WO2020087130A1 - Prognosis and treatment of inflammatory bowel disease - Google Patents

Abstract

Provided herein are methods and kits for determining a prognosis and/or treatment of a subject with IBD. Methods and kits for assessing susceptibility to and/or determining the presence and/or treatment of dysbiosis in a subject are also described herein.

Description

TITLE

PROGNOSIS AND TREATMENT OF INFLAMMATORY BOWEL DISEASE

TECHNICAL FIELD

THIS INVENTION relates to the prognosis and/or treatment of an inflammatory bowel disease. More particularly, this invention relates to determining the presence or absence of a risk variant in an IL23R gene or protein correlated with an inflammatory bowel disease in a biological sample from a subject.

BACKGROUND

The development of Crohn’s disease (CD), one of the major forms of inflammatory bowel disease (IBD), is determined by a number of factors including genetic predisposition, environment, lifestyle and altered gut microbiota¹. Genetic studies have identified over 200 susceptibility risk loci for IBD ^1-5 with the NOD2 gene being the first discovered and most studied susceptibility gene for CD ⁶. Other major CD- susceptibility genes include autophagy-related protein 16-1 (. ATG16L1 ), and interleukin 23 receptor ( IL23R ). Altered gut microbiota or dysbiosis has been consistently reported in paediatric, early or new-onset disease and in adult CD populations ⁷’⁸. IBD gut microbiota dysbiosis in CD compared to healthy individuals includes decreased numbers of Firmicutes species, particularly a reduction in Faecalibacterium prausnitzii ⁹, and increased abundance of Proteobacteria phylum members specifically the families Enter obacteriaceae, Pasteur ellaceae , and Neisseriaceae and the species Escherichia coli ^8,1°. An enrichment of Proteobacteria in the gut is associated with chronic inflammation ^{7 U}. In a healthy state, the relative abundance of Proteobacteria represents 2.5-4.6% of the human gut phylum, however, Proteobacteria is unstable, and exploits changes to the gut environment via facultative metabolism ^{12 15}. This selective advantage allows Proteobacteria to outcompete the obligate anaerobes Firmicutes and Bacteroidetes, and occupy inflamed intestinal niches.

Inflammation in Crohn’s disease most commonly involves the terminal ileum with up to 75% of patients having ileal disease with or without colonic involvement ^{18 20}. Studies investigating the association between inflammation and the mucosal microbiota in CD subjects showed inconsistencies in the results ^21-25 and are limited by a low sample size. Accordingly, there remains a need for protective markers that are useful in the prognosis and/or therapeutic decision making in patients with IBD. Specifically, the use of such protective markers may address the outstanding clinical needs for prognostic tools to guide treatment decisions for patients with advanced or more sever disease.

SUMMARY

The invention is broadly directed to a method of prognosis and/or treatment of an inflammatory bowel disease (IBD), such as Crohn’s disease (CD). Additionally, the present invention is broadly directed towards a method of diagnosis, prognosis and/or treatment of dysbiosis.

More particularly, the inventors have discovered that a specific protective variant in the Interleukin 23 receptor (IL23R) can prove to be useful in the prognosis and/or treatment of IBD. Subsequently, methods have been developed to provide an indication of disease prognosis. Furthermore, the inventors have discovered that the protective variant of IL23R may serve as a prognostic marker with respect to treatment response.

In a first aspect, the invention provides a method of determining a prognosis of a subject with an inflammatory bowel disease (IBD), the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, to thereby determine the prognosis of the IBD in the subject.

In one embodiment, determination of the presence of the polymorphic marker is indicative of a positive prognosis.

In certain embodiments, the present method further includes the step of determining whether the subject would benefit from a treatment of the IBD based, at least in part, on the prognosis.

In some embodiments, the present method further includes the step of determining whether the subject would benefit from a treatment of dysbiosis based, at least in part, on the prognosis.

In particular embodiments, the method of the current aspect further includes the step of determining suitability of the subject for a treatment based, at least in part, on the prognosis.

In one embodiment, the current method further includes the step of developing a treatment strategy for the subject based, at least in part, on the prognosis.

In a second aspect, the invention provides a method of treating IBD in a subject, the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, before, during and/or after treatment of IBD, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, and based on the determination made, initiating, continuing, modifying or discontinuing a treatment of IBD.

In one embodiment, the present method further includes the step of selecting the treatment of IBD based on the presence or absence of the polymorphic marker.

For the aforementioned aspects, the treatment is suitably selected from the group consisting of an IL23R antagonist, an IL23 antagonist, a microbiome modulator, a dietary supplement, a probiotic composition, a faecal transplant and any combination thereof.

In a third aspect, the invention provides a kit for determining a prognosis and/or treatment of a subject with IBD, the kit comprising:

reagents for selectively detecting a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, and

a collection of data comprising correlation data between the polymorphic marker and prognosis and/or treatment of IBD.

Suitably, the kit of the present aspect is for use in the method of the first and/or second aspects.

Suitably, the collection of data is on a computer-readable medium.

In a fourth aspect, the invention provides a method of determining a susceptibility to dysbiosis in a subject, the method including the step of determining the presence or absence of a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, and wherein determination of the presence or absence of the risk variant is indicative of the susceptibility to dysbiosis in the subject.

In a fifth aspect, the invention provides a method of determining whether or not a subject has dysbiosis, the method including the step of determining the presence or absence of a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, and wherein determination of the presence or absence of the risk variant is indicative of the subject having dysbiosis.

Suitably, determination of the absence of the polymorphic marker is indicative of the subject being susceptible to or having dysbiosis. In a sixth aspect, the invention provides a method of treating dysbiosis in a subject, the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, before, during and/or after treatment of dysbiosis, and based on the determination made, initiating, continuing, modifying or discontinuing a treatment of dysbiosis.

In one embodiment, the present method further includes the step of selecting the treatment of dysbiosis based on the presence or absence of the polymorphic marker.

In particular embodiments, the treatment is selected from the group consisting of an IL23R antagonist, an IL23 antagonist, a microbiome modulator, a dietary supplement, a probiotic composition, a faecal transplant and any combination thereof.

In particular embodiments of the first, second, fourth, fifth and sixth aspects, the method of the invention further includes the step of determining a microbiome in the subject. In this regard, the step of determining a microbiome suitably includes analysing a variable region, such as a V3-V4 hypervariable region, of a 16S ribosomal subunit of a microbe. In one particular embodiment, the variable region is analysed using one or more primers that comprise the nucleotide sequence set forth in SEQ ID NO: l and/or SEQ ID NO:2 or a fragment, variant or derivative thereof.

With respect to the methods of the fourth, fifth and sixth aspects, the polymorphic marker suitably is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith.

Referring to the first, second, fourth, fifth and sixth aspects, the method suitably further includes the step of determining an expression level of a protein, miRNA or nucleic acid biomarker.

In a seventh aspect, the invention provides a kit for assessing susceptibility to and/or determining the presence and/or treatment of dysbiosis in a subject, the kit comprising:

reagents for detecting a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, and

a collection of data comprising correlation data between the polymorphic marker and susceptibility to and/or presence of dysbiosis.

Suitably, the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith. In one embodiment of the third and seventh aspects, the kit further includes one or more primers for determining a microbiome in the subject. More particularly, the one or more primers are suitably for analysing a variable region of the 16S ribosomal subunit of a microbe. To this end, the one or more primers may comprise the nucleotide sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO:2 or a fragment, variant or derivative thereof.

In one embodiment, the present kit is for use in the method of the fourth, fifth and/or sixth aspects.

Suitably, the collection of data is on a computer-readable medium.

With respect to the above aspects, the inflammatory bowel disease is suitably Crohn’s disease or ulcerative colitis. Preferably, the inflammatory bowel disease is Crohn’s disease.

Suitably, the subject referred to herein is a mammal. Preferably, the subject is a human.

As used herein, except where the context requires otherwise, the term“ comprise” and variations of the term, such as“ comprising’’,“ comprises” and“ comprised’, are not intended to exclude further elements, components, integers or steps but may include one or more unstated further elements, components, integers or steps.

It will be appreciated that the indefinite articles“a” and“aw” are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example,“a” cell includes one cell, one or more cells and a plurality of cells.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Impact of IL23R genotype on the global microbial community composition, taxa at OTU level and microbial richness in the ileum. All samples are grouped as wild- type (wt, GG genotype) and heterozygote (het, GA genotype) forms of the IL23R coding variant, R381Q. (A) Supervised RDA analysis at OTU level using gender, age and IL23R genotype as the covariates revealed clustering according to IL23R genotype in healthy subjects. (B) Unsupervised PCoA analysis of the IL23R gene in healthy and CD subjects. (C) Differences in the mucosal-associated microbiota at OTU level between wt and het forms of the IL23R gene in healthy subjects. Significant differences adjusted for gender and age using multiple linear regression model are indicated as follows * P < 0.05, ** P < 0.01. (D) Decreased bacterial richness at OTU level is observed in healthy and CD subjects with the IL23R rsl 1209026 wild-type (GG) genotype. Richness is adjusted for gender and age.

Figure 2. PCoA and heatmap of ileal CD samples based on the OTU level. (A) Samples from the same subject clustered together in a PCoA plot.

Figure 3. Comparative analysis of microbial composition and microbial diversity between healthy and ileal CD patients. (A) Ileal samples from CD patients and healthy controls show a tendency to cluster separately using PCoA. The microbiome of CD patients and healthy controls differs significantly in abundance of bacteria at (B) family and (C) OTU level. Significant differences based on multiple linear regression adjusted for age and gender are indicated with asterisks, *** P < 0.001, ** P < 0.01. The microbial community of healthy subjects has a higher (D) Shannon diversity and (E) richness as compared to ileal CD subjects. Shannon index and richness were adjusted for age and gender using a multiple linear regression model and tested using ANOVA.

Figure 4. Correlation network showing co-occurring taxa and the explanatory variables disease status and diversity in healthy controls and CD patients. Nodes represent OTUs (coloured by phylum assignments) and explanatory variables (white nodes) predicted in all samples including inflamed and non-inflamed CD subject and healthy controls. Edges indicate positive (red) and negative (blue) associations as calculated by Pearson correlation. Healthy status, diversity and richness correlate positively (indicated by red edges) with OTUs belonging to Firmicutes (orange nodes) and Bacteroides (blue nodes) species. These OTUs correlate negatively (blue edges) with disease status and OTUs assigned to Proteobacteria (yellow nodes) and Fusobacteria (green nodes). Only correlation of at least 0.25 similarity are shown.

Figure 5. Significant differences between the microbiome in ileum and rectum in healthy control subjects. (A) Shannon diversity, (B) richness and taxa at (C-G) family and (H) OTU level differed between ileum and rectum. E-values are based on mixed effect linear regression.

