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Chronic lung allograft dysfunction (CLAD) is a major complication after lung transplantation that results from a complex interplay of innate inflammatory and alloimmune factors, culminating in parenchymal and/or obliterative airway fibrosis. Excessive IL-17A signaling and chronic inflammation have been recognized as key factors in these pathological processes. Herein, we developed a model of repeated airway inflammation in mouse minor alloantigen-mismatched single-lung transplantation. Repeated intratracheal LPS instillations augmented pulmonary IL-17A expression. LPS also increased acute rejection, airway epithelial damage, and obliterative airway fibrosis, similar to human explanted lung allografts with antecedent episodes of airway infection. We then investigated the role of donor and recipient IL-17 receptor A (IL-17RA) in this context. Donor IL-17RA deficiency significantly attenuated acute rejection and CLAD features, whereas recipient IL-17RA deficiency only slightly reduced airway obliteration in LPS allografts. IL-17RA immunofluorescence positive staining was greater in human CLAD lungs compared with control human lung specimens, with localization to fibroblasts and myofibroblasts, which was also seen in mouse LPS allografts. Taken together, repeated airway inflammation after lung transplantation caused local airway epithelial damage, with persistent elevation of IL-17A and IL-17RA expression and particular involvement of IL-17RA on donor structural cells in development of fibrosis.

Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here, we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independently of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.

Aberrant fibroblast function plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, a devastating disease of unrelenting extracellular matrix deposition in response to lung injury. Platelet-derived growth factor α–positive (Pdgfra+) lipofibroblasts (LipoFBs) are essential for lung injury response and maintenance of a functional alveolar stem cell niche. Little is known about the effects of lung injury on LipoFB function. Here, we used single-cell RNA-Seq (scRNA-Seq) technology and PdgfraGFP lineage tracing to generate a transcriptomic profile of Pdgfra+ fibroblasts in normal and injured mouse lungs 14 days after bleomycin exposure, generating 11 unique transcriptomic clusters that segregated according to treatment. While normal and injured LipoFBs shared a common gene signature, injured LipoFBs acquired fibrogenic pathway activity with an attenuation of lipogenic pathways. In a 3D organoid model, injured Pdgfra+ fibroblast–supported organoids were morphologically distinct from those cultured with normal fibroblasts, and scRNA-Seq analysis suggested distinct transcriptomic changes in alveolar epithelia supported by injured Pdgfra+ fibroblasts. In summary, while LipoFBs in injured lung have not migrated from their niche and retain their lipogenic identity, they acquire a potentially reversible fibrogenic profile, which may alter the kinetics of epithelial regeneration and potentially contribute to dysregulated repair, leading to fibrosis.

Secondary lung infection by inhaled Staphylococcus aureus (SA) is a common and lethal event for individuals infected with influenza A virus (IAV). How IAV disrupts host defense to promote SA infection in lung alveoli, where fatal lung injury occurs, is not known. We addressed this issue using real-time determinations of alveolar responses to IAV in live, intact, perfused lungs. Our findings show that IAV infection blocked defensive alveolar wall liquid (AWL) secretion and induced airspace liquid absorption, thereby reversing normal alveolar liquid dynamics and inhibiting alveolar clearance of inhaled SA. Loss of AWL secretion resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in the alveolar epithelium, and airspace liquid absorption was caused by stimulation of the alveolar epithelial Na+ channel (ENaC). Loss of AWL secretion promoted alveolar stabilization of inhaled SA, but rescue of AWL secretion protected against alveolar SA stabilization and fatal SA-induced lung injury in IAV-infected mice. These findings reveal a central role for AWL secretion in alveolar defense against inhaled SA and identify AWL inhibition as a critical mechanism of IAV lung pathogenesis. AWL rescue may represent a new therapeutic approach for IAV-SA coinfection.

The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl– into the airway surface liquid through cystic fibrosis transmembrane conductance regulator (CFTR) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl– channels in their basolateral membrane. Disrupting barttin/Cl– channel function impaired liquid absorption, and overexpressing barttin/Cl– channels increased absorption. Together, apical CFTR and basolateral barttin/Cl– channels provide an electrically conductive pathway for Cl– flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.

A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ are implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrated that Yap and Taz were normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2-to-AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to an LPS injury prevented AT1 cell regeneration, led to intraalveolar collagen deposition, and resulted in persistent innate inflammation. These findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling.

