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Arthritis is an autoimmune disease that adversely affects the quality of life of patients. Treatment options for autoimmunity include the use of chemical and biologic agents that suppress inflammation. However, these can result in patients being vulnerable to opportunistic infections. Adoptive T regulatory (Treg) cell transfer can specifically block auto-inflammatory immune cells in an antigen-specific manner while maintaining an intact immune response to pathogens. However, isolating sufficient Treg cells for adoptive cell transfer therapy is challenging; therefore, improving Treg cell survival and function could have important impacts on adoptive Treg transfer therapy. To explore the use of bacterial vaccines as autoimmune therapies, we engineered an attenuated brucellosis vaccine (Brucella melitensis ΔvjbR Tna) to produce indole, a bacterial metabolite that induces Treg expansion. In the collagen induced arthritis (CIA) murine model, Treg transfer following BmΔvjbR Tna treatment decreased arthritis incidence and score. CD4+FoxP3+ T regulatory cells and CD8+ T cells in spleen and lymph nodes were increased in BmΔvjbR Tna treated CIA mice. BmΔvjbR Tna injection also promoted CD4+ cell proliferation, and increased IL-2 and IFN-g expression. The levels of some pro-inflammatory cytokines (IL-1β and IL-6) were also dramatically reduced in BmΔvjbR Tna infected bone marrow derived macrophages (BMDMs). Taken together, these findings indicate that the indole-producing BmΔvjbR Tna strain increases Treg cell populations and proliferation, suppresses proinflammatory cytokine secretion, and reduces autoimmune responses.

A significant hallmark of the chronic inflammatory disease, atherosclerosis, is the accumulation of pro-inflammatory M1-like macrophages (macs) and dendritic cells (DCs) in coronary arteries that respond to pro-atherogenic stimuli and fail to digest lipids that contribute to the formation of foam cells in atherosclerotic plaques. Mechanisms that reduce Mac/DC inflammation, increase lipid degradation, and prevent foam cell formation are likely to decrease progression of atherosclerosis. Mechanisms that regulate autophagy in macs and DCs will likely reveal targets for the prevention of atherosclerosis. Mouse models of high-fat diets demonstrate a link between microbiota composition and the development of obesity, diabetes, and atherosclerosis through several mechanisms best demonstrated by the microbiota-dependent production of TMAO that promotes atherosclerosis. We have identified a tryptophan-derived microbiota metabolite, indole, that has novel immunomodulatory properties. This strong association between obesity, inflammation, and the microbiota has led us to the hypothesis that microbiota-derived metabolites in the GI tract might regulate dietary saturated fat-induced inflammation. To test this hypothesis, we generated bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) and tested the effects of indole on these cells in the context of saturated fatty acid-induced inflammation. We investigated pro-inflammatory cytokine production, M1 macrophage development, and metabolic flux. Here, we demonstrate that indole reduces saturated fatty acid-derived inflammation in BMDCs and BMDMs and regulates metabolic programming of phagocytes by inducing autophagy on a level similar to rapamycin.

It is established that the gut microbiota plays an important role in Non-Alcoholic Fatty Liver Disease (NAFLD) and different bacterial taxa have been correlated to disease. However, the specific metabolites through which the microbiota exert their effects and the target pathways in host cells are not fully understood. We used liquid chromatography-mass spectrometry metabolomics to identify two tryptophan-derived metabolites – tryptamine (TA) and indole-3-acetate (I3A) – that require the microbiota and are depleted in high-fat diet mice compared to low fat diet mice. Both I3A and TA reduced palmitate and LPS induced production of pro-inflammatory cytokines (TNFα, IL-1β and MCP-1) in RAW 264.7 macrophages, as well as inhibited macrophage migration toward the chemokine MCP-1. We identified that both palmitate and LPS reduced levels of p-AMPK and the addition of I3A and TA reversed p-AMPK reduction. Since the p-AMPK activator AICAR reduced palmitate and LPS induced inflammatory cytokine production, we propose that I3A and TA modulate inflammation through modulating p-AMPK levels.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in western countries. While it is established that the gut microbiota plays a role in NAFLD, the identities of microbiota metabolites involved and their target pathways in host cells is poorly understood. Based on prior work showing that the microbiota metabolite indole attenuates indicators of inflammation in intestinal epithelial cells, we hypothesized that microbiota metabolites modulate macrophage inflammation in the liver. Mass spectrometry analysis of microbiota metabolites in serum and liver of mice identified tryptamine (TA) and indole-3-acetate (I3A) as depleted in mice on a high-fat diet compared to low-fat diet. Therefore, we investigated the effect of I3A and TA on macrophage inflammation and migration. RAW 264.7 murine macrophages were treated with different concentrations of I3A and TA for 4 hours, stimulated with palmitate for 18 hours followed by LPS for 6 hours in the presence of metabolites. Our results show that treatment with I3A significantly attenuated the induction of the pro-inflammatory cytokines TNFa, IL-1b and MCP-1 by palmitate and LPS in a dose dependent manner at both mRNA and protein levels. Unlike I3A, TA only decreased the gene expression of IL-1b but significantly decreased TNFa and MCP-1 protein levels. Both I3A and TA significantly decreased bone marrow derived macrophage migration towards MCP-1 in a dose dependent manner. Together, our results suggest that microbiota metabolites can modulate NAFLD by directly acting on macrophages. Ongoing work focuses on investigating the mechanisms underlying the anti-inflammatory activity of I3A and TA, which could provide new strategies to treat NAFLD.