IL-1ß-dependent extravasation of preexisting lung-restricted autoantibodies during lung transplantation activates complement and mediates primary graft dysfunction.

Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1ß-mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective.

CD11b suppresses TLR activation of nonclassical monocytes to reduce primary graft dysfunction after lung transplantation.

Primary graft dysfunction (PGD) is the leading cause of postoperative mortality in lung transplant recipients and the most important risk factor for development of chronic lung allograft dysfunction. The mechanistic basis for the variability in the incidence and severity of PGD between lung transplant recipients is not known. Using a murine orthotopic vascularized lung transplant model, we found that redundant activation of Toll-like receptors 2 and 4 (TLR2 and -4) on nonclassical monocytes activates MyD88, inducing the release of the neutrophil attractant chemokine CXCL2. Deletion of Itgam (encodes CD11b) in nonclassical monocytes enhanced their production of CXCL2 and worsened PGD, while a CD11b agonist, leukadherin-1, administered only to the donor lung prior to lung transplantation, abrogated CXCL2 production and PGD. The damage-associated molecular pattern molecule HMGB1 was increased in peripheral blood samples from patients undergoing lung transplantation after reperfusion and induced CXCL2 production in nonclassical monocytes via TLR4/MyD88. An inhibitor of HMGB1 administered to the donor and recipient prior to lung transplantation attenuated PGD. Our findings suggest that CD11b acts as a molecular brake to prevent neutrophil recruitment by nonclassical monocytes following lung transplantation, revealing an attractive therapeutic target in the donor lung to prevent PGD in lung transplant recipients.

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.

Crosstalk between nonclassical monocytes and alveolar macrophages mediates transplant ischemia-reperfusion injury through classical monocyte recruitment.

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1ß production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1ß production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R-dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1ß-dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1ß, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

Impaired phagocytic function in CX3CR1+ tissue-resident skeletal muscle macrophages prevents muscle recovery after influenza A virus-induced pneumonia in old mice.

Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individuals in whom it causes substantial morbidity. We found that skeletal muscle recovery was impaired in old compared with young mice after influenza A virus-induced pneumonia. In young mice, recovery of muscle loss was associated with expansion of tissue-resident skeletal muscle macrophages and downregulation of MHC II expression, followed by a proliferation of muscle satellite cells. These findings were absent in old mice and in mice deficient in Cx3cr1. Transcriptomic profiling of tissue-resident skeletal muscle macrophages from old compared with young mice showed downregulation of pathways associated with phagocytosis and proteostasis, and persistent upregulation of inflammatory pathways. Consistently, skeletal muscle macrophages from old mice failed to downregulate MHCII expression during recovery from influenza A virus-induced pneumonia and showed impaired phagocytic function in vitro. Like old animals, mice deficient in the phagocytic receptor Mertk showed no macrophage expansion, MHCII downregulation, or satellite cell proliferation and failed to recover skeletal muscle function after influenza A pneumonia. Our data suggest that a loss of phagocytic function in a CX3CR1+ tissue-resident skeletal muscle macrophage population in old mice precludes satellite cell proliferation and recovery of skeletal muscle function after influenza A pneumonia.

Residual endotoxin induces primary graft dysfunction through ischemia/reperfusion-primed alveolar macrophages.

Despite the widespread use of antibiotics, bacterial pneumonias in donors strongly predispose to the fatal syndrome of primary graft dysfunction (PGD) following lung transplantation. We report that bacterial endotoxin persists in human donor lungs after pathogen is cleared with antibiotics and is associated with neutrophil infiltration and PGD. In mouse models, depletion of tissue-resident alveolar macrophages (TRAMs) attenuated neutrophil recruitment in response to endotoxin as shown by compartmental staining and intravital imaging. Bone marrow chimeric mice revealed that neutrophils were recruited by TRAM through activation of TLR4 in a MyD88-dependent manner. Intriguingly, low levels of endotoxin, insufficient to cause donor lung injury, promoted TRAM-dependent production of CXCL2, increased neutrophil recruitment, and led to PGD, which was independent of donor NCMs. Reactive oxygen species (ROS) increased in human donor lungs starting from the warm-ischemia phase and were associated with increased transcription and translocation to the plasma membrane of TLR4 in donor TRAMs. Consistently, scavenging ROS or inhibiting their production to prevent TLR4 transcription/translocation or blockade of TLR4 or coreceptor CD14 on donor TRAMs prevented neutrophil recruitment in response to endotoxin and ameliorated PGD. Our studies demonstrate that residual endotoxin after successful treatment of donor bacterial pneumonia promotes PGD through ischemia/reperfusion-primed donor TRAMs.

High CO2 Levels Impair Lung Wound Healing.

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-κB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-κB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, CXCL12 transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.

Elevated CO2 Levels Delay Skeletal Muscle Repair by Increasing Fatty Acid Oxidation.