Figure 6. Impact of IL23R genotype on microbial diversity in the rectum and the rectal microbiome at family level in healthy subjects. (A) Decreased microbial Shannon diversity and (B) richness are measured in healthy subjects with the IL23R (RS11209026) wildtype G allele. All samples are grouped as wildtype (wt, GG genotype) and heterozygote (het, GA genotype) forms of the IL23R gene. Shannon diversity and richness were adjusted for gender and age. (C) Differences in the rectal microbiome between wt and het forms of the IL23R gene in healthy subjects. Significant differences using Wilcox on rank test are indicated as follows * P < 0.05.

Figure 7. Impact of NOD2 (R702W, G908R, Leul007fsinsC) and ATG16L1 (rs224l880) genotype on the gut microbiome. (A) All samples obtained from CD patients were grouped as wildtype (wt), heterozygote (het) and homozygote (horn) forms of the NOD2 gene (R702W, G908R, Leul007fsinsC). Unsupervised analysis revealed no clustering of the samples by wildtype and variant genotypes. (B) The family Bifidobacteriaceae tended to be increased in CD patients carrying the wt NOD2 genotype. (C) Decreased Shannon diversity and (D) richness (corrected for age and gender using multivariable linear regression) were observed in CD subjects with the homozygote form of NOD2. (E) No differences in Shannon and (F) richness were identified between the ATG16L1 genotypes. Figure 8. Impact of immunosuppression therapy on the ileal microbial diversity and composition at OTU level in CD patients. (A) Shannon diversity and (B) Chaol richness were decreased in the non-inflamed tissue samples from patients on immunosuppression therapy (1ST). (C) Differences in abundance of mucosal-associated OTUs between patients on immunosuppression therapy (1ST) and without (no 1ST) based on LEfSe analysis.

Figure 9. Bar chart of the mucosa-associated microbiota in ileal CD patients on phylum (A) and family (B) level. The samples are grouped by individual with the non-inflamed samples on the left, and inflamed samples on the right. Only the top 15 families are shown.

Figure 10. OTUs differing between inflamed and non-inflamed ileal tissues from patients with CD using a mixed effect linear regression model. (A) OTU related to Lachnospiraceae was decreased, (B) Bacteroides, (C) Eubacterum, (D) Bacteroides were increased in non-inflamed as compared to inflamed tissue samples.

Figure 11. (A) Shannon diversity and (B) richness in inflamed and non-inflamed samples from CD subjects with mild and moderate disease. Diversity and richness were significantly different between mild and moderate disease in non-inflamed samples. Anova test, P-values * < 0.05. (C) PCoA of inflamed CD samples coloured according to inflammation degree.

Figure 12. (A) Heatmap of the 50 most abundant OTUs predicted in microbial samples obtained from inflamed and non-inflamed ileal tissues of CD patients. Subjects with moderate CD and high abundance of Enterobacteriaceae tended to aggregate in cluster 1, and mild CD subjects and different OTUs including Bacteroides and Faecalibacterium formed cluster 2. (B) Heatmap of the ileal CD and healthy control with the dendrogram indicating relationships between samples. Dendrogram is based on all OTUs, but only top 12 are shown. Each sample is coloured by disease status, Shannon diversity and richness. The cluster associated with healthy status was characterized by high microbial diversity, high abundance of Faecalibacterium sp. and Bacteroides sp. and low abundance of Enterobacteriaceae sp. (p. Proteobacteria). A second cluster composed of both healthy control and ileal CD subjects showed low diversity.

Figure 13. Proteobacteria and diversity indices stratified by CRP and fecal calprotectin.

(A) Proteobacteria abundance in healthy control subjects with normal and high CRP and

(B) fecal calprotectin. (C) Diversity and (D) richness in healthy subjects with high and normal fecal calprotectin values (E-values corrected for gender and age).

Figure 14. Heatmap of the composition of phylotypes predicted in the ileal and rectal microbiota of healthy control subjects. Dendrograms indicate relationships between samples (top) and OTUs (left). All OTUs were included for the clustering (complete clustering method, Euclidian distance). The samples are coloured by subject id and sampling site.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 Forward Primer (341F) in Table 7

SEQ ID NO:2 Reverse Primer (806R) in Table 7

DETAILED DESCRIPTION

The present invention is predicated, at least in part, on the surprising discovery that a protective variant in IL23R may provide a prognostic and/or predictive treatment response marker for IBD patients, particularly those patients with associated dysbiosis. Additionally, the inventors demonstrate herein that the protective variant in IL23R may be used to diagnose and/or determine the susceptibility of subjects to dysbiosis. Accordingly, the protective variant disclosed herein may also have utility in methods of treating dysbiosis.

In an aspect, the invention provides a method of determining a prognosis of a subject with an inflammatory bowel disease (IBD), the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, to thereby evaluate the prognosis of the IBD in the subject.

Suitably, determination of the presence of the polymorphic marker in the subject is indicative of a positive prognosis, such as the presence of mild or less severe IBD and/or the presence of low or absence of microbial dysbiosis (e.g., a relatively high microbial diversity and/or richness in the subject’s microbiome).

As used herein, “7 L23R” refers to a protein or nucleic acid encoded by a mammalian IL23R gene. Also contemplated are fragments and/or variants of IL23R nucleic acids and proteins. Non-limiting examples of a nucleotide sequence of human IL23R and/or its encoded protein, include NM_l4470l, AAH16829.1, AAH40720.1, HGNC: 19100, Entrez Gene: 149233, Ensembl: ENSG00000162594, OMIM: 607562, UniProtKB: Q5VWK5, XP_0l 1539092.1, XP Ol 1539093.1, NP_653302.2 and

XP 005270573.1. The nucleotide and/or protein sequences provided by these accessions is incorporated by reference herein.

The terms“ prognosis” and“ prognostic” are used herein to include making a prognosis, which can provide for predicting a clinical outcome (with or without medical treatment), selecting an appropriate course of treatment (or whether treatment would be effective) and/or monitoring a current treatment and potentially changing the treatment. This may be at least partly based on determining the presence or absence of a polymorphic marker of an IL23R gene or protein, as are known in the art, by the methods of the invention, which may be in combination with determining the expression levels of additional protein, miRNA and/or nucleic acid biomarkers. A prognosis may also include a prediction, forecast or anticipation of any lasting or permanent physical or psychological effects of IBD suffered by the subject after the IBD has been successfully treated or otherwise resolved. Furthermore, prognosis may include one or more of therapeutic responsiveness, implementing appropriate treatment regimes, determining the probability, likelihood or potential for IBD recurrence after therapy and prediction of development of resistance to established therapies. It would be appreciated that a positive prognosis typically refers to a beneficial clinical outcome or outlook, such as disease regression or remission, whereas a negative prognosis typically refers to a negative clinical outcome or outlook, such as disease recurrence or progression.

As used herein, the term " inflammatory bowel diseases " or "IBD" includes art- recognized forms of a group of related inflammatory gastrointestinal conditions. Several major forms of IBD are known, such as Crohn's disease (regional bowel disease, inclusive of inactive and active forms) and ulcerative colitis (inclusive of inactive and active forms). In addition, IBD encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis. Other less common forms of IBD include indeterminate colitis, infectious colitis (viral, bacterial or protozoan, e.g. amoebic colitis) (e.g., Clostridium dificile colitis), pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet's disease, sarcoidosis, scleroderma, IBD- associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.

In one preferred embodiment, the IBD is or comprises Crohn's disease.

Accordingly, it will be appreciated that IBD represents chronic, inflammatory diseases of the gastrointestinal tract. IBD can be characterized by abdominal pain, diarrhoea (often bloody), a variable group of '"extra-intestinal ^"' manifestations (such as arthritis, uveitis, skin changes, etc.) and the accumulation of inflammatory cells within the small intestine and/or colon. Additional signs or symptoms of IBD include malabsorption of food, altered bowel motility, infection, fever, rectal bleeding, weight loss, signs of malnutrition, perianal disease, abdominal mass, and growth failure, as well as intestinal complications such as stricture, fistulas, toxic megacolon, perforation, and cancer, and including endoscopic findings, such as friability, aphthous and linear ulcers, cobblestone appearance, pseudopolyps and rectal involvement.

The terms“ polymorphism” or“ polymorphic” refer to the coexistence of more than one form of a gene, a protein or a portion thereof. By way of example, a portion of a gene in which there are at least two different forms, i.e., two different nucleotide sequences, can be referred to as a“polymorphic region of a gene.” A polymorphic locus can be a single nucleotide, the identity of which differs in the other alleles. A polymorphic locus can also be more than one nucleotide long. The allelic form occurring most frequently in a selected population is typically referred to as the reference and/or wild-type form. Other allelic forms are typically designated as alternative or variant alleles. Diploid organisms may be homozygous or heterozygous for a polymorphic marker.

For the aforementioned aspects, the terms“ polymorphic nucleotide/amino acid’ or “ polymorphic marker” refer to one or more nucleotides and/or amino acids in the IL23R gene and/or protein which can be used, for example, to determine a prognosis and/or a response to therapy for subjects with IBD and/or determine a diagnosis and/or the susceptibility of a subject to dysbiosis. The polymorphic marker may be a SNP. To this end, the polymorphic marker is preferably rsl 1209026, inclusive of markers in linkage disequilibrium therewith.

In this regard, the polymorphic marker is identified based on a change in the nucleotide and/or amino acid sequence from a consensus sequence, or the“reference sequence”. As used herein, the reference sequence of IL23R can be any nucleotide and/or amino acid sequence known in the art, such as those hereinbefore described. To identify the location of the polymorphic markers of the present invention, a specific nucleotide or amino acid residue in a reference sequence is listed for the polymorphism, where nucleotide or amino acid residue number 1 is the first nucleotide (i.e., 5') or amino acid (i.e., N-terminal) in each reference sequence.

The nucleic acid molecules of the invention can be double- or single-stranded. Accordingly, the invention further provides for the complementary nucleic acid strands comprising the polymorphic marker provided herein.

In certain embodiments, the polymorphic marker is or comprises a nucleic acid variant in an IL23R gene that includes a guanine to adenine variation at position 1142 thereof (i.e., H42G>A of GenBank accession: NM_l4470l, GenelD: 149233).

In particular embodiments, the polymorphic marker is or comprises an amino acid variant in an encoded IL23R protein that comprises an arginine to glutamine variation at position 381 (i.e., R381Q).

The term“ linkage” describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome. It can be measured by percent recombination between the two genes, alleles, loci, or genetic markers. The term“ linkage disequilibrium” refers to a greater than random association between specific alleles at two marker loci within a particular population. In general, linkage disequilibrium decreases with an increase in physical distance. If linkage disequilibrium exists between two polymorphic markers, or SNPs, then the genotypic information at one marker, or SNP, can be used to make probabilistic predictions about the genotype of the second marker.

