The consequence of lymphatic disruption during transplantation of solid organs remains unknown. Long-term survival after organ transplantation is limited by chronic rejection, a poorly understood process involving fibrotic remodeling and functional decline of the graft. Here, we found that transplanted human lungs and hearts with chronic rejection exhibited fibrosis distributed along dysmorphic lymphatics in areas densely concentrated with hyaluronan, an interstitial glycosaminoglycan that depends on lymphatic drainage for clearance. We illustrated similar findings in transplanted mouse lungs and hearts, which were accompanied by lymphographic findings of graft lymphedema. Using unsupervised clustering, we found a subset of stromal cells present in fibrotic syngeneic mouse lung grafts and human lung and heart grafts with chronic rejection that coexpressed hyaluronan synthase 1 and interleukin-1 receptor 1. Shortly after reperfusion of syngeneic mouse lung grafts, we identified neutrophilic expression of interleukin-1β (Il1b) as a driver of hyaluronan synthase 1 up-regulation. We found interleukin-1-mediated hyaluronan accumulation as a mechanism driving fibrosis that occurred independent of alloimmunity in the setting of lymphatic disruption after transplantation. Development of fibrotic remodeling in transplanted mouse lungs was inhibited by preventing hyaluronan synthesis through the administration of 4-methylumbilliferone, accelerating lymphangiogenesis with pharmacologic activation of VEGF (vascular endothelial growth factor) receptor-3, or inhibiting interleukin-1 receptor 1 signaling in the graft. These therapeutic interventions lay the foundation for future clinical strategies to prevent chronic rejection.

Small extracellular vesicles (sEVs) isolated from plasma of lung transplant recipients (LTRs) with chronic lung allograft dysfunction (CLAD) contain increased levels of lung associated self-antigens, Kα1 tubulin and collagen V, and decreased expression of the tumor suppressor liver kinase B1 (LKB1). In this study, sEVs were isolated from plasma collected from LTRs with or without cystic fibrosis (CF) from multiple centers at the onset of CLAD and 6 and 12 months before clinical diagnosis of CLAD (n = 32) as well as from time-matched stable controls (n = 25). sEVs were analyzed for Kα1 tubulin, collagen V, and LKB1 by western blot. Exoview R200, a functionalized microarray chip was employed to characterize the LKB1 in sEVs. EVs from non-CF LTRs had higher levels of lung self-antigens (p < 0.05) and lower levels of LKB1 (p = 0.024) 12 months before CLAD diagnosis than those from time-matched stable LTRs; however, in CF LTRs, only LKB1 levels were lower (p = 0.0005) 6 months before diagnosis. Further characterization of sEVs 6 months before CLAD in CF LTRs also demonstrated significantly lower numbers of LKB1 and LKB1/CD9 + sEV particles. Reduced LKB1 in circulating sEVs offers a potential biomarker for the risk of CLAD in LTRs with CF.

How pathogens inhibit transplant tolerance remains unclear. Here, we found that Pseudomonas aeruginosa infection, but not other common bacterial respiratory infections, increases antibody-mediated rejection (AMR) risk in recipients of lung transplants. To explore this relationship, we performed orthotopic lung transplants in mice, infected recipients with P. aeruginosa, and observed for the development of AMR. Intravital two-photon microscopy showed that P. aeruginosa rapidly invaded bronchial-associated lymphoid tissues, which resulted in acute lymphocytotoxicity, including the death of forkhead box P3 (Foxp3)+CD4+ T cells that are required to suppress AMR. P. aeruginosa-mediated AMR required expression of the type III secretion system (T3SS), which injects exotoxins into the cell cytoplasm. Through a combination of mutagenesis and epitope tagging experiments, we revealed that T3SS exotoxin T ADP ribosyl-transferase activity was sufficient for graft-resident Foxp3+CD4+ T cell apoptosis, leading to myeloid differentiation primary response 88 (Myd88)-dependent generation of T-box expressed in T cells (T-bet)- and C-X-C motif chemokine receptor 3 (CXCR3)-positive germinal center and memory B cells with high donor antigen avidity. We also found that T-bet+ and CXCR3+ B cells were elevated in biopsies from recipients of lung transplants who were diagnosed with AMR. In mice, CXCR3 deficiency restricted to B cells or CXCR3 blockade prevented AMR despite P. aeruginosa infection. Our work has identified a previously unrecognized role of bacterial virulence in lung allograft rejection and suggests potential strategies to prevent AMR for those at high risk of P. aeruginosa infection after transplant.

