Lipoxin A 4 /FPR2 signaling mitigates ferroptosis of alveolar epithelial cells via NRF2-dependent pathway during lung ischemia-reperfusion injury.

BACKGROUND: Post-lung transplantation (LTx) injury can involve sterile inflammation due to ischemia-reperfusion injury (IRI). We investigated the cell-specific role of ferroptosis (excessive iron-mediated cell death) in mediating lung IRI and determined if specialized pro-resolving mediators such as Lipoxin A4 (LxA 4 ) can protect against ferroptosis in lung IRI. METHODS: Single-cell RNA sequencing of lung tissue from post-LTx patients was analyzed. Lung IRI was evaluated in C57BL/6 (WT), formyl peptide receptor 2 knockout ( Fpr2 -/- ) and nuclear factor erythroid 2-related factor 2 knockout ( Nrf2 -/- ) mice using a hilar-ligation model with or without LxA 4 administration. Furthermore, the protective efficacy of LxA 4 was evaluated employing a murine orthotopic LTx model and in vitro studies using alveolar type II epithelial (ATII) cells. RESULTS: Differential expression of ferroptosis-related genes was observed in post-LTx patient samples compared to healthy controls. A significant increase in the levels of oxidized lipids and reduction in the levels of intact lipids were observed in mice subjected to IRI compared to shams. Furthermore, pharmacological inhibition of ferroptosis with liproxstatin-1 mitigated lung IRI and lung dysfunction. Importantly, LxA 4 treatment attenuated pulmonary dysfunction, ferroptosis and inflammation in WT mice subjected to lung IRI, but not in Fpr2 -/- or Nrf2 -/- mice, after IRI. In the murine LTx model, LxA 4 treatment increased PaO 2 levels and attenuated lung IRI. Mechanistically, LxA 4 -mediated protection involves increase in NRF2 activation and glutathione concentration as well as decrease in MDA levels in ATII cells. CONCLUSIONS: LxA 4 /FPR2 signaling on ATII cells mitigates ferroptosis via NRF2 activation and protects against lung IRI.

MerTK-dependent efferocytosis by monocytic-MDSCs mediates resolution of post-lung transplant injury.

Rationale: Patients with end stage lung diseases require lung transplantation (LTx) that can be impeded by ischemia-reperfusion injury (IRI) leading to subsequent chronic lung allograft dysfunction (CLAD) and inadequate outcomes. Objectives: We examined the undefined role of MerTK (receptor Mer tyrosine kinase) on monocytic myeloid-derived suppressor cells (M-MDSCs) in efferocytosis (phagocytosis of apoptotic cells) to facilitate resolution of lung IRI. Methods: Single-cell RNA sequencing of lung tissue and BAL from post-LTx patients was analyzed. Murine lung hilar ligation and allogeneic orthotopic LTx models of IRI were used with Balb/c (WT), cebpb -/- (MDSC-deficient), Mertk -/- or MerTK-CR (cleavage resistant) mice. Lung function, IRI (inflammatory cytokine and myeloperoxidase expression, immunohistology for neutrophil infiltration), and flow cytometry of lung tissue for efferocytosis of apoptotic neutrophils were assessed in mice. Measurements and Main Results: A significant downregulation in MerTK-related efferocytosis genes in M-MDSC populations of CLAD patients compared to healthy subjects was observed. In the murine IRI model, significant increase in M-MDSCs, MerTK expression and efferocytosis was observed in WT mice during resolution phase that was absent in cebpb -/- Land Mertk -/- mice. Adoptive transfer of M-MDSCs in cebpb -/- mice significantly attenuated lung dysfunction, and inflammation leading to resolution of IRI. Additionally, in a preclinical murine orthotopic LTx model, increases in M-MDSCs were associated with resolution of lung IRI in the transplant recipients. In vitro studies demonstrated the ability of M-MDSCs to efferocytose apoptotic neutrophils in a MerTK-dependent manner. Conclusions: Our results suggest that MerTK-dependent efferocytosis by M-MDSCs can significantly contribute to the resolution of post-LTx IRI.

Resolution of post-lung transplant ischemia-reperfusion injury is modulated via Resolvin D1-FPR2 and Maresin 1-LGR6 signaling.

