Nanoparticle targeting of neutrophil glycolysis prevents lung ischemia-reperfusion injury.

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.

Avoid being trapped by your liver: ischemia-reperfusion injury in liver transplant triggers S1P-mediated NETosis.

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.

Reprogramming alveolar macrophage responses to TGF-ß reveals CCR2+ monocyte activity that promotes bronchiolitis obliterans syndrome.

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-ß bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes were primarily engulfed by alveolar macrophages (AMs), which were reprogrammed to become less responsive to TGF-ß and reduce TGF-ß bioavailability through secretion of the TGF-ß antagonist decorin. In untreated recipients, high airway TGF-ß activity stimulated AMs to express CCL2, leading to CCR2+ monocyte-driven BOS development. Moreover, we found TGF-ß receptor 2-dependent differentiation of CCR2+ monocytes was required for the generation of monocyte-derived AMs, which in turn promoted BOS by expanding tissue-resident memory CD8+ T cells that inflicted 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-ß-dependent network that couples CCR2+ monocyte recruitment and differentiation to alloimmunity and BOS.

The role of neutrophil extracellular traps in acute 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.

Necroptosis triggers spatially restricted neutrophil-mediated vascular damage during lung ischemia reperfusion 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.

Circulating mitochondrial DNA is an early indicator of severe illness and mortality from COVID-19.

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.

Pericardial Mitochondrial DNA Levels Are Associated With Atrial Fibrillation After Cardiac Surgery.

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 inflammation that may contribute to POAF, and may offer novel therapeutic targets.

Full-Length TrkB Variant in NSCLC Is Associated with Brain Metastasis.

Despite remarkable therapeutic advances have been made in the last few decades, non-small cell lung cancer (NSCLC) is still one of the leading causes of death worldwide. Brain metastases are a common complication of a wide range of human malignancies and in particular NSCLC. Brain-derived neurotrophic factor (BDNF), binding its high-affinity tyrosine kinase B receptor, has been shown to promote cancer progression and metastasis. We hereby investigated the expression of the BDNF and its TrkB receptor in its full-length and truncated isoform T1, in samples from primary adenocarcinomas (ADKs) of the lung and in their metastasis to evaluate if their expression was related to preferential tumor entry into the central nervous system (CNS). By immunohistochemistry, 80% of the ADKs that metastasize to central nervous system expressed TrkB receptor compared to 33% expressing of ADKs without CNS metastasis. Moreover, ADKs with CNS metastasis showed an elevated expression of the full-length TrkB receptor. The TrkB receptor FL/T1 ratio was statistically higher in primary ADKs with brain metastasis compared to ADKs without brain metastasis. Our data indicate that TrkB full-length isoform expression in primary ADK cells may be associated with higher risk to develop brain metastasis. Therefore, TrkB receptor may possess prognostic and therapeutic implications in lung ADK.

Circulating Mitochondrial DNA is an Early Indicator of Severe Illness and Mortality from COVID-19.

Mitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether the appearance of cell-free MT-DNA is linked to poor COVID-19 outcomes remains undetermined. Here, we quantified circulating MT-DNA in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at the time of hospital presentation. Circulating MT-DNA were sharply elevated in patients who eventually died, required ICU admission or intubation. Multivariate regression analysis revealed that high circulating MT-DNA levels is an independent risk factor for all of these outcomes after adjusting for age, sex and comorbidities. Additionally, we found that circulating MT-DNA has a similar or superior area-under-the curve when compared to clinically established measures of systemic inflammation, as well as emerging markers currently of interest as investigational targets for COVID-19 therapy. These results show that high circulating MT-DNA levels is a potential indicator for poor COVID-19 outcomes.

Recent advances into the role of pattern recognition receptors in transplantation.

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.

Protective role of brain derived neurotrophic factor (BDNF) in obstructive sleep apnea syndrome (OSAS) patients.

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.

Lung transplant outcomes are influenced by severity of neutropenia and granulocyte colony-stimulating factor treatment.

