Mitochondrial Dysfunction and Alloimmunity in Accelerated Bronchiolitis Obliterans After Lung Transplantation

Purpose Bronchiolitis obliterans (BOS) remains a major cause of death after lung transplantation limiting the life of our lung transplant patients. Unfortunately, the mechanisms by which BOS develops remain poorly understood. We previously observed that human lung recipient carriers of a mutation in ATG16L1 (rs2241880), an autophagy related protein, developed earlier BOS relative to non-carriers. Prior work has shown that this mutation leads to protein instability resulting in deficiency of ATG16L1 in monocyte-derived (Mo-) macrophages. Although it is known that genetically encoded deficiencies in mitophagy, a specialized form of autophagy that targets the removal of damaged mitochondria promote clinical disease, it is not known if they play a role in BOS. To study this, we analyzed the effects of ATG16L1 deficiency in Mo-CD11c+ cells in a mouse orthotopic lung transplant model of BOS. Methods CD11cCreATG16L1flox/flox (ATG16L1Δ/Δ) and control CD11cCre recipients received major MHC-mismatched FVB lungs, with immunosuppression to induce acceptance. Following induced epithelial injury, allografts were assessed for obliterative airway lesions (OB) for up to 28d. Intragraft Mo-CD11c+ cells were analyzed for bulk RNA sequencing and by FACS for mitochondrial mass and ROS production. Mitophagic flux was visualized by confocal microscopy using Mt-kiema mitophagy reporter mice. Mo-CD11c+ cells were characterized metabolically using a Seahorse analyzer. Alloimmunity was examined via T cell proliferation assays and ELISA for IL1-β. Results Indeed, our mouse ATG16L1Δ/Δ recipients also showed accelerated OB compared to controls. ATG16L1Δ/Δ Mo-CD11c+ cells showed higher mitochondrial mass, elevated ROS production as well as loss of transcripts encoding subunits of mitochondria electron complexes. Confocal analysis revealed attenuated mitophagic flux. In line with these, were decreased maximal mitochondrial respiratory capacity, lower mitochondrial ATP production and increased utilization of glycolysis, as well as increased T cell proliferation and higher IL1-β suggesting metabolic adaptation of an M1 inflammatory phenotype. Conclusion These data reveal a new role for ATG16L1 as a regulator of mitochondrial quality control and alloimmunity with implication for lung transplant recipients that carry the ATG16L1 rs2241880 mutation. Bronchiolitis obliterans (BOS) remains a major cause of death after lung transplantation limiting the life of our lung transplant patients. Unfortunately, the mechanisms by which BOS develops remain poorly understood. We previously observed that human lung recipient carriers of a mutation in ATG16L1 (rs2241880), an autophagy related protein, developed earlier BOS relative to non-carriers. Prior work has shown that this mutation leads to protein instability resulting in deficiency of ATG16L1 in monocyte-derived (Mo-) macrophages. Although it is known that genetically encoded deficiencies in mitophagy, a specialized form of autophagy that targets the removal of damaged mitochondria promote clinical disease, it is not known if they play a role in BOS. To study this, we analyzed the effects of ATG16L1 deficiency in Mo-CD11c+ cells in a mouse orthotopic lung transplant model of BOS. CD11cCreATG16L1flox/flox (ATG16L1Δ/Δ) and control CD11cCre recipients received major MHC-mismatched FVB lungs, with immunosuppression to induce acceptance. Following induced epithelial injury, allografts were assessed for obliterative airway lesions (OB) for up to 28d. Intragraft Mo-CD11c+ cells were analyzed for bulk RNA sequencing and by FACS for mitochondrial mass and ROS production. Mitophagic flux was visualized by confocal microscopy using Mt-kiema mitophagy reporter mice. Mo-CD11c+ cells were characterized metabolically using a Seahorse analyzer. Alloimmunity was examined via T cell proliferation assays and ELISA for IL1-β. Indeed, our mouse ATG16L1Δ/Δ recipients also showed accelerated OB compared to controls. ATG16L1Δ/Δ Mo-CD11c+ cells showed higher mitochondrial mass, elevated ROS production as well as loss of transcripts encoding subunits of mitochondria electron complexes. Confocal analysis revealed attenuated mitophagic flux. In line with these, were decreased maximal mitochondrial respiratory capacity, lower mitochondrial ATP production and increased utilization of glycolysis, as well as increased T cell proliferation and higher IL1-β suggesting metabolic adaptation of an M1 inflammatory phenotype. These data reveal a new role for ATG16L1 as a regulator of mitochondrial quality control and alloimmunity with implication for lung transplant recipients that carry the ATG16L1 rs2241880 mutation.