The term “ determining’ includes any form of measurement, and includes determining if an element, such as a polymorphic marker or protective variant, is present or not. As used herein, the terms“ determining’ “measuring ,“evaluating",“assessing" and “ assaying” are used interchangeably and include quantitative and qualitative determinations. Determining may be relative or absolute.“ Determining the presence of’ includes determining the amount of something present ( e.g ., an miRNA, nucleic acid and/or protein biomarker), and/or determining whether it is present or absent.

The term“ control sample" typically refers to a biological sample from a healthy or non-diseased individual not having IBD. In one embodiment, the control sample may be from a subject known to be free of IBD. Alternatively, the control sample may be from a subject in remission from IBD. The control sample may be a pooled, average or an individual sample. An internal control is a marker from the same biological sample being tested.

It will be appreciated that the methods of the invention can include the additional step of determining the expression level of the one or more biomarkers, such as miRNA, nucleic acid and/or protein biomarkers, which have also been identified as being prognostic for IBD, in combination with determining the presence or absence of the rsl 1209026 protective variant of IL23R. In certain embodiments, the expression level of one or more biomarkers, such as protein and/or nucleic acid biomarkers, may also be determined. In this regard, the one or more biomarkers may include, for example, C- reactive protein (CRP), erythrocyte sedimentation rate (ESR), platelet count, mean platelet volume (MPV), red blood cell distribution (RDW), faecal calprotectin, faecal lactoferrin, faecal neopterin, S100A12, adenosine deaminase, lipopolysaccharide-binding protein and CD 14, Mopterin, Soluble ST2, Nitric Oxide, Soluble triggering receptor expressed on myeloid cells-l (sTREM-l), substance P, activated thrombin activatable fibrinolysis inhibitor (TAFIa), Chitinase 3 -like- 1 (CHI3L1/YKL-40) and angiogenin, albeit without limitation thereto. Additional diagnostic and prognostic biomarkers for IBD have been described previously, as reviewed in BIOMARKERS OF INFLAMMATORY BOWEL DISEASE, Yi Fengming and Wu Jianbing, (. Disease Markers , Volume 2014, Article ID 710915), which is incorporated by reference herein.

The expression level of the one or more biomarkers may be determined by any method known in the art. By way of example, the expression level of miRNA and nucleic acid biomarkers may be determined by hybridization-based techniques (e.g., Northern blots, in situ hybridization, RT-PCR, and microarrays), amplification-based techniques (e.g, real-time quantitative PCR; gold nanoparticle-initiated silver enhancement) and cloning-based techniques (e.g, miRAGE). Further, the expression level of protein biomarkers may be determined by antibody-based detection methods that use one or more antibodies which bind the protein, electrophoresis, isoelectric focussing, protein sequencing, chromatographic techniques and mass spectroscopy and combinations of these, although without limitation thereto. Antibody-based detection may include flow cytometry using fluorescently-labelled antibodies that bind the protein, ELISA, immunoblotting, immunoprecipitation, in situ hybridization, immunohistochemistry and immuncytochemistry, although without limitation thereto. Suitable techniques may be adapted for high throughput and/or rapid analysis such as using protein arrays such as a TissueMicroArray™ (TMA), MSD MultiArrays™ and multiwell ELISA, although without limitation thereto.

As would be understood by the skilled person, the expression level of the one or more miRNA, nucleic acid and/or protein biomarkers is deemed to be“ altered " or “ modulated’ when the amount or expression level of the respective biomarker is increased or up regulated or decreased or down regulated, as defined herein.

In one embodiment, a prognosis or treatment response for IBD correlates with the one or more miRNA, nucleic acid and/or protein biomarkers when they are at a reduced level, down regulated or absent in the biological sample. In an alternative embodiment, a prognosis or treatment response for IBD correlates with the one or more miRNA biomarkers when they are at an increased level, up regulated or present in the biological sample.

By“ enhanced “ increased” or“ up regulated’ as used herein to describe the expression level of miRNA, protein and/or nucleic acid biomarkers, refers to the increase in and/or amount or level of one or more miRNA, protein and/or nucleic acid biomarkers, including variants, in a biological sample when compared to a control or reference sample or a further biological sample from a subject. The expression level of a biomarker may be relative or absolute. In some embodiments, the expression level of the one or more miRNA, protein and/or nucleic acid biomarkers is increased if its level of expression is more than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400% or at least about 500% higher than the level of expression of the corresponding miRNA, protein and/or nucleic acid biomarkers in a control sample or further biological sample from a subject.

The terms, “ reduced’ and “ down regulated as used herein to describe the expression level of miRNA, protein and/or nucleic acid biomarkers, refer to a reduction in and/or amount or level of one or more miRNA, protein and/or nucleic acid biomarkers, including variants, in a biological sample when compared to a control or reference sample or further biological sample from a subject. The expression level of a biomarker may be relative or absolute. In some embodiments, the expression level of one or more miRNA, protein and/or nucleic acid biomarkers is reduced or down regulated if its level of expression is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level of expression of the corresponding miRNA, protein and/or nucleic acid biomarkers in a control sample or further biological sample from a subject.

In particular embodiments, the biological sample comprises tissue, blood, serum, plasma or cerebrospinal fluid. Typically, the polymorphic marker described herein is obtainable from a cellular source. Accordingly, the biological sample is, comprises, or is obtained from a cellular source. To this end, the biological sample may be tissue or blood, although without limitation thereto.

In some embodiments, the method of determining whether or not a subject has the protective variant of IL23R, such as rsl 1209026 ( i.e ., c. H42G>A, p.Arg38lGln) may be performed in“high throughput” prognostic tests or procedures such as performed by commercial pathology laboratories or in hospitals. Furthermore or alternatively, the method of the present aspect may be used to confirm a prognosis of an IBD, including Crohn’s disease, such as that initially tested by a different or alternative prognostic test or procedure.

It would be further appreciated, that such methods of determining the presence or absence of a polymorphic marker of IL23R in the biological sample from an IBD patient may have potential utility in selecting patients for particular treatments.

Accordingly, in some embodiments, the method of the present aspect further includes the step of determining whether the subject would benefit from a treatment of the IBD based, at least in part, on the prognosis.

Further, in certain embodiments, the present method further includes the step of determining whether the subject would benefit from a treatment of dysbiosis based, at least in part, on the prognosis. In this regard, determination of the absence of the polymorphic marker is suitably indicative that the subject would benefit from the treatment of dysbiosis, such as those described herein.

In one embodiment, the method of the current aspect further includes the step of determining suitability of the subject for a treatment based, at least in part, on the prognosis.

In another embodiment, the present method further includes the step of developing a treatment strategy for the subject based, at least in part, on the prognosis. Again, a determination of the presence or absence of the polymorphic marker in the IL23R gene and/or protein may indicate that the subject would benefit from a particular treatment. By way of example, determination of the absence of the rsl 1209026 polymorphic marker in a IL23R gene and/or protein suitably indicates that the subject may benefit from a treatment of dysbiosis and/or an antagonist of IL23R signalling (e.g., an IL23R antagonist, a IL23 antagonist), inclusive of those treatments known in the art and described herein. Without being bound by any theory, it is believed that the rsl 1209026 polymorphic marker results in a reduced functionality of the IL23R, with disease progression and/or dysbiosis presumably being driven by IL23/IL23R signalling.

In particular embodiments, the treatment, inclusive of treatments of IBD and/or dysbiosis, is selected from the group consisting of an IL23R antagonist, an IL23 antagonist, a microbiome modulator, a dietary supplement, a probiotic composition, a faecal transplant and any combination thereof.

The IL23R antagonist may be an antibody or small molecule inhibitor, such as PTG-200.

It will be appreciated that Interleukin-23 is a heterodimer with a 19 000 molecular weight fourfold helical core a subunit (IL-23pl9), disulphide linked to an additional 40 000 molecular weight distinct b subunit (IL-l2p40). Accordingly, the IL23 antagonists described herein may be configured to target one or both of the pl9 and p40 subunits of the IL23 molecule. Non-limiting examples of IL23 antagonists or inhibitors include ustekinumab (Stelara), risankizumab, briakinumab, MP-196, FM-202, FM-303, ADC- 1012, LY-2525623 and Apilimod (any others). Accordingly, the IL23 antagonist can be an antibody or a small molecule inhibitor.

The term“ microbiome modulator” refers to an agent that is capable of altering the gut associated microbial community or microbiome of a subject administered the composition. Nonlimiting examples of microbiome modulators include biguanidines (e.g., metformin), beta-glucan, polyphenolic compounds and an indigestible carbohydrate (e.g., inulin, oligofructoses, fructo-oligosaccharides, lactulose, xylo-oligosaccharides, resistant starches and short-chain galactooligosaccharides).

As generally used herein, the term“ probiotic” or“ probiotic microorganism’’ refers to one or more live microorganisms that when administered in adequate amounts to a subject may confer a health benefit to said animal. This health benefit is typically the result of the probiotic beneficially modulating the subject’s gastrointestinal microbial balance or flora. Suitably, the probiotic microorganism is a bacterium or a fungus. Broadly, probiotic microorganisms may be of genera selected from the group consisting of Lactobacillus , Bifidobacterium , Enterococcus , Streptococcus , Bacillus , Propionibacterium , Enterococcus , Streptococcus , Pediococcus , Clostridium , Aspergillus , Candida , Saccharomyces and Megasphaera, although without limitation thereto.

Suitably, the probiotic composition described herein comprises one or more bacterial strains that stimulate the growth or activity of one or more bacterial taxa which are under-represented in microbiota of the subject as compared to a healthy control (e.g., a control subject without IBD), such as those of the Firmicutes spp. (e.g., Faecalibacterium prausnitzii ) and Bacteroidetes spp.. Additionally or alternatively, the probiotic composition suitably inhibits growth or activity of one or more bacterial taxa which are over-represented in microbiota of the subject as compared to a healthy control (e.g., a control subject without IBD), such as those of the Proteobacteria phylum (e.g., Enter obacteriaceae, Pasteur ellaceae , and Neisseriaceae and Escherichia coli).

As will be appreciated, faecal transplantation is when a doctor transplants faeces from a healthy donor into another person to at least partly restore the balance of bacteria in their gut. In some embodiments, the faecal transplant is processed faecal material (e.g., a faecal filtrate) having reduced volume and/or faecal aroma relative to unprocessed faecal material. In certain embodiments, the faecal transplant is a faecal bacterial sample. The process may be also referred to as faecal microbiota transplantation (FMT), stool transplant or bacteriotherapy.

In a further aspect, the invention provides a method of treating IBD in a subject, the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, before, during and/or after treatment of IBD, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, and based on the determination made, initiating, continuing, modifying or discontinuing a treatment of IBD.