Although neutropenia is a common complication after lung transplant, its relationship with recipient outcomes remains understudied. We evaluated a retrospective cohort of 228 adult lung transplant recipients between 2008 and 2013 to assess the association of neutropenia and granulocyte colony-stimulating factor (GCSF) treatment with outcomes. Neutropenia was categorized as mild (absolute neutrophil count 1000-1499), moderate (500-999), or severe (<500) and as a time-varying continuous variable. Associations with survival, acute rejection, and chronic lung allograft dysfunction (CLAD) were assessed with the use of Cox proportional hazards regression. GCSF therapy impact on survival, CLAD, and acute rejection development was analyzed by propensity score matching. Of 228 patients, 101 (42.1%) developed neutropenia. Recipients with severe neutropenia had higher mortality rates than those of recipients with no (adjusted hazard ratio [aHR] 2.97, 95% confidence interval [CI] 1.05-8.41, P = .040), mild (aHR 14.508, 95% CI 1.58-13.34, P = .018), or moderate (aHR 3.27, 95% CI 0.89-12.01, P = .074) neutropenia. Surprisingly, GCSF treatment was associated with a higher risk for CLAD in mildly neutropenic patients (aHR 3.49, 95% CI 0.93-13.04, P = .063), although it did decrease death risk in severely neutropenic patients (aHR 0.24, 95% CI 0.07-0.88, P = .031). Taken together, our data point to an important relationship between neutropenia severity and GCSF treatment in lung transplant outcomes.

An obligatory role for club cells in preventing obliterative bronchiolitis in lung transplants.

Obliterative bronchiolitis (OB) is a poorly understood airway disease characterized by the generation of fibrotic bronchiolar occlusions. In the lung transplant setting, OB is a pathological manifestation of bronchiolitis obliterans syndrome (BOS), which is a major impediment to long-term recipient survival. Club cells play a key role in bronchiolar epithelial repair, but whether they promote lung transplant tolerance through preventing OB remains unclear. We determined if OB occurs in mouse orthotopic lung transplants following conditional transgene-targeted club cell depletion. In syngeneic lung transplants club cell depletion leads to transient epithelial injury followed by rapid club cell-mediated repair. In contrast, allogeneic lung transplants develop severe OB lesions and poorly regenerate club cells despite immunosuppression treatment. Lung allograft club cell ablation also triggers the recognition of alloantigens, and pulmonary restricted self-antigens reported associated with BOS development. However, CD8+ T cell depletion restores club cell reparative responses and prevents OB. In addition, ex-vivo analysis reveals a specific role for alloantigen-primed effector CD8+ T cells in preventing club cell proliferation and maintenance. Taken together, we demonstrate a vital role for club cells in maintaining lung transplant tolerance and propose a new model to identify the underlying mechanisms of OB.

Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance.

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse-1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2-photon microscopy, we analyzed the effects of DNAse-1 on NETs in mouse orthotopic lung allografts damaged by ischemia-reperfusion injury. Although DNAse-1 treatment rapidly degrades intragraft NETs, the consequential release of NET fragments induces prolonged interactions between infiltrating CD4+ T cells and donor-derived antigen presenting cells. DNAse-1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen-specific CD4+ T cell proliferation through activating toll-like receptor (TLR) - Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil-specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse-1 administration to wild-type recipients promotes the recognition of allo- and self-antigens and prevents immunosuppression-mediated lung allograft acceptance through a MyD88-dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.

Mitochondrial damage-associated molecular patterns released by lung transplants are associated with primary graft dysfunction.

Primary graft dysfunction (PGD) is a major limitation in short- and long-term lung transplant survival. Recent work has shown that mitochondrial damage-associated molecular patterns (mtDAMPs) can promote solid organ injury, but whether they contribute to PGD severity remains unclear. We quantitated circulating plasma mitochondrial DNA (mtDNA) in 62 patients, before lung transplantation and shortly after arrival to the intensive care unit. Although all recipients released mtDNA, high levels were associated with severe PGD development. In a mouse orthotopic lung transplant model of PGD, we detected airway cell-free damaged mitochondria and mtDNA in the peripheral circulation. Pharmacologic inhibition or genetic deletion of formylated peptide receptor 1 (FPR1), a chemotaxis sensor for N-formylated peptides released by damaged mitochondria, inhibited graft injury. An analysis of intragraft neutrophil-trafficking patterns reveals that FPR1 enhances neutrophil transepithelial migration and retention within airways but does not control extravasation. Using donor lungs that express a mitochondria-targeted reporter protein, we also show that FPR1-mediated neutrophil trafficking is coupled with the engulfment of damaged mitochondria, which in turn triggers reactive oxygen species (ROS)-induced pulmonary edema. Therefore, our data demonstrate an association between mtDAMP release and PGD development and suggest that neutrophil trafficking and effector responses to damaged mitochondria are drivers of graft damage.

Spleen-derived classical monocytes mediate lung ischemia-reperfusion injury through IL-1ß.

