Abstract Rationale Chronic lung allograft dysfunction (CLAD) is a progressive complication post-lung transplant with patients unresponsive to immunosuppression and presenting relentless fibrosis. Our prior studies identified JNK as an upstream regulator of both isoforms of the mammalian target of rapamycin complex (mTORC1/2), and collagen I expression in mesenchymal cells (MCs) during fibrotic activation. While oxidative stress is a key regulator of JNK activation, single-cell sequencing analyses of murine lungs showed C3 as a top gene specifically expressed in bronchovascular bundle MCs and further upregulated in allografts, driving oxidative stress and pro-fibrotic phenotype. We investigated the role of JNK activation, mediated by oxidative stress, in accelerating the pro-fibrotic transformation regulated by intracellular C3 signaling in CLAD MCs. Methods Human non-CLAD MCs were pre-treated with JNK inhibitor or Mito Temp and exposed to H2O2 or exogenous C3a, respectively. 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. C3aR blockade was examined in vivo using JR14a (10 mg/kg; intra-peritoneal injections) in a unilateral left lung transplant CLAD model with B6D2F1/J as a donor and either C57BL/6J (allograft) or B6D2F1/J (isograft) as a recipient. Mice were sacrificed at day 28, and lungs were processed for histology and hydroxyproline assessment. Results Pharmacologic blockade of JNK abrogated H2O2 -induced phosphorylation of JNK (Thr183/Tyr185) and eIF4E-S209. Endogenous lentiviral-C3/C3a overexpression and non-CLAD MCs exposed to exogenous C3a lead to robust JNK phosphorylation and collagen l expression, which were attenuated by Mito Temp (superoxide dismutase mimetic that acts as a mitochondria-specific antioxidant). CLAD MCs subjected to RNAi silencing or pharmacological blockade of C3 or C3AR1, demonstrated reduced phosphorylation of JNK and eIF4E-S209. Concomitant decrease in substrate phosphorylation of mTORC1 (pS6K and 4EBP1) and mTORC2 (AKT-Ser473) was observed. Key pro-fibrotic secretory proteins, collagen I and autotaxin, shown to be translationally regulated in activated MCs, were also significantly decreased. Histopathological examination of mice treated with JR14a revealed significantly lower fibrosis and collagen deposition as observed by hematoxylin and eosin staining and Masson’s trichrome staining, respectively. Quantitative hydroxyproline assay and Col1a1 mRNA expression confirmed decreased collagen content in the JR14a-treated allografts. Conclusions Our findings suggest that endogenous C3 expression and C3a/C3aR signaling sustains translational activation and fibrogenic transformation of CLAD MCs in a JNK-dependent manner, and that C3a receptor may serve as a potential therapeutic candidate in alleviating chronic rejection pathology. 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.