δPKC-Mediated DRP1 Phosphorylation Impacts Macrophage Mitochondrial Function and Inflammatory Response to Endotoxin

Abstract

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 (dPKC)-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 dPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit dPKC activation (dV1-1) and dPKC-Drp1 interaction (cDrp1), we determined whether dPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response. Results: Our results demonstrated that dPKC-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 dPKC phosphorylation of Drp1 (cDrp1). Interestingly, limiting excessive mitochondrial fission using cDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-kB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1b, TNFa, IL-6). Conclusion: These data suggest that inhibiting Drp1 phosphorylation by dPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting dPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, dPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.

Pretreatment with Kahweol Attenuates Sepsis‐Induced Acute Lung Injury via Improving Mitochondrial Homeostasis in a CaMKKII/AMPK‐Dependent Pathway

“…Targeting mitochondrial fission pharmacologically or genetically can reduce LPS‐triggered proinflammatory responses. [ 32,39 ] In this study, we observed that LPS‐induced mitochondrial fission slightly increased mitophagy and caused significant oxidative stress and inflammation in both macrophages and mouse lung tissue. Interestingly, Kahweol inhibited mitochondrial fission and enhanced mitophagy to remove dysfunctional mitochondria, thus preventing oxidative stress and inflammation.…”

Mitochondrial PGAM5−Drp1 signaling regulates the metabolic reprogramming of macrophages and regulates the induction of inflammatory responses

“…Previous studies demonstrated that LPS-induced activation of Drp1 regulates the mitochondrial fission and subsequent inflammatory responses of macrophages and contributes to the promotion of vascular remodeling after injury ( 14 , 15 ). Furthermore, PKCδ-dependent phosphorylation of Drp1 or Stat2-dependent phosphorylation of Drp1-dependent mitochondrial mass increase might be an upstream mechanism that regulates the induction of proinflammatory responses in macrophages ( 16 , 17 ), suggesting that Drp1 plays a crucial role in connecting the changes in mitochondrial dynamics and innate immunity. In contrast, a study showed that KD of Drp1 augmented the activation of NLRP3 inflammasome activation without changing mtROS production or mitochondrial damage ( 41 ).…”

“…Activation of Drp1 is determined by the phosphorylation status of Ser residues, as phosphorylation at Ser 616 and dephosphorylation at Ser 637 implicate the Drp1 activation. Previous studies showed that Stat2 phosphorylates Drp1 at serine 616 for Drp1-mediated mitochondrial fission ( 44 ) and that protein kinase C (PKC) δ regulates the mitochondrial fission by phosphorylating Drp1 at Ser 616 ( 16 ). Additionally, phosphorylation of Ser 637 in HEK 293T cells is mediated by PKA ( 44 ).…”

“…Accumulating data suggest that dynamin-related protein 1 (Drp1), one of the dynamin-related GTPases, plays a role in disease development (11)(12)(13), and Drp1 regulates the mitochondrial fission and subsequent inflammatory responses of macrophages (14,15). Furthermore, PKCd-or Stat2-dependent activation of Drp1 regulates the induction of proinflammatory responses in macrophages (16,17), suggesting that Drp1 plays a crucial role in connecting alterations in mitochondrial dynamics and innate immunity. PGAM5 (phosphoglycerate mutase 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1 (10,18).…”