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1ß production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.

Naive CD4+ T Cells Carrying a TLR2 Agonist Overcome TGF-ß-Mediated Tumor Immune Evasion.

TLR agonists are effective at treating superficial cancerous lesions, but their use internally for other types of tumors remains challenging because of toxicity. In this article, we report that murine and human naive CD4+ T cells that sequester Pam3Cys4 (CD4+ TPam3) become primed for Th1 differentiation. CD4+ TPam3 cells encoding the OVA-specific TCR OT2, when transferred into mice bearing established TGF-ß-OVA-expressing thymomas, produce high amounts of IFN-γ and sensitize tumors to PD-1/programmed cell death ligand 1 blockade-induced rejection. In contrast, naive OT2 cells without Pam3Cys4 cargo are prone to TGF-ß-dependent inducible regulatory Foxp3+ CD4+ T cell conversion and accelerate tumor growth that is largely unaffected by PD-1/programmed cell death ligand 1 blockade. Ex vivo analysis reveals that CD4+ TPam3 cells are resistant to TGF-ß-mediated gene expression through Akt activation controlled by inputs from the TCR and a TLR2-MyD88-dependent PI3K signaling pathway. These data show that CD4+ TPam3 cells are capable of Th1 differentiation in the presence of TGF-ß, suggesting a novel approach to adoptive cell therapy.

BDNF/TrkB axis activation promotes epithelial-mesenchymal transition in idiopathic pulmonary fibrosis.

BACKGROUND: Neurotrophins (NT) belongs to a family of growth factors which promotes neurons survival and differentiation. Increasing evidence show that NT and their receptor are expressed in lung tissues suggesting a possible role in lung health and disease. Here we investigated the expression and functional role of the TrkB/BDNF axis in idiopathic pulmonary fibrotic lung (myo)fibroblasts. METHODS: Lung fibroblast were isolated from IPF patients and characterized for the expression of mesenchymal markers in comparison to normal lung fibroblasts isolated from non-IPF controls. RESULTS: BDNF treatment promoted mesenchymal differentiation and this effect was counteracted by the TrkB inhibitor K252a. In this regard, we showed that K252a treatment was able to control the expression of transcription factors involved in epithelial to mesenchymal transition (EMT). Accordingly, K252a treatment reduced matrix metalloproteinase-9 enzyme activity and E-cadherin expression while increased cytoplasmic ß-catenin expression. CONCLUSIONS: Our results suggest that BDNF/TrkB axis plays a role in EMT promoting the acquisition of (myo)fibroblast cell phenotype in IPF. Targeting BDNF/TrkB seems to represent a viable approach in order to prevent EMT dependent lung fibrosis.

The Role of Neutrophils in Transplanted Organs.

Neutrophils are often viewed as nonspecialized effector cells whose presence is a simple indicator of tissue inflammation. There is new evidence that neutrophils exist in subsets and have specialized effector functions that include extracellular trap generation and the stimulation of angiogenesis. The application of intravital imaging to transplanted organs has revealed novel requirements for neutrophil trafficking into graft tissue and has illuminated direct interactions between neutrophils and other leukocytes that promote alloimmunity. Paradoxically, retaining some neutrophilia may be important to induce or maintain tolerance. Neutrophils can stimulate anti-inflammatory signals in other phagocytes and release molecules that inhibit T cell activation. In this article, we will review the available evidence of how neutrophils regulate acute and chronic inflammation in transplanted organs and discuss the possibility of targeting these cells to promote tolerance.

Mechanisms of graft rejection after lung transplantation.

PURPOSE OF REVIEW: To date, outcomes after lung transplantation are far worse than after transplantation of other solid organs. New insights into mechanisms that contribute to graft rejection and tolerance after lung transplantation remain of great interest. This review examines the recent literature on the role of innate and adaptive immunity in shaping the fate of lung grafts. RECENT FINDINGS: Innate and adaptive immune cells orchestrate allograft rejection after transplantation. Innate immune cells such as neutrophils are recruited to the lung graft early after reperfusion and subsequently promote allograft rejection. Although it is widely recognized that CD4 T lymphocytes in concert with CD8 T cells promote graft rejection, regulatory Foxp3 CD4 T, central memory CD8 T cells, and natural killer cells can facilitate tolerance. SUMMARY: This review highlights interactions between innate and adaptive immune pathways and how they contribute to lung allograft rejection. These findings lay a foundation for the design of new therapeutic strategies that target both innate and adaptive immune responses.