IgG hexamers initiate complement-dependent acute lung injury.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, in reactions to transfusions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. Harmful antibodies often activate the complement cascade. A model for how IgG antibodies trigger complement activation involves interactions between IgG Fc domains driving the assembly of IgG hexamer structures that activate C1 complexes. The importance of IgG hexamers in initiating injury responses was not clear, so we tested their relevance in a mouse model of alloantibody- and complement-mediated acute lung injury. We used 3 approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer "decoy" therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate an in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

IgG hexamers initiate acute lung injury.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, transfusion reactions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. A previously overlooked step in complement activation by IgG antibodies has been elucidated involving interactions between IgG Fc domains that enable assembly of IgG hexamers, which can optimally activate the complement cascade. Here, we tested the in vivo relevance of IgG hexamers in a complement-dependent alloantibody model of acute lung injury. We used three approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from Staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer 'decoy' therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate a direct in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

Decoding functional hematopoietic progenitor cells in the adult human lung.

The bone marrow is the main site of blood cell production in adults, however, rare pools of hematopoietic stem and progenitor cells with self-renewal and differentiation potential have been found in extramedullary organs. The lung is primarily known for its role in gas exchange but has recently been described as a site of blood production in mice. Here, we show that functional hematopoietic precursors reside in the extravascular spaces of the human lung, at a frequency similar to the bone marrow, and are capable of proliferation and engraftment. The organ-specific gene signature of pulmonary and medullary CD34+ hematopoietic progenitors indicates greater baseline activation of immune, megakaryocyte/platelet and erythroid-related pathways in lung progenitors. Spatial transcriptomics mapped blood progenitors in the lung to a vascular-rich alveolar interstitium niche. These results identify the lung as a pool for uniquely programmed blood stem and progenitor cells with the potential to support hematopoiesis in humans.

NKG2D receptor activation drives primary graft dysfunction severity and poor lung transplantation outcomes.

Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first posttransplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. NK cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. These data support an aberrant NK cell/neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.

ADAM8 signaling drives neutrophil migration and ARDS severity.

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8-/- mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

Complement activation on endothelium initiates antibody-mediated acute lung injury.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.

Cystic fibrosis transmembrane conductance regulator dysfunction in platelets drives lung hyperinflammation.

Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in CF, and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets and TRPC6 are what we believe to be novel therapeutic targets in the treatment of CF lung disease.

Mitochondrial DNA Stimulates TLR9-Dependent Neutrophil Extracellular Trap Formation in Primary Graft Dysfunction.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.