As used herein, “ treating ^r”, “ treaf or “ treatment’ refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of IBD or dysbiosis after the disease and/or its symptoms have at least started to develop. As used herein, preventing",“ prevent’ or“ prevention” refers to a therapeutic intervention, course of action or protocol initiated prior to the onset of IBD, dysbiosis and/or a symptom thereof so as to prevent, inhibit or delay development or progression of the IBD, dysbiosis or the symptom thereof. In particular embodiments, the present method further includes the step of selecting the treatment of IBD based on the presence or absence of the polymorphic marker.

In certain embodiments, the expression level of one or more miRNA, protein and/or nucleic acid biomarkers, as herein before described, may also be determined.

In particular embodiments, the biological sample comprises tissue, blood, serum, plasma or cerebrospinal fluid. Preferably, the biological sample comprises tissue and/or blood, although without limitation thereto.

In one embodiment, the method further comprises selecting a treatment for IBD based on the presence or absence of the polymorphic marker.

It will be appreciated that the method of treating IBD may include administration of one or more other therapeutic agents that facilitate IBD treatment or prevention. By way of example only, these include: an anti-inflammatory agent (e.g., NSAIDs, corticosteroids, aminosalicylates, an IL23R antagonist, an IL23 antagonist, a TNF-alpha antagonist), balsalazide (Colazal) and olsalazine (Dipentum)), an immunosuppressive agent (e.g., azathioprine, mercaptopurine, cyclosporine and methotrexate), a microbiome modulator, a dietary supplement (e.g., psyllium powder, methylcellulose, an iron supplement, a calcium supplement, a vitamin D supplement), a probiotic composition, an antibiotic a faecal transplant, although without limitation thereto.

Suitably, the treatment of the present aspect is selected from the group consisting of an IL23R antagonist, an IL23 antagonist, a microbiome modulator, a dietary supplement, a probiotic composition, a faecal transplant and any combination thereof.

It will be appreciated that administration of a therapeutic agent can occur through any route, means or method known in the art. In some embodiments the treatment can be administered orally, for example, through a capsule, pill, powder, tablet, gel, or liquid, designed to release the composition in the gastrointestinal tract of the subject. In certain embodiments, administration of a therapeutic agent occurs by a suppository and/or by enema. In other embodiments, a combination of administration routes is utilized.

In another aspect, the invention resides in a kit for determining a prognosis and/or treatment of a subject with IBD, the kit comprising:

reagents for selectively detecting a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, and a collection of data comprising correlation data between the polymorphic marker and prognosis and/or treatment of IBD.

Suitably, the kit of the present aspect is for use in the method of aforementioned aspects.

Suitably, the collection of data is on a computer-readable medium (e.g., software embodying or utilised by any one or more of the methodologies or functions described herein). The computer-readable medium can be included on a storage device, such as a computer memory (e.g., hard disk drives or solid state drives) and preferably comprises computer readable code components that when selectively executed by a processor implements one or more aspects of the present invention.

In one embodiment, the kit further includes one or more primers for determining a microbiome, such as a gut microbiome, in the subject. More particularly, the one or more primers are suitably for use in analysing, amplifying and/or sequencing a variable region of the 16S ribosomal subunit of a microbe. In one particular embodiment, the one or more primers comprise, consist of or consist essentially of the nucleotide sequence set forth in SEQ ID NO: l and/or SEQ ID NO:2 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto.

A“ primer” is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid “template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.

Suitably, primer or nucleic acid variants share at least 40%, 45%, 50%, 55%, 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more preferably at least 80%, 81%, 82%, 83%, 84%, or 85%, and even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with an isolated nucleic acid or primer described herein, inclusive of SEQ ID NOs: l and 2, inclusive of nucleic acids complementary thereto.

A“fragment” is a segment, domain, portion or region of a nucleic acid, which constitutes less than 100% of the nucleotide sequence of the nucleic acid.

In general, fragments may comprise up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 nucleotides or any range therein of a nucleic acid or primer sequence, such as SEQ ID NOS: 1 and 2.

As used herein,“ derivative” nucleic acids have been altered, for example by conjugation or complexing with other chemical moieties. By way of example, the invention further provides use of modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (for example, thiouridine and methylcytosine) in nucleic acids or primers described herein.

In a further aspect, the invention provides a method of determining a susceptibility to dysbiosis in a subject, the method including the step of determining the presence or absence of a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, and wherein determination of the presence or absence of the risk variant is indicative of the susceptibility to dysbiosis in the subject.

In a related aspect, the invention provides a method of determining whether or not a subject has dysbiosis (i.e., diagnosing dysbiosis), the method including the step of determining the presence or absence of a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, and wherein determination of the presence or absence of the risk variant is indicative of the subject having dysbiosis.

Suitably, for the two aforementioned aspects, the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith.

As used herein, the term“ dysbiosis” refers to a condition of microbial (e.g., bacterial, yeast, viral, parasite, etc.) imbalance within the body, such as when the symbiosis of the gut microbiota is dysregulated or disrupted. Dysbiosis can often produce harmful effects via (a) qualitative and quantitative changes in the content or amount of the microbiota itself, (b) changes in their metabolic activities; and/or (c) changes in their local distribution.

In particular embodiments, wherein determination of the absence of the polymorphic marker, such as rsl 1209026, is indicative of the subject being susceptible to or having dysbiosis.

In another aspect, the invention provides a method of treating dysbiosis in a subject, the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, before, during and/or after treatment of dysbiosis, and based on the determination made, initiating, continuing, modifying or discontinuing a treatment of dysbiosis.

Suitably, the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith.

In one embodiment, the method further includes the step of selecting the treatment of dysbiosis based on the presence or absence of the polymorphic marker. Suitably, the treatment is selected from the group consisting of an IL23R antagonist, an IL23 antagonist, a microbiome modulator, a dietary supplement, a probiotic composition, a faecal transplant and any combination thereof.

Suitably, the method of the present aspect further includes the step of administering the treatment or therapeutic agent to the subject. In this regard, administration of a therapeutic agent can occur through any route, means or method known in the art, such as those hereinbefore described.

Referring to the aforementioned aspects, the methods of the invention may further include the step of determining a microbiome or a microbiome profile in the subject. To this end, the microbiome may be used, for example, to detect and/or confirm a diagnosis of dysbiosis in the subject and/or for assisting in determining a prognosis and/or treatment regime for IBD in the subject.

As used herein, an attempt has been made to use the term“ microbiota” to indicate the collections of organisms inhabiting a site, such as the gut, whereas the term “ microbiome” refers, collectively, to the entirety of microbes, their genetic elements (genomes), and environmental interactions, found in association with a higher organism, such as a human. Accordingly, the term microbiome is generally used to indicate the genetic information available in a sample, such as a faecal sample, containing microorganisms. Additionally, the microbiome is a characterization of a microorganism environment, such as the gastrointestinal system, through the corresponding genetic content of those organisms, and includes phylogenetic markers, such as l6SrRNA or HSP60. The microbiota (and hence microbiome) may comprise various different bacteria, protists, archea, viruses, and fungi.

In some embodiments, a subject's microbiome or microbiome profile can be determined from one or a plurality of microbial habitats of the subject. For example, a subject's microbiome or microbiome profile can be determined from an oral microbiome, an oesophageal microbiome, an intestinal microbiome, a gastric microbiome, a colonic microbiome and/or a gut microbiome.

A microbiome or microbiome profile can be assessed using any suitable detection means that can analyse, measure or quantify the genetic material of one or more microbes (bacteria, fungi, viruses and archaea) that comprise the microbiome. Nucleic acid sample prepared from a biological sample, such as a faecal sample or a biopsy, can be subjected to a detection method to generate a profile of the microbiome associated with the sample. Profiling of a microbiome can comprise one or more detection methods known in the art, such as analysis or sequencing of a 16S ribosomal subunit, a 23 S ribosomal subunit, intergenic regions, and other genetic elements of the microbiota. Suitable detection methods can be chosen to provide sufficient discriminative power in a particular microbe in order to identify informative microbiome profiles.

In some embodiments, the entire genomic region of the 16S or 23 S ribosomal subunit of the microbe is analysed or sequenced to determine a subject's microbiome. In other embodiments, a variable region of the 16S and/or 23S ribosomal subunit of the microbe are analysed or sequenced to determine a subject's microbiome. In one specific embodiment, the step of determining a microbiome suitably includes amplifying, analysing and/or sequencing a V3-V4 hypervariable region of a 16S ribosomal subunit of a microbe.

In certain embodiments, the entire genome of the microbe is analysed or sequenced to determine a subject's microbiome. In other applications, the variable regions of the microbe's genome are analysed or sequenced to determine a subject's microbiome. For example, genetic variation in the genome can include restriction fragment length polymorphisms, single nucleotide polymorphisms, insertions, deletions, indels (insertions- deletions), microsatellite repeats, minisatellite repeats, short tandem repeats, transposable elements, randomly amplified polymorphic DNA, amplification fragment length polymorphism or a combination thereof.

Primers used in the disclosure can be prepared by any suitable method, for example, cloning of appropriate sequences and direct chemical synthesis. Primers can also be obtained from commercial sources. In addition, computer programs can be used to design primers. Primers can contain unique barcode identifiers. In one specific embodiment, the primers for use in determining a subject’s microbiome comprise the nucleotide sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO:2 or a fragment, variant or derivative thereof or a nucleotide sequence complementary thereto.

Microbiome profiling can further comprise use of, for example, a nucleic acid microarray, a biochip, a protein microarray, an analytical protein microarray, reverse phase protein microarray (RPA), a digital PCR device, and/or a droplet digital PCR device.

In yet another aspect, the invention resides in a kit for assessing susceptibility to and/or determining the presence and/or treatment of dysbiosis in a subject, the kit comprising: reagents for selectively detecting a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, and

a collection of data comprising correlation data between the polymorphic marker and susceptibility to and/or presence of dysbiosis

Suitably, the collection of data is on a computer-readable medium.

Preferably, the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith.

Suitably, the present kit is for use in the method of three aforementioned aspects.

In one embodiment, the kit further includes one or more primers for determining a microbiome, such as a gut microbiome, in the subject. More particularly, the one or more primers are suitably for use in analysing, amplifying and/or sequencing a variable region of the 16S ribosomal subunit of a microbe. In one particular embodiment, the one or more primers comprise, consist of or consist essentially of the nucleotide sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO:2 or a fragment, variant or derivative thereof.

It would be understood that the methods described herein may be applicable to any mammal. In particular embodiments, the term“ mammaF includes but is not limited to humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Preferably, the subject is a human.

So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples.