Muscle dysfunction often occurs in patients with chronic obstructive pulmonary diseases (COPD) and affects ventilatory and non-ventilatory skeletal muscles. We have previously reported that hypercapnia (elevated CO2 levels) causes muscle atrophy through the activation of the AMPKα2-FoxO3a-MuRF1 pathway. In the present study, we investigated the effect of normoxic hypercapnia on skeletal muscle regeneration. We found that mouse C2C12 myoblasts exposed to elevated CO2 levels had decreased fusion index compared to myoblasts exposed to normal CO2. Metabolic analyses of C2C12 myoblasts exposed to high CO2 showed increased oxidative phosphorylation due to increased fatty acid oxidation. We utilized the cardiotoxin-induced muscle injury model in mice exposed to normoxia and 10% CO2 for 21 days and observed that muscle regeneration was delayed. High CO2-delayed differentiation in both mouse C2C12 myoblasts and skeletal muscle after injury and was restored to control levels when cells or mice were treated with a carnitine palmitoyltransfearse-1 (CPT1) inhibitor. Taken together, our data suggest that hypercapnia leads to changes in the metabolic activity of skeletal muscle cells, which results in impaired muscle regeneration and recovery after injury.

Elevated CO2 regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans.

Carbon dioxide (CO2) is sensed by cells and can trigger signals to modify gene expression in different tissues leading to changes in organismal functions. Despite accumulating evidence that several pathways in various organisms are responsive to CO2 elevation (hypercapnia), it has yet to be elucidated how hypercapnia activates genes and signaling pathways, or whether they interact, are integrated, or are conserved across species. Here, we performed a large-scale transcriptomic study to explore the interaction/integration/conservation of hypercapnia-induced genomic responses in mammals (mice and humans) as well as invertebrates (Caenorhabditis elegans and Drosophila melanogaster). We found that hypercapnia activated genes that regulate Wnt signaling in mouse lungs and skeletal muscles in vivo and in several cell lines of different tissue origin. Hypercapnia-responsive Wnt pathway homologues were similarly observed in secondary analysis of available transcriptomic datasets of hypercapnia in a human bronchial cell line, flies and nematodes. Our data suggest the evolutionarily conserved role of high CO2 in regulating Wnt pathway genes.

Clinical relevance of lung-restricted antibodies in lung transplantation.

Lung transplant is a definitive treatment for several end-stage lung diseases. However, the high incidence of allograft rejection limits the overall survival following lung transplantation. Traditionally, alloimmunity directed against human leukocyte antigens (HLA) has been implicated in transplant rejection. Recently, the clinical impact of non-HLA lung-restricted antibodies (LRA) has been recognized and extensive research has demonstrated that they may play a dominant role in the development of lung allograft rejection. The immunogenic lung-restricted antigens that have been identified include amongst others, collagen type I, collagen type V, and k-alpha 1 tubulin. Pre-existing antibodies against these lung-restricted antigens are prevalent in patients undergoing lung transplantation and have emerged as one of the predominant risk factors for primary graft dysfunction which limits short-term survival following lung transplantation. Additionally, LRA have been shown to predispose to chronic lung allograft rejection, the predominant cause of poor long-term survival. This review will discuss ongoing research into the mechanisms of development of LRA as well as the pathogenesis of associated lung allograft injury.

Cardiac glycosides decrease influenza virus replication by inhibiting cell protein translational machinery.

Cardiac glycosides (CGs) are used primarily for cardiac failure and have been reported to have other effects, including inhibition of viral replication. Here we set out to study mechanisms by which CGs as inhibitors of the Na-K-ATPase decrease influenza A virus (IAV) replication in the lungs. We found that CGs inhibit influenza virus replication in alveolar epithelial cells by decreasing intracellular potassium, which in turn inhibits protein translation, independently of viral entry, mRNA transcription, and protein degradation. These effects were independent of the Src signaling pathway and intracellular calcium concentration changes. We found that short-term treatment with ouabain prevented IAV replication without cytotoxicity. Rodents express a Na-K-ATPase-α1 resistant to CGs. Thus we utilized Na-K-ATPase-α1-sensitive mice, infected them with high doses of influenza virus, and observed a modest survival benefit when treated with ouabain. In summary, we provide evidence that the inhibition of the Na-K-ATPase by CGs decreases influenza A viral replication by modulating the cell protein translational machinery and results in a modest survival benefit in mice.

Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1.

Muscle dysfunction is common in patients with adult respiratory distress syndrome and is associated with morbidity that can persist for years after discharge. In a mouse model of severe influenza A pneumonia, we found the proinflammatory cytokine IL-6 was necessary for the development of muscle dysfunction. Treatment with a Food and Drug Administration-approved Ab antagonist to the IL-6R (tocilizumab) attenuated the severity of influenza A-induced muscle dysfunction. In cultured myotubes, IL-6 promoted muscle degradation via JAK/STAT, FOXO3a, and atrogin-1 upregulation. Consistent with these findings, atrogin-1+/- and atrogin-1-/- mice had attenuated muscle dysfunction following influenza infection. Our data suggest that inflammatory endocrine signals originating from the injured lung activate signaling pathways in the muscle that induce dysfunction. Inhibiting these pathways may limit morbidity in patients with influenza A pneumonia and adult respiratory distress syndrome.

