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Abstract
Butyrate is a gut microbiome-derived short chain fatty acid with pleiotropic favorable effects on inflammation and metabolism. In murine models of atherosclerosis, butyrate has been shown to significantly reduce atherosclerotic lesions, rectify routine metabolic parameters such as low-density lipoprotein cholesterol (LDL-C), and reduce systemic inflammation. However, its foul odor, rapid metabolism in the gut and thus low systemic bioavailability, limit its therapeutic effectiveness. Our laboratory has engineered an ester-linked L serine-conjugate to butyrate (SerBut) to mask its taste and odor and to co-opt amino acid transporters in the gut to increase its systemic bioavailability as determined by tissue measurements of free butyrate, produced by hydrolysis of SerBut. In vitro, SerBut maintains the anti-inflammatory, NFκB-suppressing capacity of butyrate while allowing higher dosing without cytotoxicity. In an apolipoprotein-E (ApoE)-/- mouse model of atherosclerosis, Ser-But reduced systemic LDL-C and pro-inflammatory cytokines, plaque burden in the aortic root, and monocytes in the aorta. In addition, SerBut suppressed circulating liver injury markers and early steatosis in the liver. SerBut overcomes several barriers to the translation of butyrate and shows superior promise in slowing atherosclerosis and liver injury than equidosed sodium butyrate. Efferocytosis maintains homeostasis by suppressing secondary necrosis and promotes immune tolerance through propagation of TAM receptor signaling. Growth arrest-specific 6 (GAS6), a bridge protein mediating efferocytosis through TAM receptors, has been implicated in regulation of inflammatory cycles. However, the mechanism of tolerogenic antigen presentation is incompletely understood. To unravel the interplay between an efferocytic antigen, the dendritic cell (DC) collecting it, and the T cell engaged in that immune synapse, we engineered a GAS6 bridge protein by fusing it to the model antigen ovalbumin yielding GAS6-OVA (GO), providing an experimental tool enabling characterization of efferocytic myeloid-lymphoid signaling. A non-PS-binding proteoform of GO (ΔGO) distinguished between effects mediated by TAM receptor signaling alone or associated with apoptotic debris. GO induced tolerogenic antigen presentation by both MHC-I and MHC-II under both homeostatic and inflammatory conditions, resulting in antigen-specific tolerance including Treg proliferation and terminal exhaustion of CD8+ T cells. Flow cytometry revealed reciprocal tolerogenic polarization by both myeloid and lymphoid sides of the immune synapse. GO pre-exposure ameliorated inflammatory airway pathology in a murine model, demonstrating functional potency of the GAS6-mediated tolerogenic pathway with an endogenous repertoire. Thus, when antigen is collected and presented via the GAS6-mediated pathway of efferocytosis, reciprocal tolerogenic antigen presentation occurs by both MHC-I and MHCII in a PS binding dependent manner regardless of inflammatory conditions.