BACKGROUND: Dysregulation of inflammation-resolution pathways leads to postlung transplant (LTx) ischemia-reperfusion (IR) injury and allograft dysfunction. Our hypothesis is that combined treatment with specialized pro-resolving lipid mediators, that is, Resolvin D1 (RvD1) and Maresin-1 (MaR1), enhances inflammation-resolution of lung IR injury. METHODS: Expression of RvD1 and MaR1 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on days 0, 1, and 7 post-LTx. Lung IR injury was evaluated in C57BL/6 (WT), FPR2-/-, and LGR6 siRNA treated mice using a hilar-ligation model with or without administration with RvD1 and/or MaR1. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by RvD1 and MaR1 against lung IR injury. In vitro studies analyzed alveolar macrophages and type II epithelial cell activation after treatment with RvD1 or MaR1. RESULTS: RvD1 and MaR1 expressions in BAL from post-LTx patients was significantly increased on day 7 compared to days 0 and 1. Concomitant RvD1 and MaR1 treatment significantly mitigated early pulmonary inflammation and lung IR injury in WT mice, which was regulated via FPR2 and LGR6 receptors. In the murine orthotopic donation after cardiac death LTx model, RvD1 and MaR1 treatments significantly attenuated lung IR injury and increased PaO2 levels compared to saline-treated controls. Mechanistically, RvD1/FPR2 signaling on alveolar macrophages attenuated HMGB1 and TNF-α secretion and upregulated uptake of macrophage-dependent apoptotic neutrophils (efferocytosis), whereas MaR1/LGR6 signaling mitigated CXCL1 secretion by epithelial cells. CONCLUSIONS: Bioactive proresolving lipid mediator-dependent signaling that is, RvD1/FPR2 and MaR1/LGR6- offers a novel therapeutic strategy in post-LTx injury.

Modulating donor mitochondrial fusion/fission delivers immunoprotective effects in cardiac transplantation.

Early insults associated with cardiac transplantation increase the immunogenicity of donor microvascular endothelial cells (ECs), which interact with recipient alloreactive memory T cells and promote responses leading to allograft rejection. Thus, modulating EC immunogenicity could potentially alter T cell responses. Recent studies have shown modulating mitochondrial fusion/fission alters immune cell phenotype. Here, we assess whether modulating mitochondrial fusion/fission reduces EC immunogenicity and alters EC-T cell interactions. By knocking down DRP1, a mitochondrial fission protein, or by using the small molecules M1, a fusion promoter, and Mdivi1, a fission inhibitor, we demonstrate that promoting mitochondrial fusion reduced EC immunogenicity to allogeneic CD8+ T cells, shown by decreased T cell cytotoxic proteins, decreased EC VCAM-1, MHC-I expression, and increased PD-L1 expression. Co-cultured T cells also displayed decreased memory frequencies and Ki-67 proliferative index. For in vivo significance, we used a novel murine brain-dead donor transplant model. Balb/c hearts pretreated with M1/Mdivi1 after brain-death induction were heterotopically transplanted into C57BL/6 recipients. We demonstrate that, in line with our in vitro studies, M1/Mdivi1 pretreatment protected cardiac allografts from injury, decreased infiltrating T cell production of cytotoxic proteins, and prolonged allograft survival. Collectively, our data show promoting mitochondrial fusion in donor ECs mitigates recipient T cell responses and leads to significantly improved cardiac transplant survival.

In Situ Pre-Treatment of Vascularized Composite Allografts With a Targeted Complement Inhibitor Protects Against Brain Death and Ischemia Reperfusion Induced Injuries.

Introduction: Donor brain death (BD) is an unavoidable component of vascularized composite allograft (VCA) transplantation and a key contributor to ischemia-reperfusion injury (IRI). Complement is activated and deposited within solid organ grafts as a consequence of BD and has been shown to exacerbate IRI, although the role of BD and complement in VCA and the role it plays in IRI and VCA rejection has not been studied. Methods: BD was induced in Balb/c donors, and the VCA perfused prior to graft procurement with UW solution supplemented with or without CR2-Crry, a C3 convertase complement inhibitor that binds at sites of complement activation, such as that induced on the endothelium by induction of BD. Following perfusion, donor VCAs were cold stored for 6 hours before transplantation into C57BL/6 recipients. Donor VCAs from living donors (LD) were also procured and stored. Analyses included CR2-Crry graft binding, complement activation, toxicity, injury/inflammation, graft gene expression and survival. Results: Compared to LD VCAs, BD donor VCAs had exacerbated IRI and rejected earlier. Following pretransplant in-situ perfusion of the donor graft, CR2-Crry bound within the graft and was retained post-transplantation. CR2-Crry treatment significantly reduced complement deposition, inflammation and IRI as compared to vehicle-treated BD donors. Treatment of BD donor VCAs with CR2-Crry led to an injury profile not dissimilar to that seen in recipients of LD VCAs. Conclusion: Pre-coating a VCA with CR2-Crry in a clinically relevant treatment paradigm provides localized, and therefore minimally immunosuppressive, protection from the complement-mediated effects of BD induced exacerbated IRI.

