Delta PKC-MEDIATED DRP1 PHOSPHORYLATION IMPACTS MACROPHAGE MITOCHONDRIAL FUNCTION AND INFLAMMATORY RESPONSE TO ENDOTOXIN

Background: Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms that link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if Protein Kinase C, subunit delta (delta PKC)-mediated phosphorylation of dynamin-related protein 1 (Drp1), a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation. Methods: Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in delta PKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit delta PKC activation (delta V1-1) and delta PKC-Drp1 interaction (psi Drp1), we determined whether delta PKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response. Results: Our results demonstrated that delta PKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally designed peptide that selectively inhibits delta PKC phosphorylation of Drp1 (psi Drp1). Interestingly, limiting excessive mitochondrial fission using psi Drp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-kappa B nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1 beta, TNF alpha, IL-6). Conclusion: These data suggest that inhibiting Drp1 phosphorylation by delta PKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting delta PKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, delta PKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.