Mito-inflammation and sepsis-induced acute lung injury - SUMMARY Our prior work has shown that TLR4-mediated increases in mitochondrial (mt)-ROS play an essential role in the inflammatory phase of acute lung injury (ALI) by stimulating NF-kB and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activity. However, the molecular mechanisms by which mt-ROS stimulates inflammatory response are unresolved and are the focus of our application. The mitochondrial network constantly forms elongated tubes through fusion and splits into small, less-connected mitochondria through fission. Recent work has highlighted the importance of pathological mitochondrial network remodeling in human disease. Thus, optimizing mitochondrial network dynamics is critical for mitochondrial homeostasis. We recently discovered that TLR4 activation increases mt-ROS in lung EC by stimulating mitochondrial fission. Based on these exciting findings, this proposal will test the hypothesis that TLR4-mediated pathological mitochondrial network remodeling is a previously undescribed mechanism that prolongs the inflammatory phase during sepsis- mediated ALI. Specific Aim (SA) #1 will define how TLR4-mediated mitochondrial fission requires RhoA/ROCK- dependent cytoskeletal remodeling and evaluate the role of increased activation of the fission protein, Drp1, and reductions in the levels of the fusion proteins MFN1 and MFN2. The involvement of nitration-mediated post- translational modifications (PTMs) in RhoA will also be evaluated. The effect of fission-mediated mt-ROS on NF- kB activity and NLRP3 inflammasome assembly and activation will also be evaluated. Mechanistically, we will focus on the mt-ROS-mediated inhibition of the NF-kB regulatory protein IkBa in the sustained activation of NF- kB. SA #1 will also delineate the involvement of JNK as a downstream target of ROCK signaling in Drp1 activation and the decrease in MFN1/2 levels. The inhibitory effect of pathologic mitochondrial network remodeling on the autophagy/mitophagy response in the inflammatory response will also be evaluated. Using multiple pre-clinical mouse models, SA #2 will test new therapeutic strategies to preserve/restore the mitochondrial network balance and determine their efficacy in attenuating the inflammatory injury associated with sepsis-induced ALI. Our explorations are highly innovative, as they will define a concept-advancing interplay between TLR4 activation, pathologic mitochondrial network remodeling, autophagy/mitophagy inhibition, and the inflammatory response, opening a new avenue for therapeutic development in lung injury. Finally, these findings are highly significant, as they will promote a more thorough understanding of sepsis pathobiology and highlight the application of novel therapies for the critically ill.