Background Primary Graft Dysfunction (PGD) is an early post-lung transplant (LTx) inflammatory condition primarily driven by lung ischemia-reperfusion injury (LIRI). Neutrophils are key mediators of LIRI, but their phenotypic diversity and maturation state remain poorly characterized. In other inflammatory settings, early expansion of immature neutrophils has been linked to increased tissue injury and worse clinical outcomes. Whether immature neutrophils increase following LTx and contribute to PGD severity remains unclear. Methods Circulating neutrophil heterogeneity was analyzed by flow cytometry in 20 LTx candidates with advanced lung disease and 30 LTx recipients. Matched plasma samples were used for cytokine profiling. The role of immature neutrophils in LIRI was studied using a murine left pulmonary hilar clamp model with or without anti-G-CSF treatment. The differentiation and effector functions of immature neutrophils derived from murine hematopoietic progenitors were studied in vitro. Results LTx recipients exhibited an early rise in circulating immature neutrophils, correlated with higher G-CSF levels and PGD severity. In mice, LIRI was linked to increased G-CSF levels, significant mobilization, and lung infiltration of immature neutrophils with an activated, ROS-producing phenotype. These cells showed prolonged survival, strong ROS activity, but impaired phagocytosis. Preoperative anti-G-CSF treatment decreased lung injury while reducing immature neutrophil mobilization and recruitment to the lung. Conclusions Our findings underscore the clinical significance of neutrophil heterogeneity in the early perioperative setting following LTx. Targeting the G-CSF-immature neutrophil axis may offer a novel strategy to improve early lung allograft outcomes.

BACKGROUND:Severe primary graft dysfunction (PGD) after lung transplantation (LTx) is a significant risk factor for the development of bronchiolitis obliterans syndrome (BOS). Recent data from our group demonstrated that small extracellular vesicles (sEVs) isolated from the plasma of LTx recipients with BOS have reduced levels of tumor suppressor gene liver kinase B1 ( LKB1 ) and promote epithelial-to-mesenchymal transition (EMT) and fibrosis. Here, we hypothesized that early inflammatory responses associated with severe PGD (PGD2/3) can downregulate LKB1 levels in sEVs, predisposing to the development of chronic lung allograft dysfunction (CLAD). METHODS:sEVs were isolated from the plasma of human participants by Exosome Isolation Kit followed by 0.20-µm filtration and characterized by NanoSight and immunoblotting analysis. Lung self-antigens (K alpha 1 tubulin, Collagen V), LKB1 , nuclear factor kappa B, and EMT markers in sEVs were compared by densitometry analysis between PGD2/3 and no-PGD participants. Neutrophil-derived factors and hypoxia/reperfusion effects on LKB1 levels and EMT were analyzed in vitro using quantitative real-time polymerase chain reaction and Western blotting. RESULTS:LKB1 was significantly downregulated in PGD2/3 sEVs compared with no-PGD sEVs. Within PGD2/3 participants, lower post-LTx LKB1 was associated with CLAD development. Hypoxia/reperfusion downregulates LKB1 and is associated with markers of EMT in vitro. Finally, lower LKB1 levels in PGD2/3 are associated with increased markers of EMT. CONCLUSIONS:Our results suggest that in post-LTx recipients with PGD2/3, downregulation of LKB1 protein levels in sEVs is associated with increased EMT markers and may result in the development of CLAD. Our results also suggest that ischemia/reperfusion injury during LTx may promote CLAD through the early downregulation of LKB1 .

Neutrophils exacerbate pulmonary ischemia-reperfusion injury (IRI) resulting in poor short and long-term outcomes for lung transplant recipients. Glycolysis powers neutrophil activation, but it remains unclear if neutrophil-specific targeting of this pathway will inhibit IRI. Lipid nanoparticles containing the glycolysis flux inhibitor 2-deoxyglucose (2-DG) were conjugated to neutrophil-specific Ly6G antibodies (NP-Ly6G(2-DG)). Intravenously administered NP-Ly6G(2-DG) to mice exhibited high specificity for circulating neutrophils. NP-Ly6G(2-DG)-treated neutrophils were unable to adapt to hypoglycemic conditions of the lung airspace environment as evident by the loss of demand-induced glycolysis, reductions in glycogen and ATP content and an increased vulnerability to apoptosis. NP-Ly6G(2-DG) treatment inhibited pulmonary IRI following hilar occlusion and orthotopic lung transplantation. IRI protection was associated with less airspace neutrophil extracellular trap generation, reduced intragraft neutrophilia and enhanced alveolar macrophage efferocytotic clearance of neutrophils. Collectively, our data show that pharmacologically targeting glycolysis in neutrophils inhibits their activation and survival leading to reduced pulmonary IRI.