Lung contusion and gastric aspiration (LC and GA) are major risk factors for developing acute respiratory distress following trauma. Hypoxia from lung injury is mainly regulated by hypoxia-inducible factor 1α (HIF-1α). Published data from our group indicate that HIF-1α regulation in airway epithelial cells (AEC) drives the acute inflammatory response following LC and GA. Metabolomic profiling and metabolic flux of Type II AEC following LC revealed marked increases in glycolytic and TCA intermediates in vivo and in vitro that were HIF-1α dependent. GLUT-1/4 expression was also increased in HIF-1α+/+ mice, suggesting that increased glucose entry may contribute to increased intermediates. Importantly, lactate incubation in vitro on Type II cells did not significantly increase the inflammatory byproduct IL-1β. Contrastingly, succinate had a direct proinflammatory effect on human small AEC by IL-1β generation in vitro. This effect was reversed by dimethylmalonate, suggesting an important role for succinate dehydrogenase in mediating HIF-1α effects. We confirmed the presence of the only known receptor for succinate binding, SUCNR1, on Type II AEC. These results support the hypothesis that succinate drives HIF-1α–mediated airway inflammation following LC. This is the first report to our knowledge of direct proinflammatory activation of succinate in nonimmune cells such as Type II AEC in direct lung injury models.

Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), cause severe endothelial dysfunction in the lung, and vascular endothelial growth factor (VEGF) is elevated in ARDS. We found that the levels of a VEGF-regulated microRNA, microRNA-1 (miR-1), were reduced in the lung endothelium after acute injury. Pulmonary endothelial cell–specific (EC-specific) overexpression of miR-1 protected the lung against cell death and barrier dysfunction in both murine and human models and increased the survival of mice after pneumonia-induced ALI. miR-1 had an intrinsic protective effect in pulmonary and other types of ECs; it inhibited apoptosis and necroptosis pathways and decreased capillary leak by protecting adherens and tight junctions. Comparative gene expression analysis and RISC recruitment assays identified miR-1 targets in the context of injury, including phosphodiesterase 5A (PDE5A), angiopoietin-2 (ANGPT2), CNKSR family member 3 (CNKSR3), and TNF-α–induced protein 2 (TNFAIP2). We validated miR-1–mediated regulation of ANGPT2 in both mouse and human ECs and found that in a 119-patient pneumonia cohort, miR-1 correlated inversely with ANGPT2. These findings illustrate a previously unknown role of miR-1 as a cytoprotective orchestrator of endothelial responses to acute injury with prognostic and therapeutic potential.

Reducing inflammatory damage and improving alveolar epithelium regeneration are two key approaches to promoting lung repair in acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Stimulation of cholinergic α7 nicotinic acetylcholine receptor (α7nAChR, coded by Chrna7) signaling could dampen lung inflammatory injury. However, whether activation of α7nAChR in alveolar type II (AT2) cells promotes alveolar epithelial injury repair and underlying mechanisms is elusive. Here, we found that α7nAChR was expressed on AT2 cells and was upregulated in response to LPS-induced ALI. Meanwhile, deletion of Chrna7 in AT2 cells impeded lung repair process and worsened lung inflammation in ALI. Using in vivo AT2 lineage–labeled mice and ex vivo AT2 cell–derived alveolar organoids, we demonstrated that activation of α7nAChR expressed on AT2 cells improved alveolar regeneration by promoting AT2 cells to proliferate and subsequently differentiate toward alveolar type I cells. Then, we screened out the WNT7B signaling pathway by the RNA-Seq analysis of in vivo AT2 lineage–labeled cells and further confirmed its indispensability for α7nAChR activation–mediated alveolar epithelial proliferation and differentiation. Thus, we have identified a potentially unrecognized pathway in which cholinergic α7nAChR signaling determines alveolar regeneration and repair, which might provide us a novel therapeutic target for combating ALI.

