Abstract Rationale Chronic lung allograft dysfunction(CLAD) is a fatal complication of lung-transplant with ineffective therapies. These patients demonstrate increased complement activation evidenced by single-cell and ATAC sequencing analyses revealing high C3 expression in allograft-derived mesenchymal cells(MCs), high extracellular C3a in BAL, and high intracellular C3/C3a and mitochondrial C3a receptor expression in CLAD MCs. Given mitochondrial dysfunction is linked to ‘Warburg’-induced pathological transformations, and STAT3 is a direct C3aR target implicated in CLAD pathology, we investigated the intracellular C3-STAT3 signaling in glycolytic reprogramming and mitochondrial alterations in CLAD MCs. Methods Human CLAD MCs were targeted for C3 or C3AR1 inhibition using RNA interference or antagonists specific to Cathepsin L (to inhibit intracellular C3a generation) or C3a receptor. Inhibition of STAT3 and aberrant glycolysis-related hexokinase-2 (indirect STAT3 target) was performed via JAK1/2-related-Ruxolitinib or JAK2-related-NVP-BSK, and Lonidamine, respectively. Non-CLAD MCs were subjected to lentiviral-C3 overexpression and treated with above inhibitors; or pre-treated with C3AR1 antagonist prior to exogenous C3a. Secreted lactate and IL-6 levels were measured by ELISA. Mitochondrial dysfunction and oxidative stress was assessed via the Seahorse XF Cell Mito Stress Test and MitoTrackerTM-based imaging, respectively. Results CLAD MCs compared to non-CLAD controls demonstrated aberrant glycolysis and mitochondrial bioenergetics demonstrating higher oxygen consumption rates through basal and maximal respiration. Metabolic shift toward aerobic glycolysis was evidenced by elevated proton leak and extracellular acidification rates with concurrently high secreted lactate levels. Lentiviral-C3 overexpressing non-CLAD MCs(C3-OE) mimicked these effects. Further, CLAD MCs and C3-OE cells demonstrated increased hexokinase-2 expression and mitochondrial oxidative stress evidenced by superoxide and hydrogen peroxide generation. Blocking C3 and C3AR1 in CLAD MCs or intracellular C3a in C3-OE cells, reversed the above effects to levels resembling non-CLAD controls. Receptor blockade mitigated exogenous C3a-induced hexokinase-2 and oxidative stress-related NOX4 expression. CLAD MCs demonstrated constitutive STAT3-Y705 phosphorylation and secreted IL-6, which was abrogated by C3 and C3AR1 silencing. Comparable to CLAD MCs, C3-OE cells demonstrated increased STAT3-Y705 phosphorylation and secreted IL-6, also observed due to exogenous C3a. These effects were reversed by blocking intracellular C3a or JAK-STAT. Pharmacologic blockade of JAK/STAT or hexokinase-2 signaling in CLAD MCs or exogenous C3a-treated non-CLAD controls led to a decrease in mitochondrial stress-related respiration parameters and collagen 1 expression. Conclusions C3/C3a-induced STAT3 signaling and downstream hexokinase-2 with associated mitochondrial dysfunction is an important under-studied pathway. Our findings demonstrate a link between intracellular C3 activation and downstream C3a signaling to metabolic rewiring causing fibrotic activation of CLAD MCs. This abstract is funded by: National Institutes of Health Grants R01 HL118017 and R01 HL094622, and Cystic Fibrosis Foundation LAMA21AB0
Vittal et al. (Fri,) studied this question.