Liver transplantation can be a life-saving treatment for end-stage hepatic disease. Unfortunately, some recipients develop ischemia-reperfusion injury (IRI) that leads to poor short- and long-term outcomes. Recent work has shown neutrophils contribute to IRI by undergoing NETosis, a form of death characterized by DNA ejection resulting in inflammatory extracellular traps. In this issue of the JCI, Hirao and Kojima et al. report that sphingosine-1-phosphate (S1P) expression induced by liver transplant–mediated IRI triggers NETosis. They also provide evidence that neutrophil expression of the carcinoembryonic antigen–related cell adhesion molecule-1 (CC1) long isoform inhibited NETosis by controlling S1P receptor–mediated autophagic flux. These findings suggest stimulating regulatory mechanisms that suppress NETosis could be used to prevent IRI.

Acute lung injury (ALI) is a heterogeneous inflammatory condition associated with high morbidity and mortality. Neutrophils play a key role in the development of different forms of ALI, and the release of neutrophil extracellular traps (NETs) is emerging as a common pathogenic mechanism. NETs are essential in controlling pathogens, and their defective release or increased degradation leads to a higher risk of infection. However, NETs also contain several pro-inflammatory and cytotoxic molecules than can exacerbate thromboinflammation and lung tissue injury. To reduce NET-mediated lung damage and inflammation, DNase is frequently used in preclinical models of ALI due to its capability of digesting NET DNA scaffold. Moreover, recent advances in neutrophil biology led to the development of selective NET inhibitors, which also appear to reduce ALI in experimental models. Here we provide an overview of the role of NETs in different forms of ALI discussing existing gaps in our knowledge and novel therapeutic approaches to modulate their impact on lung injury.

Acute lung injury (ALI) is a heterogeneous inflammatory condition associated with high morbidity and mortality. Neutrophils play a key role in the development of different forms of ALI, and the release of neutrophil extracellular traps (NETs) is emerging as a common pathogenic mechanism. NETs are essential in controlling pathogens, and their defective release or increased degradation leads to a higher risk of infection. However, NETs also contain several pro-inflammatory and cytotoxic molecules than can exacerbate thromboinflammation and lung tissue injury. To reduce NET-mediated lung damage and inflammation, DNase is frequently used in preclinical models of ALI due to its capability of digesting NET DNA scaffold. Moreover, recent advances in neutrophil biology led to the development of selective NET inhibitors, which also appear to reduce ALI in experimental models. Here we provide an overview of the role of NETs in different forms of ALI discussing existing gaps in our knowledge and novel therapeutic approaches to modulate their impact on lung injury.

Ischemia reperfusion injury represents a common pathological condition that is triggered by the release of endogenous ligands. While neutrophils are known to play a critical role in its pathogenesis, the tissue-specific spatiotemporal regulation of ischemia-reperfusion injury is not understood. Here, using oxidative lipidomics and intravital imaging of transplanted mouse lungs that are subjected to severe ischemia reperfusion injury, we discovered that necroptosis, a nonapoptotic form of cell death, triggers the recruitment of neutrophils. During the initial stages of inflammation, neutrophils traffic predominantly to subpleural vessels, where their aggregation is directed by chemoattractants produced by nonclassical monocytes that are spatially restricted in this vascular compartment. Subsequent neutrophilic disruption of capillaries resulting in vascular leakage is associated with impaired graft function. We found that TLR4 signaling in vascular endothelial cells and downstream NADPH oxidase 4 expression mediate the arrest of neutrophils, a step upstream of their extravasation. Neutrophil extracellular traps formed in injured lungs and their disruption with DNase prevented vascular leakage and ameliorated primary graft dysfunction. Thus, we have uncovered mechanisms that regulate the initial recruitment of neutrophils to injured lungs, which result in selective damage to subpleural pulmonary vessels and primary graft dysfunction. Our findings could lead to the development of new therapeutics that protect lungs from ischemia reperfusion injury.

Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-β bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes are engulfed primarily by alveolar macrophages (AM), which become reprogrammed to become less responsive to TGF-β and reduce TGF-β bioavailability through secretion of the TGF-β antagonist Decorin. In untreated recipients, high airway TGF-β activity stimulates AM to express CCL2 leading to CCR2+ monocyte-driven BOS development. Moreover, we find TGF-β receptor 2-dependent differentiation of CCR2+ monocytes is required for the generation of monocyte-derived AM, which in turn promote BOS by expanding tissue-resident memory CD8+ T cells that inflict airway injury through Blimp-1-mediated Granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-β-dependent network, which couples CCR2+ monocyte recruitment and differentiation to alloimmunity and BOS. Alveolar macrophage plasticity can be harnessed to prevent Bronchiolitis Obliterans Syndrome.

BackgroundMitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether circulating cell-free MT-DNA quantitation could be used to predict the risk of poor COVID-19 outcomes remains undetermined.MethodsWe measured circulating MT-DNA levels in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at hospital presentation. Our primary outcome was mortality. Intensive care unit (ICU) admission, intubation, vasopressor, and renal replacement therapy requirements were secondary outcomes. Multivariate regression analysis determined whether MT-DNA levels were independent of other reported COVID-19 risk factors. Receiver operating characteristic and area under the curve assessments were used to compare MT-DNA levels with established and emerging inflammatory markers of COVID-19.ResultsCirculating MT-DNA levels were highly elevated in patients who eventually died or required ICU admission, intubation, vasopressor use, or renal replacement therapy. Multivariate regression revealed that high circulating MT-DNA was an independent risk factor for these outcomes after adjusting for age, sex, and comorbidities. We also found that circulating MT-DNA levels had a similar or superior area under the curve when compared against clinically established measures of inflammation and emerging markers currently of interest as investigational targets for COVID-19 therapy.ConclusionThese results show that high circulating MT-DNA levels are a potential early indicator for poor COVID-19 outcomes.FundingWashington University Institute of Clinical Translational Sciences COVID-19 Research Program and Washington University Institute of Clinical Translational Sciences (ICTS) NIH grant UL1TR002345.

Background. Postoperative atrial fibrillation (POAF) is the most common complication after cardiac surgery, and is associated with increased morbidity and mortality. Inflammation has been implicated as an etiology of POAF. Mitochondrial DNA (mtDNA) has been shown to initiate inflammation. This study analyzed inflammatory mechanisms of POAF by evaluating mtDNA, neutrophils, and cytokines/chemokines in the pericardial fluid and blood after cardiac surgery. Methods. Blood and pericardial fluid from patients who underwent coronary artery bypass or heart valve surgery, or both, were collected intraoperatively and at 4, 12, 24, and 48 hours postoperatively. Real-time polymerase chain reaction was used to quantify mtDNA in the pericardial fluid and blood. A Luminex (Luminex Corp, Austin, TX) assay was used to study cytokine and chemokine levels. Flow cytometry was used to analyze neutrophil infiltration and activation in the pericardial fluid. Results. Samples from 100 patients were available for analysis. Postoperatively, mtDNA and multiple cytokine levels were higher in the pericardial fluid versus blood. Patients who had POAF had significantly higher levels of mtDNA in the pericardial fluid compared with patients who did not (P < .001, area under the curve 0.74). There was no difference in the mtDNA concentration in the blood between the POAF group and non-POAF group (P = .897). Neutrophil concentration increased in the pericardial fluid over time from a baseline of 0.8% to 56% at 48 hours (P < .01). Conclusions. The pericardial space has a high concentration of inflammatory mediators postoperatively. Mitochondrial DNA in the pericardial fluid was strongly associated with the development of POAF. This finding provides insight into a possible mechanism of inflam-mation that may contribute to POAF, and may offer novel therapeutic targets. (C) 2021 by The Society of Thoracic Surgeons

Pattern recognition receptors (PRRs) are germline-encoded sensors best characterized for their critical role in host defense. However, there is accumulating evidence that organ transplantation induces the release or display of molecular patterns of cellular injury and death that trigger PRR-mediated inflammatory responses. There are also new insights that indicate PRRs are able to distinguish between self and non-self, suggesting the existence of non-clonal mechanisms of allorecognition. Collectively, these reports have spurred considerable interest into whether PRRs or their ligands can be targeted to promote transplant survival. This review examines the mounting evidence that PRRs play in transplant-mediated inflammation. Given the large number of PRRs, we will focus on members from four families: the complement system, toll-like receptors, the formylated peptide receptor, and scavenger receptors through examining reports of their activity in experimental models of cellular and solid organ transplantation as well as in the clinical setting.

Obstructive sleep apnea syndrome (OSAS) is a common disorder characterized by repeated episodes of upper airways collapse during the sleep. The following intermittent hypoxia triggers a state of chronic inflammation, which also interests the nervous system leading to neuronal damage and increased risk of cognitive impairment. Brain derived neurotrophic factor (BDNF) is a growth factor often associated with neuroplasticity and neuroprotection whose levels increase in several condition associated with neuronal damage. However, whether patients affected by OSAS have altered BDNF levels and whether such alteration may be reflective of their cognitive impairment is still controversial. Here we show that, when compared to healthy control volunteers, OSAS patients have increased serum levels of BDNF. Moreover, OSAS patients with the higher levels of BDNF also have reduced neurocognitive impairment as measured by The Montreal Cognitive Assessment (MoCA) questionnaire. Treatment with standard non-invasive mechanical ventilation (CPAP) also was able to ameliorate the level of cognitive impairment. Altogether our results indicate that BDNF levels represent a neuroprotective response to intermittent hypoxia in OSAS patients.