EXAMPLE 1

Inflammation in Crohn’s disease most commonly involves the terminal ileum with up to 75% of patients having ileal disease with or without colonic involvement ^{18 20}. Studies investigating the association between inflammation and the mucosal microbiota in CD subjects showed inconsistencies in the results ^21-25 and are limited by a low sample size. The present Example investigates this comparative analysis further by examining mucosa-associated microbiota in a larger patient cohort including ileal CD patients (matched non-inflamed and inflamed ileum) and non-IBD controls (matched ileum and rectum). The strength of the present Example is the use of healthy subjects, ascertained from the same catchment area as the CD case population, without any clinical IBD symptoms for the analysis of the impact of variants within three CD susceptible genes, namely NOD2, IL23R and ATG16L1, on the ileal microbiota.

MATERIALS AND METHODS

Study Participants

Samples were collected from 15 CD patients and 58 healthy individuals (Table 1). Colonoscopy was performed on patients with a diagnosis of ileal CD, and from healthy controls undergoing colorectal cancer family history screening. All subjects gave written informed consent, and the study was approved by the Human Research Ethics Committees of the Royal Brisbane and Women’s Hospital, Brisbane, Australia and the QIMR Berghofer Medical Research Institute, Brisbane, Australia. Pinch biopsy samples were retrieved for DNA extraction from both inflamed ileum (affected CD), non-inflamed ileum (unaffected, CD and healthy controls), and non-inflamed rectum (healthy controls) (Table 2). Additional biopsy samples were taken from within these same areas and placed in formalin for histological examination by a pathologist. Samples for DNA extraction were immediately snap-frozen in dry ice following withdrawal of the endoscope, and then stored at -80°C until use. DNA samples were genotyped as previously described using Taqman technology^1,26’²⁷. Inflammation was assessed using C-reactive protein (in all participating subjects) and calprotectin (healthy control subjects only). The cut-off for high faecal calprotectin and CPR levels were defined as > 50 ug/g and 5 mg/L, respectively.

The CD patient group (median 30, range 19-54) were significantly younger than the healthy control group (median 52.5, range 27 - 82; t-test, P < 0.001), but were similar in BMI (CD, median: 24.1, range: 20 - 33.5; control, median: 26.9, range: 15.1 - 43.9) and smoking prevalence at the time of colonoscopy (CD, 20% yes; control, 29% yes).

DNA Extraction, 16S rRNA analysis

DNA was isolated from mucosal pinch biopsies using DNeasy Blood and Tissue Kit (QIAGEN) following a tissue homogenisation step using Precellys Lysing Kit tubes containing 1.4 mm ceramic beads. DNA was quantitated using Nanodrop 2000 (Thermo Scientific). The V3-V4 hypervariable region of the 16S rRNA gene was PCR amplified for sequencing and sequenced on a MiSeq sequencer (Australian Genome Research Facility (AGRF), Brisbane, Australia). The primers used for this region are shown in Table 7. Raw sequenced reads were merged using PEAR vO.9.6 ²⁸ and quality filtered in the Quantitative Insights Into Microbial Ecology (QIIME) ²⁹ software vl.9. l using default settings. Filtered sequences were clustered into Operational Taxonomic Units (OTUs) using Greengenes database vl3.5 ³⁰ as a reference and a 97% sequence identity cut-off with the UCLUST software v8.0.1623 ³¹. Sequences without matches to the reference database were clustered de-novo (97% sequence identity). The seed sequence of each cluster was subsequently assigned to a taxonomic lineage using the UCLUST software. Seed sequences that are identified as chimeric with UCHIME were removed. Also singleton OTUs were discarded from the downstream analysis.

Statistical analysis

Using Calypso v8.20 ³², we analysed the impact of the patient genotype and sample collection, i.e. non-inflamed vs inflamed ileum in CD patients and non-inflamed ileum vs non-inflamed rectum in non-IBD control subjects, on the intestinal microbiota. Briefly, data was imported using total sum normalization with square root transformation. Multivariable linear regression with genotype, gender and age as independent and taxa abundances (family or OTU level) and diversity (Shannon diversity, microbial richness) as dependent variables were applied to identify genotype-associated taxa and diversity changes. In addition, Redundancy Analysis (RDA) including genotype, age and gender as explanatory variables was carried out to assess associations between the microbial (OTU) and host genetic compositions. Analysis of similarity (ANOSIM) and a multivariate ANOVA based on dissimilarity test (ADONIS) were applied to analyse the impact of each demographic variable on the overall composition at OTU level of the ileal microbiota in inflamed or non-inflamed subjects. Hierarchical clustering, RDA, Principal Coordinates Analysis (PCoA) and multivariable linear regression were executed in Calypso using default settings to study disease-associated changes in OTUs between healthy controls and CD subjects. Lasso regularized regression was used to identify a phylotype signature describing the microbial data in health and disease. Correlations between OTUs and explanatory variables (diversity, richness, inflammation and disease group) were identified using network analysis. Hierarchical clustering, PCoA and RDA including inflammation status and patient identifier as explanatory variables were run on the OTU profiles of CD subjects using Bray-Curtis distance. Multivariable linear regression analysis with diversity and richness as dependent variable and age, gender, inflammation degree and treatment as independent variables were used to assess impact of the inflammation on microbial diversity in CD patients. Differences in the abundance of taxa, Shannon diversity and richness within individuals between non-inflamed and inflamed CD samples were assessed by mixed effects linear regression and paired Wilcoxon rank test. The same methods were applied to analyse taxa and diversity changes between rectum and ileum samples from healthy participants /’-values were corrected using False Discovery Rate (FDR).

RESULTS

Study design

The study cohort included 15 individuals with mild or moderate CD and 58 healthy control individuals (Table 2). A total of 13,990,816 paired end reads underwent 16S sequencing. After quality filtering a total of 12,218,964 sequences were used for downstream analysis (median 95,476, range: 5,816 - 492,046). In total, 15 CD patients with mild or moderate disease, matched ileal (non-inflamed and inflamed mucosal biopsy tissue) samples and paired biopsies from 56 healthy non-IBD controls (ileum, rectum) were analysed.

Genotype-microbiome interactions

IL23R R381Q variant impacts the ileal microbiome in healthy subjects towards a favourable microbial composition and higher diversity

The majority of healthy individuals (n=46) were wild-type (wt, genotype GG) for the IL23R gene variant rsl 1209026, while 12 individuals were heterozygous (het, genotype AG), carrying the protective A allele. Ileal samples were ordinated by RDA (Fig. 1A), which tend to separate samples by the IL23R gene alleles (RDA including age, gender as environmental factor, P = 0.026). Unsupervised ordination methods indicated that subjects who were wt genotype (GG), had a heterogeneous microbial community, which overlaps with the stable microbiome of subjects with the het genotype (AG) and with the microbiome of CD subjects (Fig. IB). The commensal family Christensenellaceae was decreased in the wt group as compared to the het group carrying the protective A allele (multiple linear regression, corrected for age and gender, P < 0.001, FDR < 0.001). At phylotype level, Operational Taxonomic Units (OTUs) related to Bacteroides caccae, Christensenellaceae, Anaerostipes and Oscillospira were decreased in the wt group (multiple linear regression, P < 0.005, FDR < 0.1) (Fig. 1C). The changes were not significant after /^J- value correction using false discovery rate, but remained significant when the healthy and CD cohorts (non-inflamed tissue samples) were included (multiple linear regression, corrected for disease status, age, gender, p < 0.001, FDR < 0.05). Healthy subjects carrying the IL23R wt genotype (GG) had a reduced Shannon diversity and richness compared to the het genotype (ANOVA, corrected for age and gender using multivariable linear regression, P < 0.05), (Fig. ID). Shannon diversity and richness were also associated with the IL23R genotype using a linear regression model with the risk genotype, gender and age as dependent variables ( P < 0.05), but not the NOD2 and ATG16Ll genotypes (P > 0.4). Unlike in the ileum, the overall rectal microbial community (RDA, corrected for age and gender, P = 0.1) was not associated with the IL23R genotype. OTU diversity (ANOVA, corrected for age and gender P = 0.08), richness ( P = 0.07), and the abundance of family Christensenellaceae (multiple linear regression, P = 0.012, FDR = 0.5) tended to be decreased in healthy individuals with the wt genotype who lacked the IL23R rsl 1209026 protective allele (Figure 6A-C).

NOD2 and ATG16L1 genotypes were not associated with microbial composition and diversity

No association between NOD2 mutations (R702W rs2066844, G908R rs2066845 and Leul007fsinsC rs5743293), (wt, n=47; het and horn combined, n=9; not defined, n=2) and the global microbial community composition was recorded in the healthy cohort (RDA, including genotype, age, gender as covariates, P > 0.7). Also no significant differences following /^J- value correction at the family and OTU level (multiple linear regression, FDR > 0.3), or in OTU diversity and richness (ANOVA corrected for age, gender, P > 0.5) were detected between the microbiomes of healthy subjects with NOD2 wt and mutated alleles. In the CD patients, the het (n=3) and hom (n=3) NOD2 variants (R702W, G908R, Leul007fsinsC) clustered apart but overlapped with the NOD2 wild- type subjects (n=9), (Figure 7A). The family Bifidobacteriaceae tended to be increased in the wild-type form of the NOD2 gene (multiple linear regression, corrected for age, gender, P = 0.052, FDR = 0.72) (Figure 7B). Subjects with NOD2 hom variants tended to have a decreased diversity, while the diversity between NOD2 wild-type subjects and those carrying a het NOD2 variant was similar (Figure 7C-D). No association between the ATG16L1 gene genotypes (rs224l880) and microbial diversity (ANOVA, P > 0.6) (Figure 7E-F) and composition was identified in the healthy cohort.

Impact of subject clinical and demographic variables on community structure and microbial diversity in CD subjects

Univariate analysis of subject demographics (age, gender, BMI, smoking behaviour) and CD patient clinical variables (age at diagnosis, Montreal location, Montreal behaviour ever, perianal disease, Harvey Bradshaw Index score (well-being), surgery, immunosuppression use >3 months, IV steroids and treatment with biologies ever), Table 1, identified immunosuppression use >3 months at the time of biopsy as affecting the microbial community in inflamed and non-inflamed ileum samples of CD patients (ANOSIM, P < 0.05). Non-inflamed tissue samples from patients on immunosuppression therapy >3 months (n=5) had a lower Chaol richness (ANOVA, P = 0.04; ANOVA corrected for inflammation degree of adjacent inflamed tissue, P = 0.09), a higher abundance of phylotypes related to Enterobacteriaceae based on LEfSe analysis (LDA score > 3.5) and multiple linear regression using inflammation degree and immunosuppression usage at dependent variables and Enterobacteriaceae OTU abundances as dependent variables ( P < 0.03, FDR = 0.67), (Figure 8). Multivariate analysis of subject demographics and treatment indicated that gender also has an impact on the microbiota in non-inflamed (ADONIS, P < 0.05). Univariate and multivariate analysis did not indicate associations between age and microbial composition. Using a multivariable linear regression model with richness as dependent variable and demographics as independent factors revealed that gender tend to be associated with richness ( P = 0.08).