Alveolar epithelial type II (AEC2) cells strictly regulate lipid metabolism to maintain surfactant synthesis. Loss of AEC2 cell function and surfactant production are implicated in the pathogenesis of the smoking-related lung disease chronic obstructive pulmonary disease (COPD). Whether smoking alters lipid synthesis in AEC2 cells and whether altering lipid metabolism in AEC2 cells contributes to COPD development are unclear. In this study, high-throughput lipidomic analysis revealed increased lipid biosynthesis in AEC2 cells isolated from mice chronically exposed to cigarette smoke (CS). Mice with a targeted deletion of the de novo lipogenesis enzyme, fatty acid synthase (FASN), in AEC2 cells (FasniΔAEC2) exposed to CS exhibited higher bronchoalveolar lavage fluid (BALF) neutrophils, higher BALF protein, and more severe airspace enlargement. FasniΔAEC2 mice exposed to CS had lower levels of key surfactant phospholipids but higher levels of BALF ether phospholipids, sphingomyelins, and polyunsaturated fatty acid–containing phospholipids, as well as increased BALF surface tension. FasniΔAEC2 mice exposed to CS also had higher levels of protective ferroptosis markers in the lung. These data suggest that AEC2 cell FASN modulates the response of the lung to smoke by regulating the composition of the surfactant phospholipidome.

Optimal lung repair and regeneration are essential for recovery from viral infections, including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known whether the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections, including the need for hospitalization and mortality after discharge; this persists even after adjusting for common confounding factors. Furthermore, we use a combination of lung organoid assays, single-cell RNA sequencing, and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show that lung organoids have a functional circadian clock and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration — in tracheal cells via the Wnt/β-catenin pathway and through IL-1β in alveolar epithelial cells. We speculate that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.

Proline and its synthesis enzyme pyrroline-5-carboxylate reductase 1 (PYCR1) are implicated in epithelial-mesenchymal transition (EMT), yet how proline and PYCR1 function in allergic asthmatic airway remodeling via EMT has not yet been addressed to our knowledge. In the present study, increased levels of plasma proline and PYCR1 were observed in patients with asthma. Similarly, proline and PYCR1 in lung tissues were high in a murine allergic asthma model induced by house dust mites (HDMs). Pycr1 knockout decreased proline in lung tissues, with reduced airway remodeling and EMT. Mechanistically, loss of Pycr1 restrained HDM-induced EMT by modulating mitochondrial fission, metabolic reprogramming, and the AKT/mTORC1 and WNT3a/β-catenin signaling pathways in airway epithelial cells. Therapeutic inhibition of PYCR1 in wild-type mice disrupted HDM-induced airway inflammation and remodeling. Deprivation of exogenous proline relieved HDM-induced airway remodeling to some extent. Collectively, this study illuminates that proline and PYCR1 involved with airway remodeling in allergic asthma could be viable targets for asthma treatment.

During alveolar repair, alveolar type 2 (AT2) epithelial cell progenitors rapidly proliferate and differentiate into flat AT1 epithelial cells. Failure of normal alveolar repair mechanisms can lead to loss of alveolar structure (emphysema) or development of fibrosis, depending on the type and severity of injury. To test if β1-containing integrins are required during repair following acute injury, we administered E. coli lipopolysaccharide (LPS) by intratracheal injection to mice with a postdevelopmental deletion of β1 integrin in AT2 cells. While control mice recovered from LPS injury without structural abnormalities, β1-deficient mice had more severe inflammation and developed emphysema. In addition, recovering alveoli were repopulated with an abundance of rounded epithelial cells coexpressing AT2 epithelial, AT1 epithelial, and mixed intermediate cell state markers, with few mature type 1 cells. AT2 cells deficient in β1 showed persistently increased proliferation after injury, which was blocked by inhibiting NF-κB activation in these cells. Lineage tracing experiments revealed that β1-deficient AT2 cells failed to differentiate into mature AT1 epithelial cells. Together, these findings demonstrate that functional alveolar repair after injury with terminal alveolar epithelial differentiation requires β1-containing integrins.

