Modulation of Macrophage Polarization by Heterotrimeric G proteins: Implications of Gastrointestinal Inflammation

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Abstract: Chronic inflammatory disorders, whether it is arthritis, atherosclerosis, type II diabetes, or inflammatory bowel disease (IBD) are diseases that severely debilitate millions of people worldwide. Immune deregulation, microbial dysbiosis, genetics, and environmental factors, all contribute to the chronic inflammation that drives the disease. More specifically, chronic inflammatory disorders are fueled by increased expression of inflammatory cytokines and the recruitment of immune cells, including dysfunctional proinflammatory macrophages, that recruit, incite and propagate pro-inflammatory T-cell responses. In order to develop novel immunologic therapies, a deeper understanding of the mechanisms of immune homeostasis is needed, including those that address macrophage dysfunction, which plays an integral role in perpetuating inflammation. Because the molecular mechanisms that govern chronic inflammatory responses in macrophages are yet to be fully elucidated or exploited as therapeutic targets, this is a need that is both urgent and unmet. Using bioinformatics (sequence homology) we first identified GIV (a.k.a Girdin), a guanine-nucleotide exchange modulator (GEM) for trimeric G protein, Gαi, as a novel binding partner of TLR4. Subsequent work not just validated and characterized this interaction in-depth, but also revealed that GIV is a regulator of TLR4 signaling and a key determinant of macrophage polarization and inflammatory cytokine expression. GIV expression is suppressed in M1 and upregulated in M2-polarized primary macrophages, and GIV-GEM-dependent G protein signaling is required for the suppression of pro- and upregulation of anti-inflammatory cytokines in response to the ligand for TLR4, lipopolysachharide (LPS). It is hypothesized that the GIV→Gαi cascade reduces inflammation and favors healing by switching macrophages from the pro-inflammatory M1 to the anti-inflammatory M2 polarized state. This proposal seeks to elucidate how GIV-GEM regulates such a "switch" and reveal the consequences of deregulated GIV→Gαi signaling axis in the gut where macrophages within the largest immune system face-off the largest reservoir of LPS (i.e., luminal microbes). Our aims are: 1) Dissect GIV's role in shaping the dynamics of TLR4 signalosome during inflammation using a combination of in vitro protein-protein interaction assays, 3D-homology model-guided mutagenesis, immunoprecipitation assays, immunofluorescence microscopy, and specific single-AA mutants; 2) Determine GIV's ability to modulate macrophage inflammatory programs using RNA-seq transcriptome analysis, cell-based macrophage polarization assays, and assays evaluating bacterial uptake and clearance; and 3) Investigate the consequences of GIV dysregulation in intestinal inflammation using mouse models of inflammation e.g., LPS challenge; DSS-induced chemical colitis and infectious colitis (Salmonella and Citrobacter), with or without pharmacologic modulators of GIV-GEM signaling. Insights gained will help establish the TLR4-GIV signaling axis as a decisive signaling pathway in macrophage inflammatory responses and provide proof-of-principle for targeting of GIV-GEM in diseases that are fueled by chronic inflammation.