Ileal CD patients (inflamed vs. non-inflamed ileum) Community structure and microbial diversity

The phyla Bacteroidetes, Proteobacteria and Firmicutes were most abundant compared to the other main phyla in the human gut ( Actinobacteria , Fusobacteria, TM7, and Verrucomicrobid) and accounted for 85% of all sequenced reads in the ileal microbiome of CD patients (n=l5), (Figure 9A). In two CD patients in our study, however, Fusobacteria was the most abundant phylum, which when combined with Proteobacteria and Bacteroidetes contributed to at least 70% of total reads. Family members Bacteroidaceae (p. Bacteroidetes) and Enterobacteriaceae (p. Proteobacteria ) were present in considerably higher proportions in the majority (80%) of ileal (non- inflamed, inflamed) samples, and when combined together comprised approximately 50% of the ileal microbiota in the CD patient group (Figure 9B). At the phylotype level, OTUs related to Bacteroides and Enterobacteriaceae were most abundant in the ileal CD subjects.

Using mixed effects linear regression, OTUs assigned to Lachnospiraceae , Bacteroides and Eubacterium dolichum were significantly different between inflamed and non-inflamed samples from within each individual, but the changes were not significant following P-value correction ( P < 0.05, FDR = 0.08), (Figure 10). Likewise, OTUs related to Lachnospiraceae and Bacteroides differed between inflamed and non-inflamed CD tissue samples using paired Wilcoxon rank test, but no consistent significant differences were detected following C- value correction using false discovery rate (paired Wilcoxon rank test, OTU: p < 0.05, FDR > 0.9). There were no significant changes in microbial diversity and richness at the OTU level within individuals between non- inflamed and inflamed ileal matched tissue (paired Wilcoxon rank test, mixed effects linear regression, P > 0 1), (Figure 11)

Principle coordinates analysis (PCoA) (Figure 2A) of all ileal CD samples (non- inflamed, inflamed (mild and moderate)) identified high intra-individual stability and high inter-individual variability. In accordance with the observation of high intra-individual stability, multivariate and univariate statistics showed that the individual is significantly associated with the microbial community at OTU level (RDA, ANOSIM, P < 0.001), whereas biopsy inflammation status (inflamed vs non-inflamed) does not impact the microbial community composition (RDA, ANOSIM, P > 0.8).

Disease severity is associated with the ileal microbiome Moderately inflamed samples (n = 5) have a significantly lower Shannon diversity than mildly inflamed samples (n = 10), (ANOVA, P < 0.05, controlled for treatment), (Figure 11 A). Also non-inflamed tissue samples from patients with moderate inflammation differed significantly in Shannon diversity and richness as compared to the non-inflamed tissue samples from patients with mild inflammation (ANOVA, P < 0.05), (Figure 11A-B). In accordance, linear regression using inflammation degree and treatment as covariates indicated an association of the inflammation with microbial richness ( P < 0.04). Inflammation degree remained significant using age and gender as covariates in a linear regression model with richness as the dependent variable ( P < 0.05). No significant differences were identified for diversity and richness (paired Wilcoxon rank test, multivariable linear regression adjusted for treatment, P >0.1) between non- inflamed biopsies and their matched moderately inflamed tissue pairs (n = 5).

Multivariate statistics of the OTU taxonomic profile of inflamed samples using Bray-Curtis distance (n = 15, 10 mild, 5 moderate) confirmed that inflammation degree (mild versus moderate) has an impact on the microbiota (ADONIS with environmental variables inflammation degree and treatment, P = 0.045) (Figure 11C). Hierarchical cluster analysis based on the relative abundance of the 50 most abundant OTUs in the matched inflamed and non-inflamed samples identified two distinct clusters (Figure 12A). Cluster 1 (Figure 12A, left cluster) represented samples with a high abundance of OTUs assigned to Enter obacteriaceae and a low abundance of remaining OTUs. This cluster was composed of paired non-inflamed and inflamed (mild/moderate) from 7 ileal CD patients. Of note, all 5 moderately inflamed samples fell into this cluster. In contrast, cluster 2 (Figure 12A , right cluster) was characterized by a high abundance of OTUs assigned to Bacteroidetes, a low abundance of OTUs assigned to Enter obacteriaceae, and a high diversity. This cluster contained paired non-inflamed and mildly inflamed samples only.. C-reactive protein, (CRP), a marker of inflammation in blood, was not associated with the non-inflamed or inflamed ileal microbiota (RDA, P > 0.3) and with microbial Shannon diversity and richness (Muliple linear regression model, P adjusted for age, gender or BMI > 0.1). No correlation (Pearson) was found between CRP and Proteobacteria phylotypes.

The ileum microbiota in non-inflamed mucosal biopsies differs significantly between ileal CD patients and healthy control groups Community structure and microbial diversity

PCoA analysis showed that the majority of samples clustered according to disease (Figure 3A). In accordance with this observation, a heatmap of identified OTUs showed a clustering of the samples according to disease status (Figure 12B). Sample clustering according to disease status was supported by multivariate statistics using RDA indicating that CD and healthy individuals have significantly different microbial composition (RDA, controlled for age, gender, P = 0.001). On a family level, Ruminococcaceae , Christensenellaceae and Enter obacteriaceae were significantly different between non- inflamed CD and healthy subjects (P < 0.01, FDR < 0.05, corrected for gender, age), (Figure 3B)³³. In total, 11 OTUs showed a different relative abundance in both groups ( P <0.05, FDR <0.05), (Figure 3C). Most notably, Faecalibacterium prausnitzii differed between healthy control and non-inflamed CD samples (P < 0.001, FDR < 0.001)³⁴. Further OTUs significantly increased in the healthy group belonged to Lachnospiraceae , Oscillospira and Ruminococcacae ^33,34.

An optimal OTU signature for predicting disease status (non-inflamed CD vs healthy control) was determined by Lasso regularized regression and comprised of 4 OTUs, namely Faecalibacterium prausnitzii , Bacteroides ovatus, Lachnospira and Ruminococcaceae sp., which when combined achieved a notable separation (AUC of 0.95). Using only phylotype Faecalibacterium prausnitzii in Lasso regularized regression revealed an AUC of 0.88.

There were notable differences in Shannon diversity and richness between the two groups with less diversity and richness in non-inflamed ileal CD samples compared to non-inflamed healthy control samples (ANOVA, corrected for age, gender, P < 0.005), (Figure 3D-E). Network analysis of the OTUs identified in the ileal samples (CD and healthy control subjects) and environmental variables (disease status, diversity) resulted in two distinct, highly correlating clusters (Figure 4). Disease status, diversity and richness all correlated with OTUs within each cluster. Healthy status, diversity and richness correlated positively with OTUs of the phylum Firmicutes and Bacteroidetes, while disease status, regardless of inflamed or non-inflamed, was positively associated with OTUs assigned to Proteobacteria, Firmicutes and Fusobacteria.

The serum inflammatory marker CRP did not have impact on the microbial community structure in non-inflamed CD and control samples (RDA, controlled for age and gender, P > 0.1). Subjects with a high CRP tended to have a lower richness than subjects with a normal CRP ( P = 0.0693, ANOVA). There was no association between CRP and the family Enter obacteriaceae or phylum Proteobacteria (P > 0.2) (Figure 13A). Interestingly fecal calprotectin had a strong impact on the ileal microbiota in healthy subjects (RDA, corrected for gender, age, BMI, P < 0.05) and correlated positively with the phylum Proteobacteria (Partial correlation, P = 0.001) (Figure 13B) and the family Enter obacteriaceae (P = 0.001). Shannon diversity and richness were significantly lower in healthy subjects with high fecal calprotectin values than with normal calprotectin values (ANOVA, corrected for gender, age, BMI, P < 0.05), (Figure 13C-D). Samples with a high fecal calprotectin value had an increase in OTUs assigned to Enterobacteriaceae and decrease in Faecalibacterium (linear regression, P < 0.003, FDR = 0.05).

Influence of sampling site on microbial community (ileum vs rectum in healthy subjects)

Paired biopsy samples were analysed from the ileum and rectum from 56 healthy control subjects. Hierarchical cluster analysis (Figure 14) showed high intra-individual stability and high inter-individual variability for the healthy control paired samples (ileum and rectum). The overall microbial composition was significantly associated with inter individual variation (RDA, P = 0.001) and by sample site (ileum vs rectum) (RDA, P = 0.002). Of interest, microbial diversity and richness were significantly different between sampling sites (paired Wilcoxon rank test P = 0.05), with the rectum having a slightly higher diversity than the ileum (Figure 5A-B). Sample site was also associated with Shannon index and richness using mixed effects linear regression (adjusted for gender, age, P < 0.04). There were also significant differences in abundance levels in the paired ileal and rectal samples in families, specifically, Desulfovibrionaceae , Camplyobacteraceae , Bart lesie I laceae , Gemellaceae and Microcacceae (paired-Wilcoxon rank test, P < 0.05, FDR < 0.05) (Figure 5C-G). At OTU level, Oscillospira sp. and Ruminococcaceae sp. differed significantly between ileum and rectal samples (paired Wilcoxon rank test, P < 0.05, FDR < 0.05), (Figure 5H). These differences were supported using mixed effects linear regression.

DISCUSSION

The mucosa-associated microbiota is influenced by multiple environmental factors, such as diet, parasite infections and antibiotic usage, and by host genetic variation. Recently it was hypothesized that variations in IBD susceptibility genes might lead to the inability of the host to sense or respond to beneficial microbes which subsequently increases inflammation risk and IBD in the gut ³⁵. In this study, the impact of the CD risk genes NOD2 , IL23R , ATG16L1 was examined on the intestinal microbiome in healthy individuals without clinical symptoms for IBD. Individuals carrying the IL23R R381Q protective allele had an increased abundance of microbes related to Christensenellaceae , Bacteroides caccae and Oscillospira , which belong to the commensal bacteria and are reported to be enriched in non-IBD individuals ^{8 33 36·38} in particular Christensenellaceae was significantly associated with the //.23/riprotective coding variant at family level. The family Christensenellaceae was described to form a hub in a co-occurrence network with other taxa and is associated with health ³⁹. The OTU related to Oscillospira was enriched in //.23/riprotective coding variant in our data and was reported to be increased after transferring Christensenella minuta to mice ³⁹. In our study, only one of 15 ileal CD patients carried the protective R381Q protein variant of the IL23R gene, while the remaining subjects had the CD-associated wild-type form further supporting the importance of this gene in disease development.