A novel injury site-natural antibody targeted complement inhibitor protects against lung transplant injury.

Complement is known to play a role in ischemia and reperfusion injury (IRI). A general paradigm is that complement is activated by self-reactive natural IgM antibodies (nAbs), after they engage postischemic neoepitopes. However, a role for nAbs in lung transplantation (LTx) has not been explored. Using mouse models of LTx, we investigated the role of two postischemic neoepitopes, modified annexin IV (B4) and a subset of phospholipids (C2), in LTx. Antibody deficient Rag1-/- recipient mice were protected from LTx IRI. Reconstitution with either B4 or C2nAb restored IRI, with C2 significantly more effective than B4 nAb. Based on these information, we developed/characterized a novel complement inhibitor composed of single-chain antibody (scFv) derived from the C2 nAb linked to Crry (C2scFv-Crry), a murine inhibitor of C3 activation. Using an allogeneic LTx, in which recipients contain a full nAb repertoire, C2scFv-Crry targeted to the LTx, inhibited IRI, and delayed acute rejection. Finally, we demonstrate the expression of the C2 neoepitope in human donor lungs, highlighting the translational potential of this approach.

MicroRNA-206 antagomiR‒enriched extracellular vesicles attenuate lung ischemia‒reperfusion injury through CXCL1 regulation in alveolar epithelial cells.

BACKGROUND: Our hypothesis is that the immunomodulatory capacities of mesenchymal stem cell‒derived extracellular vesicles (EVs) can be enhanced by specific microRNAs (miRNAs) to effectively attenuate post-transplant lung ischemia‒reperfusion (IR) injury. METHODS: The expression of miR-206 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on Days 0 and 1 after lung transplantation. Lung IR injury was evaluated in C57BL/6 mice using a left lung hilar-ligation model with or without treatment with EVs or antagomiR-206‒enriched EVs. Murine lung tissue was used for miRNA microarray hybridization analysis, and cytokine expression, lung injury, and edema were evaluated. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by enriched EVs against lung IR injury. In vitro studies analyzed type II epithelial cell activation after coculturing with EVs. RESULTS: A significant upregulation of miR-206 was observed in the BAL fluid of patients on Day 1 after lung transplantation compared with Day 0 and in murine lungs after IR injury compared with sham. Treatment with antagomiR-206‒enriched EVs attenuated lung dysfunction, injury, and edema compared with treatment with EVs alone after murine lung IR injury. Enriched EVs reduced lung injury and neutrophil infiltration as well as improved allograft oxygenation after murine orthotopic lung transplantation. Enriched EVs significantly decreased proinflammatory cytokines, especially epithelial cell‒dependent CXCL1 expression, in the in vivo and in vitro IR injury models. CONCLUSIONS: EVs can be used as biomimetic nanovehicles for protective immunomodulation by enriching them with antagomiR-206 to mitigate epithelial cell activation and neutrophil infiltration in the lungs after IR injury.

Aneurysm of the Anomalous Splenic Artery Arising from Superior Mesenteric Artery Treated by Coil Embolization: A Report of Two Cases and Literature Review.

BACKGROUND: Aneurysm of splenic artery (SA) which arises from the superior mesenteric artery (SMA) is a very rare condition. The aim of this study was to present our experience with 2 such patients treated by coil embolization. METHODS: A 33-year-old man and a 61-year-old woman were incidentally identified with aneurysms of the SA which arose from the SMA. Endovascular therapy of coil embolization was chosen to treat the aneurysm. RESULTS: Follow-up computed tomography showed no change in the location of the coils and occlusion of majority of the aneurysm sac. The 2 patients have been doing well during a 26-month and 10-month follow-up period, respectively. CONCLUSIONS: Combined with the experience of the previous literature, we think coil embolization can be cost-effective and minimally invasive in selected cases, depending on the morphology and site of the lesion.