Hypercapnia increases airway smooth muscle contractility via caspase-7-mediated miR-133a-RhoA signaling.

The elevation of carbon dioxide (CO2) in tissues and the bloodstream (hypercapnia) occurs in patients with severe lung diseases, including chronic obstructive pulmonary disease (COPD). Whereas hypercapnia has been recognized as a marker of COPD severity, a role for hypercapnia in disease pathogenesis remains unclear. We provide evidence that CO2 acts as a signaling molecule in mouse and human airway smooth muscle cells. High CO2 activated calcium-calpain signaling and consequent smooth muscle cell contraction in mouse airway smooth muscle cells. The signaling was mediated by caspase-7-induced down-regulation of the microRNA-133a (miR-133a) and consequent up-regulation of Ras homolog family member A and myosin light-chain phosphorylation. Exposure of wild-type, but not caspase-7-null, mice to hypercapnia increased airway contraction and resistance. Deletion of the Caspase-7 gene prevented hypercapnia-induced airway contractility, which was restored by lentiviral transfection of a miR-133a antagonist. In a cohort of patients with severe COPD, hypercapnic patients had higher airway resistance, which improved after correction of hypercapnia. Our data suggest a specific molecular mechanism by which the development of hypercapnia may drive COPD pathogenesis and progression.

The Antiviral Effects of Na,K-ATPase Inhibition: A Minireview.

Since being first described more than 60 years ago, Na,K-ATPase has been extensively studied, while novel concepts about its structure, physiology, and biological roles continue to be elucidated. Cardiac glycosides not only inhibit the pump function of Na,K-ATPase but also activate intracellular signal transduction pathways, which are important in many biological processes. Recently, antiviral effects have been described as a novel feature of Na,K-ATPase inhibition with the use of cardiac glycosides. Cardiac glycosides have been reported to be effective against both DNA viruses such as cytomegalovirus and herpes simplex and RNA viruses such as influenza, chikungunya, coronavirus, and respiratory syncytial virus, among others. Consequently, cardiac glycosides have emerged as potential broad-spectrum antiviral drugs, with the great advantage of targeting cell host proteins, which help to minimize resistance to antiviral treatments, making them a very promising strategy against human viral infections. Here, we review the effect of cardiac glycosides on viral biology and the mechanisms by which these drugs impair the replication of this array of different viruses.

Inflammatory Responses Regulating Alveolar Ion Transport during Pulmonary Infections.

The respiratory epithelium is lined by a tightly balanced fluid layer that allows normal O2 and CO2 exchange and maintains surface tension and host defense. To maintain alveolar fluid homeostasis, both the integrity of the alveolar-capillary barrier and the expression of epithelial ion channels and pumps are necessary to establish a vectorial ion gradient. However, during pulmonary infection, auto- and/or paracrine-acting mediators induce pathophysiological changes of the alveolar-capillary barrier, altered expression of epithelial Na,K-ATPase and of epithelial ion channels including epithelial sodium channel and cystic fibrosis membrane conductance regulator, leading to the accumulation of edema and impaired alveolar fluid clearance. These mediators include classical pro-inflammatory cytokines such as TGF-ß, TNF-α, interferons, or IL-1ß that are released upon bacterial challenge with Streptococcus pneumoniae, Klebsiella pneumoniae, or Mycoplasma pneumoniae as well as in viral infection with influenza A virus, pathogenic coronaviruses, or respiratory syncytial virus. Moreover, the pro-apoptotic mediator TNF-related apoptosis-inducing ligand, extracellular nucleotides, or reactive oxygen species impair epithelial ion channel expression and function. Interestingly, during bacterial infection, alterations of ion transport function may serve as an additional feedback loop on the respiratory inflammatory profile, further aggravating disease progression. These changes lead to edema formation and impair edema clearance which results in suboptimal gas exchange causing hypoxemia and hypercapnia. Recent preclinical studies suggest that modulation of the alveolar-capillary fluid homeostasis could represent novel therapeutic approaches to improve outcomes in infection-induced lung injury.

Effects of hypercapnia on the lung.

Gases are sensed by lung cells and can activate specific intracellular signalling pathways, and thus have physiological and pathophysiological effects. Carbon dioxide (CO2 ), a primary product of oxidative metabolism, can be sensed by eukaryotic cells eliciting specific responses via recently identified signalling pathways. However, the physiological and pathophysiological effects of high CO2 (hypercapnia) on the lungs and specific lung cells, which are the primary site of CO2 elimination, are incompletely understood. In this review, we provide a physiological and mechanistic perspective on the effects of hypercapnia on the lungs and discuss the recent understanding of CO2 modulation of the alveolar epithelial function (lung oedema clearance), epithelial cell repair, innate immunity and airway function.