Our study indicates no association between the NOD2 gene and intestinal microbial composition in healthy individuals ¹⁰. The majority of the healthy individuals in our study cohort had either the heterozygote or wild-type NOD2 gene form, with only one subject homozygous for NOD2 (R702W), hence we might be underpowered to infer the impact of NOD2 on the microbiome. The low NOD2 allele frequencies in our healthy control population are consistent with previous observations ⁴⁰. The lack of significant microbiome variation between NOD2 wild-type and NOD2 heterozygous individuals in our dataset, however, is in agreement with a recent study, in which microbiome variation, in particular, Enterobacteriaceae distribution, was only identified in NOD 2-deficient subjects ⁴¹.

Our study examined the mucosa-associated microbiota in histologically defined matched inflamed and non-inflamed ileal biopsies in ileal CD patients with mild or moderate disease, and in matched ileal and rectal samples from healthy control subjects. 16S rRNA sequence analysis indicated within ileal CD patients, a high level of stability within the microbiota communities, particularly, at the high order taxa (phyla), in all subjects regardless of biopsy inflammation status. These findings are supported by several studies ^8,42 and demonstrate the strength and steadiness of each individual’s microbial population in the gut. Minor differences were identified at lower taxa level, but were not significant following C- value correction, which may have been a result of the small sample size. Additionally, we found a subject-specific stability in the microbiota profile between sites, ileum and rectum, in the healthy controls. Despite intra-individual stability between different sites, significant changes in OTU abundances and microbial diversity were identified. These data demonstrated, in disagreement with Gevers et al ., that biopsy site does impact the microbial composition across the human midgut and hindgut.

The microbial populations did differ substantially between CD subjects and between disease states (mild, moderate inflammation). The ileal mucosa-associated microbiota in ileal CD subjects was dominated by Bacteroidetes and Proteobacteria phyla, which were present in the majority of ileal (non-inflamed, inflamed) samples and when combined accounted for half of the ileal microbiota in the CD patient group. We examined the abundance of families in ileal CD samples stratified by inflammation status and observed a shift of families Christensenellaceae, Lachnospiraceae and Ruminococcacae (Firmicutes) towards the less diverse cluster dominated by Enterobacteriaceae (p. Proteobacteria ) in inflamed samples. Increased levels of Enterobacteriaceae have recently been detected in the gut microbiome of Proton Pump Inhibitor (PPI) users ⁴³. In our CD study cohort, only 1 of 15 patients was confirmed as a PPI user, and hence was not considered as influencing our data.

The inclusion of histologically confirmed mild and moderately inflamed mucosal biopsy samples in our ileal CD cohort identified a novel phyla signature unique to ileal CD patients with moderate disease. Notably, a dominant Proteobacteria presence was detected in moderately inflamed as well as the paired non-inflamed sample. The profound increase in Proteobacteria was associated with a significant reduction in other taxa. These data suggest a‘tipping point’ between mild and moderate Crohn’s disease severities whereby the mucosa-associated microbiota profile shifts dramatically to a less diverse, and a more pathogenic landscape. The high relative abundance of Proteobacteria has been reported in CD ³³, and other chronic disorders such as chronic obstructive pulmonary disorder and celiac disease ^44,45. This phylum, which normally represents ~5% of our human gut microbiota ¹⁵, exploits the host’s inflammatory response via anaerobic respiration gaining a fitness selection advantage over the other resident bacteria ^{12 14}. Longitudinal studies are required to determine whether the microbial diversity is restored following response to treatment in the group of ileal CD patients with moderate disease. Interestingly, the subset of healthy controls which clustered with the ileal CD group were also characterised by a high abundance of Proteobacteria in both paired ileum and rectum samples, and reduced diversity of other taxa. Detailed health/clinical histories were not collected from the healthy control subjects, but despite this limitation, our 16S data suggest profound dysbiosis of the gut microbiota in this subset of healthy controls. The positive correlation between fecal calprotectin and Proteobacteria levels in the healthy control group suggests that fecal calprotectin could potentially be useful clinically to identify individuals (patients and healthy controls) with high microbial dysbiosis (low diversity) whom may benefit from dietary and/or clinical interventions to shift the microbiota profile to a less dysbiotic state, either by the use of therapies such as metformin and/or probiotics/dietary changes to reduce host inflammation and increase the frequency of short chain fatty acid producing bacteria ⁴⁶. Similarly, fecal calprotectin could be used to exclude donors for fecal microbiota transplantation whereby elevated calprotectin levels would indicate low microbial diversity.

Two factors known to influence the gut microbial composition are antibiotic use and smoking history. Gevers et al, 2014 found that exposure to antibiotics had a strong impact on the microbial composition but a weak effect on overall species diversity. In our study, one of fifteen CD patients was on antibiotic treatment. Reanalysis based on fourteen CD patients, who did not take antibiotics one month prior sampling, supported described observations (Tables 4-5). Of the thirty-three control subjects with known antibiotic exposure details at the time of their procedure, none were on antibiotic treatment. Based on the Australian antimicrobial usage report of 2016 ⁴⁷, about 3.5% individuals per month, i.e. in our study two individuals in the sampled age group, could expect to have antibiotic exposure in the month prior to their biopsy sample being taken. We recognise antibiotic exposure as a limitation in our study as it potentially impacts the overall community structure in the healthy control data set with an increase in gut microbiota dysbiosis. A sub-analysis based on subjects without reported antibiotic exposure one month prior biopsy sampling confirmed the significant differences in the majority of genotype- and disease-associated taxa and microbial diversity (Tables 3-5). Smoking at biopsy in the ileal CD and healthy control groups did not impact microbial community, diversity, or relative abundance of Proteobacteria OTU.

In conclusion, this study shows that a microbial change has occurred in unaffected tissues in CD diagnosed individuals and in healthy subjects without a diagnosis of IBD. Genotype is highly associated with this microbial change. These data indicate that the IL23R gene affects the compositional landscape of the microbiome by shaping the ileal microbiota towards a beneficial microbial consortium with a higher diversity. However, identifying the mechanisms that are involved in modifying a healthy microbiome into a pathobiota remain a challenging task. CD is a genetically complex disease with many described genetic susceptibility loci and more research is required to understand the interaction between host genetics, microbiome and environmental factors.

Table 1: Demographic and clinical characteristics of ileal CD and non-IBD control patients.

Crohn's Healthy disease, (n=l 5) controls, (n=58)

Characteristic N (%) N(%)

Median Age at Colonoscopy ^# 30 52.5

Gender (male) 7 (47) 27(46.6)

Medi an BMI kg/ m² 24.1 * 26.9*

Smoking behaviour

Ever 9 (60) 35 (60.3)*

At time of colonoscopy 3 (20) 21 (33.3)*

Age at Diagnosis (CD)

<15 0

15-40 12 (80)

<40 3 (20)

Location (CD), At Biopsy

Ileal 9 (60)

Ileocolonic 6 (40)

Colonic 0

Behaviour (CD), Ever

Non-stricturing, non-penetrating 3 (20)

Stricturing 7 (47)

Penetrating 5 (33)

Perianal Disease (CD) Ever 4 (27)

At Biopsy 2 (13)

Harvey-Bradshaw Index score (CD)*

Remission, <5 9 (69)

Mild disease, 5-7 2 (15)

Moderate disease, 8-16 2 (15)

Severe disease, >16 0

Colectomy/Surgery Before Biopsy

(CD) 11 (73)

Antibiotics (CD) at Biopsy 1 (7)

Immunosuppression (>3 mo)(CD)

Ever 11 (73)

At Biopsy 5 (33)

IV Steroids (CD)

Ever 6 (40)

At Biopsy 0

Biologies (CD)

Ever 3 (20)

At Biopsy 1 (7)

*Data was not available for all patients

^# Variable differed significantly between the healthy control and CD group (t- test, p < 0.001) Table 2: Biopsy Characteristics

Disease (N) Location (N) Inflammation status

Non-inflamed (N) Inflamed (N)

(mild/mo derate)

CD (15) Heum (15) 15 10/5

Healthy controls (58) Heum (58) 58 0

Rectum (56) 56 0

Table 3: Associations between IL23R genotype and taxa or diversity in the ileum

¹ Based on GLM

² Based on multiple regression and aov Table 4: Associations between IL23R genotype and taxa or diversity in the rectum

¹ Based on multiple linear regression

² Based on ANOVA

Table 5: Associations between disease status and taxa or diversity in the ileum

¹ Based on multiple linear regression

² Based on ANOVA

Table 6: Associations between inflammation degree, microbial diversity and global community level

Table 7: Sequencing details

REFERENCES

1. Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012;491: 119-24.

2. Cleynen I, Boucher G, Jostins L, et al. Inherited determinants of crohn's disease and ulcerative colitis phenotypes: A genetic association study. Lancet 2016;387: 156-67.

3. de Lange KM, Moutsianas L, Lee JC, et al. Genome-wide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease. Nat Genet 2017;49:256-61.

4. Liu JZ, van Sommeren S, Huang H, et al. Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations. Nat Genet 2015;47:979-86.

5. Parkes M, Barrett JC, Prescott NJ, et al. Sequence variants in the autophagy gene irgm and multiple other replicating loci contribute to crohn's disease susceptibility. Nat Genet 2007;39:830-2.

6. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in nod2 associated with susceptibility to crohn's disease. Nature 2001;411:603-6.

7. Kaakoush NO, Day AS, Huinao KD, et al. Microbial dysbiosis in pediatric patients with crohn's disease. J Clin Microbiol 2012;50:3258-66.

8. Gevers D, Kugathasan S, Denson LA, et al. The treatment-naive microbiome in new-onset crohn's disease. Cell host & microbe 2014;15:382-92.

9. Sokol H, Pigneur B, Watterlot L, et al. Faecalibacterium prausnitzii is an anti inflammatory commensal bacterium identified by gut microbiota analysis of crohn disease patients. Proceedings of the National Academy of Sciences of the United States of

America 2008;105: 16731-6.

10. Kennedy NA, Lamb CA, Berry SH, et al. The impact of nod2 variants on fecal microbiota in crohn's disease and controls without gastrointestinal disease. Inflamm Bowel Dis 2018;24:583-92.

11. Morgan XC, Tickle TL, Sokol H, et al. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol 20l2;13:R79.

12. Winter SE, Baumler AJ. Why related bacterial species bloom simultaneously in the gut: Principles underlying the 'like will to like' concept. Cellular microbiology

2014;16: 179-84. 13. Winter SE, Baumler AJ. Dysbiosis in the inflamed intestine: Chance favors the prepared microbe. Gut microbes 2014;5:71-3.

14. Winter SE, Lopez CA, Baumler AJ. The dynamics of gut-associated microbial communities during inflammation. EMBO reports 2013;14:319-27.

