Inflammatory bowel disease (IBD) is a group of chronic inflammatory conditions in the gut. A major form of IBD is Crohn's disease (CD). Natural Killer cells are members of the innate immune system mainly known for their cytolytic abilities against infected or tumor cells. They are divided into distinct subsets comprised of licensed and unlicensed cells. NK cells are programmed at a genetic level to express discriminating Killer Immunoglobulin Receptors (KIRs) in humans and Ly49 receptors in mice. The engagement of these receptors by their cognate human leukocyte antigen (HLA) ligands induces differentiation of NK cells into a licensed state, which has functional properties distinct from the unlicensed states. It has been identified that the genetic presence of KIR2DL2/3 in the context of its ligand HLA-C1 is a risk factor for CD, yet the cellular mechanism of this genetic contribution and its contribution to immune-mediated colitis is unknown.

We used a co-culture assay to study if peripheral NK cells from CD patients with KIR2DL2/3 and HLA-C1, a genotype permits strong NK cell licensing, have a differential impact on CD4+ T cell proliferation than patients without this genotype. Using co-culture assays, flow cytometry, and a microfluidic platform for cytokine analysis, we assessed the role of KIR-HLA genetics on the immunologic functions of NK cells. To identify if KIR-HLA genetics can be used to predict thiopurine responsiveness, we stratified an independent pediatric inflammatory bowel disease cohort into licensed and unlicensed subsets, and compared the percentages of responders in the two subsets.

NK cells from CD patients with strong licensing genotype are more potent in promoting CD4+ T cell proliferation than those from patients with intermediate or low licensing genotype (p<0.005 and p<0.0005 respectively). The effect of licensed NK cell is independent on direct contact. Multiplexed analysis of bulk media and single cell NK cytokine profiles established that licensed NK cells produced higher levels pro-inflammatory cytokines including interferon-γ, tumor necrosis factor-α, and chemokines, including C-C motif ligand-5 and macrophage inflammatory protein-1β. Licensed NK cell supernatant also dramatically promotes the differentiation of TH17 cells, a signature CD4+ T helper subset in CD. Pediatric patients with licensing (strong and intermediate) genotype have a higher responder rate (64.7%) to thiopurines than those with low licensing genotype (20.0%).

Using similar methods, we demonstrated that murine licensed NK cells augment CD4+ T cell activation not only by increased secretion of proinflammatory mediators such as IL-6 and TNF-α, but also through contact-dependent mechanism, which is different from the solely cytokine-mediated mechanism seen in human licensed NK cells.

In conclusion, NK cell licensing mediated by KIR2DL2/3 and HLA-C1 elicits a novel cytokine program that induces pro-inflammatory CD4+ T cells activation and Th17 cell differentiation, thereby providing a pharmacogenomic tool for predicting responders to thiopurine treatment in inflammatory bowel diseases. Murine and human NK cell licensing share similar features, and its role in immune colitis awaits further investigation.

Inflammatory bowel disease (IBD) is a set of chronic, relapsing inflammatory diseases of the intestine. The two major subtypes of IBD are Crohn's disease (CD) and ulcerative colitis (UC). Although the pathogenesis of IBD remains largely unknown, Crohn's disease is considered to result from the interaction of environmental factors, including intestinal microbiota, with host immune mechanisms in genetically susceptible individuals. Recent advances in sequencing technologies have allowed us to characterize the IBD associated dysbiosis in unprecedented depth. However, phylogenetic profiling can only provide limited information on the functional implication of these alterations. To address this analytical challenge, we developed the novel mucosal lavage sampling approach, which enabled the profiling of multi'omic molecular features including microbiome, metaproteome and metabolome. Combined with host genomic information, these tools can provide us with unprecedented understanding of the dynamics of host-microbial interaction, and help us to investigate the pathogenesis of inflammatory bowel diseases.

Another analytical challenge to identify microbial taxa consistently representing IBD associated dysbiosis is the high complexity and low inter-individual overlap of intestinal microbial composition. This difficulty can be overcome by an ecologic analytic strategy to identify modules of interacting bacteria (rather than individual bacteria) as quantitative reproducible features of microbial composition in normal and IBD mucosa. We developed the strategy to analyze microbial composition using microbial co-occurrence network approach. This strategy uncovered 5 reproducible functional microbial communities (FMCs) detectable in the mucosa of all individuals. The quantitative levels of two FMCs were significantly associated with IBD states. Imputed metagenome analysis indicated the functional importance of the disease associated modules reflected by the enrichment of virulent and pathogenic pathways. Thus, these modules appear to define novel microbial communities within the intestinal microbial ecology, some of which are commonly and stably modified by the IBD disease state, and may be of particular relevance for microbial pathogenesis and intervention.

