Obesity is considered as a complex and multifactorial disease, the prevalence of which has significantly increased over the last decade. Obesity is associated with low-grade chronic inflammation, which is one the precipitating factors for many of its associated complications such as type 2 diabetes, cardiovascular disease, and cancer. Multiple factors that contribute to this disease have been studied; among them the association and causative role played by altered intestinal bacteria in obesity, which represents one of the astonishing finings over the past decade. The mechanisms by which the gut microbiome interacts with the host to influence the immune responses are largely undefined.
A single layer of intestinal epithelial cells are in constant interaction with the intestinal luminal contents and contributes to the host defense along with a highly specialized intestinal immune system. Together they balance steady-state tolerance of harmless antigens, rapid effector responses towards pathogens, and restoration of tissue homeostasis. We have shown one such interaction is between mucus secreting goblet cells (GCs) and antigen-presenting cells such as dendritic cells (DCs) in the lamina propria (LP) through the formation of goblet cell-associated antigen passages (GAPs). We have published that the role of GAPs extends beyond antigen delivery to imprinting DCs with tolerogenic properties and maintaining regulatory T cells (Tregs). Additionally, we observed that GAP inhibition results in the expansion of IL-17-producing Th17 cells in the small intestine (SI) LP and adipose tissue.
In order to investigate how disease-associated microbial dysbiosis contributes to intestinal and non-intestinal immune cell alteration, we established a model of colonizing mice with human microbiota from lean donors (BMI< 25) or obese donors (BMI> 30). Three weeks post colonization; we observed significant weight gain in obese donor colonized mice on a normal chow diet. Additionally, mice colonized with microbiota from obese, but not lean donors inhibit GAPs, induces the expansion of Th17 cells in the SI-LP and adipose tissue. These observations indicate that goblet cells and along with other epithelial cells play a crucial role in balancing the tolerogenic versus inflammatory axis in the SI and adipose tissue and that this axis can become unbalanced by the obesity-associated dysbiotic microbiota. Understanding how dysbiotic microbiota contribute to intestinal epithelial changes and thereby contributes to inflammation is crucial to designing appropriate therapies, to mitigate obesity-associated metabolic disorders.
During Year One of the research grant, we characterized the downstream effects of altering small intestinal GAPs on the immune cell subsets in the gut and at extra-intestinal sites. We utilized the mAChR4f/fMath1Cre*PR mouse model, where inhibition of GAP function occurs independently of goblet cell deletion. We observed that upon GAP inhibition, there is expansion of Th17 cells in the gut and expansion of Th17 cells in the adipose tissue. However, we could not observe any changes in body weight of these mice, suggesting that GAP alteration did not contribute towards changes in caloric update.
In our model of antibiotic pre-treated mice colonized with gut microbiota from obese or lean individuals, we have shown significant weight gain five weeks after colonization by mice with microbiota from obese individuals, on a normal chow diet. Additionally, these mice had increased inflammatory immune cells such as Th1 and Th17 cells in the small intestine (SI) and epididymal adipose tissue (AT); and fewer GAPs in SI compared to mice colonized with gut microbiota from lean donors (data presented last year).
To develop upon these findings, we performed RNA seq analysis on SI tissue of these mice colonized with one set of donor specimen per group. A heatmap and Venn diagrams show the unique transcriptomic signature across the groups. This initial experiment provided encouraging data suggesting pathway enrichments in T cell differentiation, metabolic changes and antigen process.
Since not all obese individuals develop insulin resistance and metabolic abnormalities, we devised studies with Dr. Samuel Klein’s laboratory which carefully characterized human subjects with known degrees of adipose tissue inflammation and insulin resistance into metabolically-healthy obese (MHO) or metabolically-unhealthy obese (MUO). C57BL/6 mice pretreated with antibiotics were colonized with MHO or MUO and maintained on normal chow diet for five weeks. Data indicate significant weight gain by mice in both MHO and MUO groups compared to non-colonized controls. However, mice with MUO microbiota demonstrated a significant reduction in number of intestinal GAPs and an increased number of proinflammatory such as Th17 cells in their SI and AT compared with MHO or controls. Additionally, significant increase in macrophage population was observed in mice with MUO donor microbiota.