BACKGROUND Despite guidelines promoting the prevention and aggressive treatment of ventilator-associated pneumonia (VAP), the importance of VAP as a driver of outcomes in mechanically ventilated patients, including patients with severe COVID-19, remains unclear. We aimed to determine the contribution of unsuccessful treatment of VAP to mortality for patients with severe pneumonia.METHODS We performed a single-center, prospective cohort study of 585 mechanically ventilated patients with severe pneumonia and respiratory failure, 190 of whom had COVID-19, who underwent at least 1 bronchoalveolar lavage. A panel of intensive care unit (ICU) physicians adjudicated the pneumonia episodes and endpoints on the basis of clinical and microbiological data. Given the relatively long ICU length of stay (LOS) among patients with COVID-19, we developed a machine-learning approach called CarpeDiem, which grouped similar ICU patient-days into clinical states based on electronic health record data.RESULTS CarpeDiem revealed that the long ICU LOS among patients with COVID-19 was attributable to long stays in clinical states characterized primarily by respiratory failure. While VAP was not associated with mortality overall, the mortality rate was higher for patients with 1 episode of unsuccessfully treated VAP compared with those with successfully treated VAP (76.4% versus 17.6%, P < 0.001). For all patients, including those with COVID-19, CarpeDiem demonstrated that unresolving VAP was associated with a transitions to clinical states associated with higher mortality.CONCLUSIONS Unsuccessful treatment of VAP is associated with higher mortality. The relatively long LOS for patients with COVID-19 was primarily due to prolonged respiratory failure, placing them at higher risk of VAP.FUNDING National Institute of Allergy and Infectious Diseases (NIAID), NIH grant U19AI135964; National Heart, Lung, and Blood Institute (NHLBI), NIH grants R01HL147575, R01HL149883, R01HL153122, R01HL153312, R01HL154686, R01HL158139, P01HL071643, and P01HL154998; National Heart, Lung, and Blood Institute (NHLBI), NIH training grants T32HL076139 and F32HL162377; National Institute on Aging (NIA), NIH grants K99AG068544, R21AG075423, and P01AG049665; National Library of Medicine (NLM), NIH grant R01LM013337; National Center for Advancing Translational Sciences (NCATS), NIH grant U01TR003528; Veterans Affairs grant I01CX001777; Chicago Biomedical Consortium grant; Northwestern University Dixon Translational Science Award; Simpson Querrey Lung Institute for Translational Science (SQLIFTS); Canning Thoracic Institute of Northwestern Medicine.

Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4+ T cell–mediated inflammation. However, the role of CD4+ T cells in promoting LTS fibrosis is unknown. The mTOR signaling pathways have been shown to regulate the T cell phenotype. Here we investigated the influence of mTOR signaling in CD4+ T cells on LTS pathogenesis. In this study, human LTS specimens revealed a higher population of CD4+ T cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and Th17 cells. Selective deletion of mTOR in CD4+ cells reduced Th17 cells and attenuated fibrosis, demonstrating CD4+ T cells’ pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased Th17 cells. In vitro, Th17 cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of Th17 cells. Collectively, mTOR signaling drove pathologic CD4+ T cell phenotypes in LTS, and targeting mTOR with sirolimus was effective at treating LTS through inhibition of profibrotic Th17 cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

Background Lung infections are among the most consequential manifestations of cystic fibrosis (CF) and are associated with reduced lung function and shortened survival. Drugs called CF transmembrane conductance regulator (CFTR) modulators improve activity of dysfunctional CFTR channels, which is the physiological defect causing CF. However, it is unclear how improved CFTR activity affects CF lung infections.Methods We performed a prospective, multicenter, observational study to measure the effect of the newest and most effective CFTR modulator, elexacaftor/tezacaftor/ivacaftor (ETI), on CF lung infections. We studied sputum from 236 people with CF during their first 6 months of ETI using bacterial cultures, PCR, and sequencing.Results Mean sputum densities of Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Achromobacter spp., and Burkholderia spp. decreased by 2–3 log10 CFU/mL after 1 month of ETI. However, most participants remained culture positive for the pathogens cultured from their sputum before starting ETI. In those becoming culture negative after ETI, the pathogens present before treatment were often still detectable by PCR months after sputum converted to culture negative. Sequence-based analyses confirmed large reductions in CF pathogen genera, but other bacteria detected in sputum were largely unchanged. ETI treatment increased average sputum bacterial diversity and produced consistent shifts in sputum bacterial composition. However, these changes were caused by ETI-mediated decreases in CF pathogen abundance rather than changes in other bacteria.Conclusions Treatment with the most effective CFTR modulator currently available produced large and rapid reductions in traditional CF pathogens in sputum, but most participants remain infected with the pathogens present before modulator treatment.Trial Registration ClinicalTrials.gov NCT04038047.Funding The Cystic Fibrosis Foundation and the NIH.