Using this experimental and bioinformatic framework, we investigated the microbial gardening effect of FUT2 gene and its link to Crohn's disease. Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease. In healthy individuals, imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism, and glycan biosynthesis and metabolism related pathways; and, the depletion of amino acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2^{-/- genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. In an extended cohort containing both healthy and CD individuals, the phylogenetic composition of intestinal mucosal microbiota was affected by an interaction of Crohn's disease status and FUT2 genotype. Decreased abundances of Firmicutes were associated with both CD and FUT2 risk allele. At metagenomic level, a distinct signature of amino acid metabolism deficiency was identified in CD and non-secretor microbiome. Such changes were also reflected at metabolomic level in the proximal gut region. Taken together, FUT2 gene increased the risk of Crohn's disease by changing the microbial composition and function to a disease-like state. The CD associated perturbations of metagenome and metabolome were driven by the FUT2 risk allele.}

The same experimental and bioinformatic approach can also be applied to study the composition and functional changes of mucosal associated microbiota in other chronic inflammatory disease, namely HIV-1 infection. In the rectal mucosa, microbial composition and imputed function in HIV-positive individuals not receiving cART was significantly different from HIV-negative individuals. Genera including Roseburia, Coprococcus, Ruminococcus, Eubacterium, Alistipes and Lachnospira were depleted in HIV-infected subjects not receiving cART, while Fusobacteria, Anaerococcus, Peptostreptococcus and Porphyromonas were significantly enriched. HIV-positive subjects receiving cART exhibited similar depletion and enrichment for these genera, but were of intermediate magnitude and did not achieve statistical significance. Imputed metagenomic functions, including amino acid metabolism, vitamin biosynthesis, and siderophore biosynthesis differed significantly between healthy controls and HIV-infected subjects not receiving cART. In the cervicovaginal mucosa, significant differences in alpha and beta diversity were observed between HIV-negative and HIV-positive women, with the latter enriched of organisms associated with bacterial vaginosis and depleted of Lactobacilli. These ecologic changes occurred concomitantly with significant metagenomic and immunologic differences. Such functional pathways may represent novel interventional targets for HIV therapy if normalizing the microbial composition or functional activity of the microbiota proves therapeutically useful.

Inflammatory bowel disease (IBD) – comprised of Crohn’s disease (CD) and ulcerative colitis (UC) – is believed to arise from a combination of genetic susceptibility and environmental factors that trigger an inappropriate mucosal immune response to constituents of the intestinal microbiome. There is now an extensive literature demonstrating that the microbiome has profound effects on immune function and, conversely, that the immune system can shape the microbiome. I hypothesized that genetic variation in mucosal immune gardening of the intestinal microbiome can result in pro-inflammatory dysbiosis, which acts as a risk factor for overt IBD. To evaluate whether individuals at risk for IBD develop dysbiosis prior to the onset of disease, a family based study was performed to characterize the microbiome and metabolome of pediatric IBD patients and their first degree relatives. These relatives are at higher risk for dysbiosis than the general population due to shared genetic and environmental factors with the IBD proband. A subset of healthy relatives in this cohort had dysbiosis with fecal metabolomic profiles (metabotypes) shared with IBD patients. The effect of the transcription factor RORgt on the intestinal microbiome was then investigated as a model of how perturbation of immune gardening could result in dysbiosis. Mice deficient in RORgt had an altered small intestinal and colonic mucosa-associated microbiome characterized by overgrowth of segmented filamentous bacteria (SFB), a microbe previously shown to promote colitis. Further knockout and cell engraftment experiments demonstrated that small intestinal gardening of SFB was mediated by RORgt-dependent T cells in a manner independent of IL-17A. The protective rs4845604 polymorphism in the RORC gene encoding RORgt was associated with altered microbial composition in mucosal wash samples from IBD patients and healthy individuals. These findings demonstrated that RORgt-dependent T cells garden the intestinal microbiome and suggest that genetic variation in this process could influence susceptibility to IBD.

