*Purpose: Sequestered self-molecules, such as HMGB1 or haptoglobin, are appreciated to stimulate the pro-inflammatory response of infiltrating innate immune cells by targeting TLR4 after their release during tissue injury. We have recently established that IL-33, a protein sequestered in the nucleus or exosome-like vesicles, acts instead as a regulatory factor that limits chronic rejection after heart transplantation by restraining the generation of pro-inflammatory macrophages in the graft. The purpose of this study was to identify the molecular mechanism underlying the regulatory capacity of IL-33 on macrophages.

*Methods: Bioenergetics of wildtype (WT) C57BL/6 (B6) macrophages exposed to IL-33 were compared to macrophages stimulated with LPS, known to generate pro-inflammatory macrophages, or IL-4, which instead generates regulatory/reparative macrophages using a Seahorse XF96e Analyzer. Macrophages from WT B6 and B6 il1rl1^-/- mice, which lack the IL-33 receptor, were assessed by RNAseq, qRT-PCR, and Western blot following treatment with IL-33, LPS, or IL-4. Macrophage functional phenotypes were also compared to mitochondrial mass, potential, and uptake of glucose and fatty acids by flow cytometry.

*Results: Unlike macrophages that responded to LPS by shifting their metabolic activity towards aerobic glycolysis, IL-33 augmented macrophage oxidative phosphorylation (OXPHOS). IL-33-stimulated macrophages also displayed increase mitochondrial potential and increased fatty acid uptake (FAU). RNAseq analysis revealed that IL-33 was a potent negative regulator of nos2, which is the message for inducible nitric oxide synthase (iNOS). Follow up studies established both that macrophage exposure to IL-33 blocked TLR4-mediated up-regulation of iNOS.

*Conclusions: Nitric oxide (NO) generated by iNOS in TLR4-stimulated macrophages has long been appreciated as a cytotoxic effector molecule, however, NO has emerged as an important regulator of mitochondrial metabolism. NO disrupts mitochondrial complex functions to generate metabolites that contribute to macrophage pro-inflammatory activity. This disruption requires macrophages to shift their metabolic activity to glycolysis. We now show that IL-33, instead blocks TLR4-induced iNOS induction and generates reparative/regulatory macrophage with augmented OXPHOS and FAU. Our data indicate that macrophage function in the graft can be shaped by local injury signals that orchestrate their metabolism.

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