Emerging data indicate an association between environmental heavy metal exposure and lung disease, including lower respiratory tract infections (LRTIs). Here, we show by single-cell RNA sequencing an increase in Pparg gene expression in lung macrophages from mice exposed to cadmium and/or infected with Streptococcus pneumoniae. However, the heavy metal cadmium or infection mediated an inhibitory posttranslational modification of peroxisome proliferator-activated receptor γ (PPARγ) to exacerbate LRTIs. Cadmium and infection increased ERK activation to regulate PPARγ degradation in monocyte-derived macrophages. Mice harboring a conditional deletion of Pparg in monocyte-derived macrophages had more severe S. pneumoniae infection after cadmium exposure, showed greater lung injury, and had increased mortality. Inhibition of ERK activation with BVD-523 protected mice from lung injury after cadmium exposure or infection. Moreover, individuals residing in areas of high air cadmium levels had increased cadmium concentration in their bronchoalveolar lavage (BAL) fluid, increased barrier dysfunction, and showed PPARγ inhibition that was mediated, at least in part, by ERK activation in isolated BAL cells. These observations suggest that impaired activation of PPARγ in monocyte-derived macrophages exacerbates lung injury and the severity of LRTIs.

BACKGROUND Lower respiratory tract infection (LRTI) is a leading cause of death in children worldwide. LRTI diagnosis is challenging because noninfectious respiratory illnesses appear clinically similar and because existing microbiologic tests are often falsely negative or detect incidentally carried microbes, resulting in antimicrobial overuse and adverse outcomes. Lower airway metagenomics has the potential to detect host and microbial signatures of LRTI. Whether it can be applied at scale and in a pediatric population to enable improved diagnosis and treatment remains unclear.METHODS We used tracheal aspirate RNA-Seq to profile host gene expression and respiratory microbiota in 261 children with acute respiratory failure. We developed a gene expression classifier for LRTI by training on patients with an established diagnosis of LRTI (n = 117) or of noninfectious respiratory failure (n = 50). We then developed a classifier that integrates the host LRTI probability, abundance of respiratory viruses, and dominance in the lung microbiome of bacteria/fungi considered pathogenic by a rules-based algorithm.RESULTS The host classifier achieved a median AUC of 0.967 by cross-validation, driven by activation markers of T cells, alveolar macrophages, and the interferon response. The integrated classifier achieved a median AUC of 0.986 and increased the confidence of patient classifications. When applied to patients with an uncertain diagnosis (n = 94), the integrated classifier indicated LRTI in 52% of cases and nominated likely causal pathogens in 98% of those.CONCLUSION Lower airway metagenomics enables accurate LRTI diagnosis and pathogen identification in a heterogeneous cohort of critically ill children through integration of host, pathogen, and microbiome features.FUNDING Support for this study was provided by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Heart, Lung, and Blood Institute (UG1HD083171, 1R01HL124103, UG1HD049983, UG01HD049934, UG1HD083170, UG1HD050096, UG1HD63108, UG1HD083116, UG1HD083166, UG1HD049981, K23HL138461, and 5R01HL155418) as well as by the Chan Zuckerberg Biohub.

Cholesterol-25-hydroxylase (CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25HC), is most highly expressed in the lung, but its role in lung biology is poorly defined. Recently, we reported that Ch25h is induced in monocyte-derived macrophages recruited to the airspace during resolution of lung inflammation and that 25HC promotes liver X receptor–dependent (LXR-dependent) clearance of apoptotic neutrophils by these cells. Ch25h and 25HC are, however, also robustly induced by lung-resident cells during the early hours of lung inflammation, suggesting additional cellular sources and targets. Here, using Ch25h–/– mice and exogenous 25HC in lung injury models, we provide evidence that 25HC sustains proinflammatory cytokines in the airspace and augments lung injury, at least in part, by inducing LXR-independent endoplasmic reticulum stress and endothelial leak. Suggesting an autocrine effect in endothelium, inhaled LPS upregulates pulmonary endothelial Ch25h, and non-hematopoietic Ch25h deletion is sufficient to confer lung protection. In patients with acute respiratory distress syndrome, airspace 25HC and alveolar macrophage CH25H were associated with markers of microvascular leak, endothelial activation, endoplasmic reticulum stress, inflammation, and clinical severity. Taken together, our findings suggest that 25HC deriving from and acting on different cell types in the lung communicates distinct, temporal LXR-independent and -dependent signals to regulate inflammatory homeostasis.