Gut microbiome has a profound effect on human health and disease. The burgeoning body of research investigating the gut microbiome has illustrated its important roles on not only the local inflammatory diseases, such as inflammatory bowel disease, but also on systemic immunological disorders, such as autoimmune diseases. Many of these associations were determined at the level of metagenome analysis. This provides categorical information on both taxonomic composition and predicted functional capacities of the microbial community. This thesis tackles the problem of how gut microbiota affect human diseases using both these categories of assessment.

Although the accumulating evidence suggested the important roles of gut microbiota in the human immune system and Crohn's disease (CD), the molecular mechanisms of how microbiome directly modulates immune responses and leads to CD progression remain unclear. To advance the current understanding of microbial roles in CD, we developed a systems biology approach to assess the human immune function in response to disease-associated microbial products. Progress in computational omics analysis of the microbiome in CD enables the functional inference and prediction of microbial metabolites. Both genetic and clinical studies suggested the involvement of human CD4+ T cells in CD progression. We therefore tabulated a set of microbial metabolites predicted to be differentially abundant in CD, and screened these microbial metabolites for their bioactivity in human CD4+ T cell functions. Our screen revealed 15 bioactive microbial metabolites, 3 previously reported and 12 unprecedented, with selective action on CD4+ T cell cytokine production. Mechanistic assessment of one novel microbe-derived metabolite, ascorbate, revealed apoptosis of activated human CD4+ T cells associated with selective inhibition of glycolytic energy metabolism. These findings suggest a substantial rate (11%) of bioactive metabolites among the predicted CD-associated metabolite reservoirs and provide evidence for novel modes of microbial activity targeting T cell metabolism.

Emerging studies have characterized the association of taxonomic composition with systemic autoimmune diseases with various clinical phenotypes and modes of pathologic immune responses. In this thesis, we assessed systemic sclerosis (SSc), a previously unstudied autoimmune disease with respect to the microbiome, notable for both systemic manifestations and gastrointestinal involvement. The metagenomic study of mucosal-luminal interface (MLI) samples revealed a unique microbial composition associated with disease state. Patients with SSc had decreased abundances of Faecalibacterium and Clostridium, and increased abundances of Bifidobacterium, Lactobacillus, Fusobacterium and γ-Proteobacteria compared with healthy controls. In addition, the increase of Fusobacterium species and the decrease of Bacteroides fragilis were associated with moderate/severe gastrointestinal symptoms. We conducted an independent study of faecal samples from two independent cohorts of patients, from United States and Norway, that revealed a similar group of microbial taxa associations, and a greater dysbiosis in patients from the United States. These studies enable the specific targets for intervention to avert or treat the gastrointestinal involvement in systemic sclerosis.

The study of gut microbiota is beginning to evolve from association toward causality. The functions of gut microbiota are not only restricted to local inflammatory responses but expanded to systemic immune regulations. We hypothesized that gut microbiome may regulate host immune cellular responses by producing metabolites. We investigated the bioactive microbial factors regulating Crohn's disease, and explored the potential microbial taxonomic biomarkers in a systemic autoimmune disorder, SSc. Those improved understandings of the microbiome activity and metabolism may inform therapeutic strategies and ultimately improve patient health.

Mucosal lymphocyte homeostasis involves the dynamic interaction of enteric microbiota, the intestinal host epithelium, and the mucosal immune system. Dysregulation of mucosal lymphocyte homeostasis results in a variety of intestinal disorders, notably inflammatory bowel diseases like ulcerative colitis and Crohn's disease. One key cellular component regulating homeostasis are B lymphocytes that reside in gut-associated lymphoid tissue. This compartment includes Peyer's patches, isolated lymphoid follicles, lamina propria, and mesenteric lymph nodes. Recent data have pointed to two new and exciting aspects of B cells in the gut. First, there has been progress on identification and functional analysis of abundant isolated lymphoid follicle B cells that are key mediators of IgA genesis. Second, several groups have now clarified the functional identification and characterization of immunoregulatory B cells in the gut. This review examines the novel aspects of these B cells, and examines how each plays a role in mediating mucosal homeostasis in this bacteria-laden compartment.