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During acute and chronic infectious diseases hosts develop complex immune responses to cope with bacterial persistence. Depending on a variety of host and microbe factors, outcomes range from peaceful co-existence to detrimental disease. Mechanisms underlying immunity to bacterial stimuli span several spatiotemporal magnitudes and the summation of these hierarchical interactions plays a decisive role in pathogenic versus tolerogenic fate for the host. This dissertation integrates diverse data from immunoinformatics analyses, experimental validation and mathematical modeling to investigate a series of hypotheses driven by computational modeling to study mucosal immunity. Two contrasting microbes, enteroaggregative Escherichia coli and Helicobacter pylori, are used to perturb gut immunity in order to discover host-centric targets for modulating the host immune system. These findings have the potential to be broadly applicable to other infectious and immune-mediated diseases and could assist in the development of antibiotic-free and host-targeted treatments that modulate tolerance to prevent disease. Ph. D.

Based on preliminary data suggesting that macrophages are critical regulators of Helicobacter pylori colonization and gastric pathology, we investigated how macrophage phenotypes shape the outcome of infection and persistence. The loss of PPARγ in the myeloid compartment favors a 5 to 10-fold decrease in bacterial loads at the expense of more severe gastric lesions. PPARγ-deficient macrophages present altered control of transcription factors such as NFκB, which results in a pro-inflammatory phenotype. Stomach lamina propria analyses at weeks 0 to 4 post-infection revealed increased levels of CD11b+ F4/80hi CD64+ CR3CR1+ macrophages in WT mice, while PPARγKO mice failed to expand and maintain such population. Macrophage depletion using clodronate liposomes resulted in a significant reduction of gastric H. pylori in WT mice, thus abrogating the differences in bacterial loads observed between WT and PPARγKO mice. In vitro co-culture of H. pylori with bone marrow derived macrophages showed that either PPARγ loss or pharmacological blockade enhances bactericidal activity, which is associated to significant differential expression of chil1, etv5, iigp1, ptger4, sqle, osm, hspa2 and rptoros levels as revealed by global transcriptome analyses. Hence, macrophages facilitate H. pylori infection by 1) serving as bacterial reservoirs and allowing intracellular replication, and 2) favoring a gastric regulatory response that favors persistence.

NLRX1 is a mitochondrial-associated NOD-like receptor that modulates antiviral immunity, cellular stress, autophagy, and reactive oxygen species (ROS). The role of NLRX1 in inflammatory bowel disease remains unknown. This study aimed to characterize NLRX1-mediated mechanisms of protection from IBD. We investigated the ability of NLRX1 to modulate gut pathology, inflammation and immunity by using DSS and CD4+CD45RBhigh transfer colitis models. Colons, spleens, and mesenteric lymph nodes were excised for characterizing immune cell subsets, histological analyses, cytokine and autophagy expression, NF-κB activity, and ROS production. The loss of NLRX1 increased severity of disease and colonic histopathology in both models of IBD. Colons of NLRX1-/- mice had significantly increased epithelial ulceration and leukocyte infiltration in the DSS model, while recipients of NLRX1-/- CD4+ T cells had increased leukocytic infiltration, proliferation, fibrosis, and crypt abscessation in both colon and ileum. The loss of NLRX1 increased numbers of Th1, Th17, and Treg in the spleen, increased colonic NF-κB activity, upregulation of IL-17, IFNγ and TNF-α production, and increased ROS production. NLRX1 ameliorates intestinal pathology during IBD by acting as an internal thermostat that modulates the balance of effector versus regulatory CD4+ T cell responses, and suppressing colonic NF-κB activity, inflammatory cytokine expression, ROS production, and autophagy.

Lanthionine synthetase C-like protein 2 (LANCL2) has emerged as a novel therapeutic target for metabolic, infectious and immune-mediated diseases. We previously demonstrated that directly targeting LANCL2 through treatment with abscisic acid or 61610 results in efficacious anti-inflammatory responses during DSS-induced colitis however molecular mechanisms underlying this immunomodulatory function remain elusive. Here we report for the first time the use of LANCL2-deficient mice to investigate the mechanism of action and cell specificity of the protein. At the cellular level, bone marrow derived macrophages (BMDM), RAW cells, and gastric epithelial cells express comparable levels of basal LANCL2 mRNA. LPS-mediated inflammation in BMDM increased both mRNA and protein levels of LANCL2 that peaked 6 hours post-challenge and subsequently returned to normal levels after 12 hours. Similarly, DSS-induced colitis induced expression of LANCL2 in the colons of wild-type (WT) mice and correlated with elevated levels of proinflammatory cytokines. Interestingly, the absence of LANCL2 did not cause significant alterations in infiltrating cellular phenotypes however LANCL2-deficiency permitted exaggerated IL17 and IFNγ expression in colonic tissue when compared to WT counterparts at day 7 post-challenge. In conclusion, our findings indicate that LANCL2 is a novel regulator of inflammation that functions predominantly at the molecular level to down-regulate excessive inflammatory responses.

Enteroaggregative Escherichia coli (EAEC) is a recognized cause of enteric disease in diverse clinical settings. Mucosal immunity towards EAEC infections is poorly understood. To better characterize immunoregulatory mechanisms underlying EAEC infections we constructed a computational model mimicking host responses to EAEC at the gut mucosa. Preliminary model calibration efforts demonstrated remarkable fitting to experimental data. Nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) has been targeted to modulate mucosal immune responses to EAEC. Wild type and conditional knockout mice lacking PPARγ in T cells were fed protein-deficient diets at weaning and challenged with 5x109cfu EAEC strain JM221. Quantitative RT-PCR data reveal that administration of GW9662, a potent PPARγ antagonist, for 7 days post infection or the deletion of PPARγ in T cells results in upregulation of pro-inflammatory cytokines including IL-6, TNF-α, and IL-1β when compared to non-treated infected animals. Histological analysis of colons divulges lower leukocyte infiltration and decreased mucosal thickness 14 days following EAEC infection after pharmacological blockade of PPARγ or deletion of PPARγ in T cells. These findings were accompanied by faster clearance of colonic EAEC in tissue-specific PPARγ null mice. In conclusion, we present a fully integrated approach to study host responses to EAEC that provides new insights on the pathogenesis and treatment of enteric infections.