15. Shin NR, Whon TW, Bae JW. Proteobacteria: Microbial signature of dysbiosis in gut microbiota. Trends in biotechnology 2015;33:496-503.

16. Dicksved J, Halfvarson J, Rosenquist M, el al. Molecular analysis of the gut microbiota of identical twins with crohn's disease. ISME J 2008;2:716-27.

17. Friswell MK, Gika H, Stratford P, el al. Site and strain-specific variation in gut microbiota profiles and metabolism in experimental mice. PLoS One 20l0;5:e8584.

18. Radford-Smith G, Pandeya N. Associations between nod2/cardl5 genotype and phenotype in crohn's disease— are we there yet? World journal of gastroenterology 2006;12:7097-103.

19. Brant SR, Picco MF, Achkar JP, et al. Defining complex contributions of nod2/cardl5 gene mutations, age at onset, and tobacco use on crohn's disease phenotypes. Inflammatory bowel diseases 2003;9:281-9.

20. Thia KT, Sandbom WJ, Harmsen WS, Zinsmeister AR, Loftus EV, Jr. Risk factors associated with progression to intestinal complications of crohn's disease in a population-based cohort. Gastroenterology 2010;139:1147-55.

21. Davenport M, Poles J, Leung JM, et al. Metabolic alterations to the mucosal microbiota in inflammatory bowel disease. Inflamm Bowel Dis 2014;20:723-31.

22. Bibiloni R, Mangold M, Madsen KL, Fedorak RN, Tannock GW. The

bacteriology of biopsies differs between newly diagnosed, untreated, crohn's disease and ulcerative colitis patients. J Med Microbiol 2006;55:1141-9.

23. Gophna U, Sommerfeld K, Gophna S, Doolittle WF, Veldhuyzen van Zanten SJ. Differences between tissue-associated intestinal microfloras of patients with crohn's disease and ulcerative colitis. J Clin Microbiol 2006;44:4136-41.

24. Sepehri S, Kotlowski R, Bernstein CN, Krause DO. Microbial diversity of inflamed and noninflamed gut biopsy tissues in inflammatory bowel disease. Inflamm Bowel Dis 2007;13:675-83.

25. Walker AW, Sanderson JD, Churcher C, et al. High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease. BMC Microbiol 2011 ; 11 : 7. 26. Doecke JD, Simms LA, Zhao ZZ, et al. Smoking behaviour modifies il23r- associated disease risk in patients with crohn's disease. Journal of gastroenterology and hepatology 2015;30:299-307.

27. Roberts RL, Gearry RB, Hollis-Moffatt JE, et al. Il23r r38lq and atg 1611 t300a are strongly associated with crohn's disease in a study of new Zealand Caucasians with inflammatory bowel disease. Am J Gastroenterol 2007;102:2754-61.

28. Zhang J, Robert K, Flouri T, Stamatakis A. Pear: A fast and accurate illumina paired-end read merger. Bioinformatics 2014;30:614-20.

29. Caporaso JG, Kuczynski J, Stombaugh J, et al. Qiime allows analysis of high- throughput community sequencing data. Nat Methods 2010;7:335-6.

30. DeSantis TZ, Hugenholtz P, Larsen N, et al. Greengenes, a chimera-checked l6s rma gene database and workbench compatible with arb. Appl Environ Microbiol

2006;72:5069-72.

31. Edgar RC. Search and clustering orders of magnitude faster than blast.

Bioinformatics 2010;26:2460-1.

32. Zakrzewski M, Proietti C, Ellis JJ, et al. Calypso: A user-friendly web-server for mining and visualizing microbiome-environment interactions. Bioinformatics

2017;33:782-3.

33. Mancabelli L, Milani C, Lugli GA, et al. Identification of universal gut microbial biomarkers of common human intestinal diseases by meta-analysis. FEMS microbiology ecology 2017; 93.

34. Tyler AD, Kirsch R, Milgrom R, et al. Microbiome heterogeneity characterizing intestinal tissue and inflammatory bowel disease phenotype. Inflammatory bowel diseases 2016;22:807-16.

35. Chu H, Khosravi A, Kusumawardhani IP, et al. Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease. Science 20l6;352: 1116-20.

36. Kverka M, Zakostelska Z, Klimesova K, et al. Oral administration of

parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition. Clin Exp Immunol 2011;163:250-9.

37. Frank DN, St Amand AL, Feldman RA, et al. Molecular-phylogenetic

characterization of microbial community imbalances in human inflammatory bowel diseases. Proceedings of the National Academy of Sciences of the United States of America 2007;104: 13780-5. 38. Pascal V, Pozuelo M, Borruel N, et al. A microbial signature for crohn's disease. Gut 2017;66:813-22.

39. Goodrich JK, Waters JL, Poole AC, et al. Human genetics shape the gut microbiome. Cell 2014;159:789-99.

40. Hugot JP, Zaccaria I, Cavanaugh J, et al. Prevalence of cardl5/nod2 mutations in

Caucasian healthy people. The American journal of gastroenterology 2007;102: 1259-67.

41. Knights D, Silverberg MS, Weersma RK, et al. Complex host genetics influence the microbiome in inflammatory bowel disease. Genome Med 20l4;6: l07.

42. Forbes JD, Van Domselaar G, Bernstein CN. Microbiome survey of the inflamed and noninflamed gut at different compartments within the gastrointestinal tract of inflammatory bowel disease patients. Inflammatory bowel diseases 2016;22:817-25.

43. Imhann F, Bonder MJ, Vich Vila A, et al. Proton pump inhibitors affect the gut microbiome. Gut 2016;65:740-8.

44. Monso E. Microbiome in chronic obstructive pulmonary disease. Annals of translational medicine 20l7;5:25l.

45. Wacklin P, Laurikka P, Lindfors K, et al. Altered duodenal microbiota composition in celiac disease patients suffering from persistent symptoms on a long-term gluten-free diet. The American journal of gastroenterology 2014;109: 1933-41.

46. Forslund K, Hildebrand F, Nielsen T, et al. Corrigendum: Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota. Nature

20l7;545: l l6.

47. Australian_Commission_on_Safety_and_Quality_in_Health_Care_(ACSQHC).

A ura 2016: First australian report on antimicrobial use and resistance in human health summary report. Sydney: ACSQHC; 2016.

Claims

1. A method of determining a prognosis of a subject with an inflammatory bowel disease (IBD), the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, to thereby evaluate the prognosis of the IBD in the subject.

2. The method of Claim 1, wherein determination of the presence of the polymorphic marker is indicative of a positive prognosis.

3. The method of Claim 1 or Claim 2, further including the step of determining whether the subject would benefit from a treatment of the IBD based, at least in part, on the prognosis.

4. The method of Claim 1 or Claim 2, further including the step of determining whether the subject would benefit from a treatment of dysbiosis based, at least in part, on the prognosis.

5. The method of Claim 1 or Claim 2, further including the step of determining suitability of the subject for a treatment based, at least in part, on the prognosis.

6. The method of any one of Claims 3 to 5, further including the step of developing a treatment strategy for the subject based, at least in part, on the prognosis.

7. A method of treating IBD in a subject, the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, before, during and/or after treatment of IBD, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, and based on the determination made, initiating, continuing, modifying or discontinuing a treatment of IBD.

8. The method of Claim 7, further including the step of selecting the treatment of IBD based on the presence or absence of the polymorphic marker.

9. The method of any one of Claims 3 to 8, wherein the treatment is selected from the group consisting of an IL23R antagonist, an IL23 antagonist, a microbiome modulator, a dietary supplement, a probiotic composition, a faecal transplant and any combination thereof.

10. The method of any one of the preceding claims, further including determining an expression level of a protein, miRNA or nucleic acid biomarker.

11. A method of determining a susceptibility to dysbiosis in a subject, the method including the step of determining the presence or absence of a polymorphic marker in an

IL23R gene and/or protein in a biological sample from the subject, and wherein determination of the presence or absence of the risk variant is indicative of the susceptibility to dysbiosis in the subject.

12. A method of determining whether or not a subject has dysbiosis, the method including the step of determining the presence or absence of a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, and wherein determination of the presence or absence of the risk variant is indicative of the subject having dysbiosis.

13. The method of any one of Claim 11 or Claim 12, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith.

14. The method of Claim 13, wherein determination of the absence of the polymorphic marker is indicative of the subject being susceptible to or having dysbiosis.

15. A method of treating dysbiosis in a subject, the method including the step of determining a presence or absence of a polymorphic marker in an interleukin-23 receptor (IL23R) gene and/or protein in a biological sample from the subject, before, during and/or after treatment of dysbiosis, and based on the determination made, initiating, continuing, modifying or discontinuing a treatment of dysbiosis.

16. The method of any one of Claim 15, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith.

17. The method of Claim 15 or 16, further including the step of selecting the treatment of dysbiosis based on the presence or absence of the polymorphic marker.

18. The method of any one of Claims 15 to 17, wherein the treatment is selected from the group consisting of an IL23R antagonist, an IL23 antagonist, a microbiome modulator, a dietary supplement, a probiotic composition, a faecal transplant and any combination thereof.

19. The method of any one of the preceding claims, wherein the subject is a mammal.

20. The method of Claim 19, wherein the subject is a human.

21. The method of any one of the preceding claims, further including the step of determining a microbiome in the subject.

22. The method of Claim 21, wherein the step of determining a microbiome includes analysing a variable region, such as a V3-V4 hypervariable region, of a 16S ribosomal subunit of a microbe.

23. The method of Claim 22, wherein the variable region is analysed using one or more primers that comprise the nucleotide sequence set forth in SEQ ID NO: l and/or SEQ ID NO: 2 or a fragment, variant or derivative thereof.

24. A kit for determining a prognosis and/or treatment of a subject with IBD, the kit comprising:

reagents for selectively detecting a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith, and

a collection of data comprising correlation data between the polymorphic marker and prognosis and/or treatment of IBD.

25. The kit of Claim 24, for use in the method of any one of Claims 1 to 10 and 19 to 23.

26. A kit for assessing susceptibility to and/or determining the presence and/or treatment of dysbiosis in a subject, the kit comprising:

reagents for selectively detecting a polymorphic marker in an IL23R gene and/or protein in a biological sample from the subject, and

a collection of data comprising correlation data between the polymorphic marker and susceptibility to and/or presence of dysbiosis.

27. The kit of Claim 26, wherein the polymorphic marker is or comprises the variant rsl 1209026 and/or a marker in linkage disequilibrium therewith.

28. The kit of Claim 26 or 27, for use in the method of any one of Claims 11 to 23.

29. The kit of any one of Claims 24 to 28, wherein the collection of data is on a computer-readable medium.

30. The kit of any one of Claims 24 to 29, further including one or more primers for determining a microbiome in the subject.