medicament with colonic epithelial cells, the elevation of the expression of IL-17D or the upregulating of IL-17D activity or the enhancing of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.
A method of screening medicament, wherein the medicament is used for treatment or prevention of dysregulation of intestinal homeostasis related diseases, wherein the dysregulation of intestinal homeostasis related disease is caused by insufficient secretion of IL-22 by ILC3 cells or low activity of ILC3, comprising:

contacting a candidate medicament with ILC3 cells, the elevation of the expression of CD93 or the upregulating of CD93 activity or the enhancing of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.
A method of screening medicament, wherein the medicament is used for treatment or prevention of dysregulation of intestinal homeostasis related diseases, wherein the dysregulation of intestinal homeostasis related disease is caused by excessive secretion of IL-22 by ILC3 cells or over activity of ILC3, comprising:

contacting a candidate medicament with colonic epithelial cells, the downregulating of the expression of IL-17D or the IL-17D activity or the weakening of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.
A method of screening medicament, wherein the medicament is used for treatment or prevention of homeostasis related diseases, wherein the intestinal homeostasis related disease is caused by excessive secretion of IL-22 by ILC3 cells or over activity of ILC3, comprising:

contacting a candidate medicament with ILC3 cells, the downregulating of the expression of CD93 or the CD93 activity or the weakening of the binding of IL-17D and CD93 after the contacting indicates that the candidate medicament is the target medicament.