Mitochondrial dysfunction at birth predicts bronchopulmonary dysplasia (BPD) in extremely low–birth weight (ELBW) infants. Recently, nebulized thyroid hormone (TH), given as triiodothyronine (T3), was noted to decrease pulmonary fibrosis in adult animals through improved mitochondrial function. In this study, we tested the hypothesis that TH may have similar effects on hyperoxia-induced neonatal lung injury and mitochondrial dysfunction by testing whether i.n. T3 decreases neonatal hyperoxic lung injury in newborn mice; whether T3 improves mitochondrial function in lung homogenates, neonatal murine lung fibroblasts (NMLFs), and umbilical cord–derived mesenchymal stem cells (UC-MSCs) obtained from ELBW infants; and whether neonatal hypothyroxinemia is associated with BPD in ELBW infants. We found that inhaled T3 (given i.n.) attenuated hyperoxia-induced lung injury and mitochondrial dysfunction in newborn mice. T3 also reduced bioenergetic deficits in UC-MSCs obtained from both infants with no or mild BPD and those with moderate to severe BPD. T3 also increased the content of peroxisome proliferator–activated receptor γ coactivator 1α in lung homogenates of mice exposed to hyperoxia as well as mitochondrial potential in both NMLFs and UC-MSCs. ELBW infants who died or developed moderate to severe BPD had lower total T4 (TT4) compared with survivors with no or mild BPD. In conclusion, TH signaling and function may play a critical role in neonatal lung injury, and inhaled T3 supplementation may be useful as a therapeutic strategy for BPD.

BACKGROUND Fibrocytes are BM-derived circulating cells that traffic to the injured lungs and contribute to fibrogenesis. The mTOR inhibitor, sirolimus, inhibits fibrocyte CXCR4 expression, reducing fibrocyte traffic and attenuating lung fibrosis in animal models. We sought to test the hypothesis that short-term treatment with sirolimus reduces the concentration of CXCR4+ circulating fibrocytes in patients with idiopathic pulmonary fibrosis (IPF).METHODS We conducted a short-term randomized double-blind placebo-controlled crossover pilot trial to assess the safety and tolerability of sirolimus in IPF. Participants were randomly assigned to sirolimus or placebo for approximately 6 weeks, and after a 4-week washout, they were assigned to the alternate treatment. Toxicity, lung function, and the concentration of circulating fibrocytes were measured before and after each treatment.RESULTS In the 28 study participants, sirolimus resulted in a statistically significant 35% decline in the concentration of total fibrocytes, 34% decline in CXCR4+ fibrocytes, and 42% decline in fibrocytes expressing α-smooth muscle actin, but no significant change in these populations occurred on placebo. Respiratory adverse events occurred more frequently during treatment with placebo than sirolimus; the incidence of adverse events and drug tolerability did not otherwise differ during therapy with drug and placebo. Lung function was unaffected by either treatment, with the exception of a small decline in gas transfer during treatment with placebo.CONCLUSION As compared with placebo, short-term treatment with sirolimus resulted in reduction of circulating fibrocyte concentrations in participants with IPF, with an acceptable safety profile.TRIAL REGISTRATION ClinicalTrials.gov, accession no. NCT01462006.FUNDING NIH R01HL098329 and American Heart Association 18TPA34170486.

BACKGROUND The fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). Th cells orchestrate immune responses against fungi, but the types of A. fumigatus–specific Th cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized.METHODS We used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls.RESULTS We show that clonally expanded, high-avidity A. fumigatus–specific effector Th cells, which were absent in healthy donors, developed in pwCF. Individual patients were characterized by distinct Th1-, Th2-, or Th17-dominated responses that remained stable over several years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2 cells that cross-recognize various filamentous fungi.CONCLUSION Our data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross-reactive Th2 cells as a potential risk factor for ABPA.FUNDING German Research Foundation (DFG), under Germany’s Excellence Strategy (EXC 2167-390884018 “Precision Medicine in Chronic Inflammation” and EXC 2051-390713860 “Balance of the Microverse”); Oskar Helene Heim Stiftung; Christiane Herzog Stiftung; Mukoviszidose Institut gGmb; German Cystic Fibrosis Association Mukoviszidose e.V; German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath (BMBF 03ZZ0838A+B).

Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a deadly disease with limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECIIs. Deficiency of CYB5R3 in AECIIs led to sustained activation of the pro-fibrotic factor TGF-β1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and the sGC/cGMP/protein kinase G axis that modulates activation of the TGF-β1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECIIs. Taken together, these results show that CYB5R3 in AECIIs is required to maintain resilience after lung injury and fibrosis and that therapeutic manipulation of the sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.

Chronic lung allograft dysfunction (CLAD) is the leading cause of death in lung transplant recipients. CLAD is characterized clinically by a persistent decline in pulmonary function and histologically by the development of airway-centered fibrosis known as bronchiolitis obliterans. There are no approved therapies to treat CLAD, and the mechanisms underlying its development remain poorly understood. We performed single-cell RNA-Seq and spatial transcriptomic analysis of explanted tissues from human lung recipients with CLAD, and we performed independent validation studies to identify an important role of Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling in airway epithelial cells that contributes to airway-specific alloimmune injury. Specifically, we established that activation of JAK-STAT signaling leads to upregulation of major histocompatibility complex 1 (MHC-I) in airway basal cells, an important airway epithelial progenitor population, which leads to cytotoxic T cell–mediated basal cell death. This study provides mechanistic insight into the cell-to-cell interactions driving airway-centric alloimmune injury in CLAD, suggesting a potentially novel therapeutic strategy for CLAD prevention or treatment.

Anti-CD36 Abs have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36 Ab–mediated TRALI, and potential therapies have not yet been identified. Here, we developed a murine model of anti-CD36 Ab–mediated TRALI to address these questions. Administration of mouse mAb against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab′)2 fragments, induced severe TRALI in Cd36+/+ male mice. Predepletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 Abs increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36–mediated TRALI. Administration of GZ1 F(ab′)2, antioxidant (N-acetyl cysteine, NAC), or C5 blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36–mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab′)2 after TRALI induction, significant improvement was achieved when mice were treated postinduction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36.

In the progression phase of idiopathic pulmonary fibrosis (IPF), the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a coculture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB populations adopted distinct pro-fibrotic cell differentiation states upon cocultivation, resembling specific cell populations that were previously identified in lungs of patients with IPF. Transcriptomic analysis revealed active NF-κB signaling early in the cocultured EC and FB, and the identified NF-κB expression signatures were found in “HAS1 High FB” and “PLIN2+ FB” populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6, interleukin-8, and CXC chemokine ligand 6 cytokine secretion, as well as collagen α-1(I) chain and α–smooth muscle actin accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.

Patients with progressive fibrosing interstitial lung diseases (PF-ILDs) carry a poor prognosis and have limited therapeutic options. A hallmark feature is fibroblast resistance to apoptosis, leading to their persistence, accumulation, and excessive deposition of extracellular matrix. A complex balance of the B cell lymphoma 2 (BCL-2) protein family controlling the intrinsic pathway of apoptosis and fibroblast reliance on antiapoptotic proteins has been hypothesized to contribute to this resistant phenotype. Examination of lung tissue from patients with PF-ILD (idiopathic pulmonary fibrosis and silicosis) and mice with PF-ILD (repetitive bleomycin and silicosis) showed increased expression of antiapoptotic BCL-2 family members in α–smooth muscle actin–positive fibroblasts, suggesting that fibroblasts from fibrotic lungs may exhibit increased susceptibility to inhibition of antiapoptotic BCL-2 family members BCL-2, BCL-XL, and BCL-W with the BH3 mimetic ABT-263. We used 2 murine models of PF-ILD to test the efficacy of ABT-263 in reversing established persistent pulmonary fibrosis. Treatment with ABT-263 induced fibroblast apoptosis, decreased fibroblast numbers, and reduced lung collagen levels, radiographic disease, and histologically evident fibrosis. Our studies provide insight into how fibroblasts gain resistance to apoptosis and become sensitive to the therapeutic inhibition of antiapoptotic proteins. By targeting profibrotic fibroblasts, ABT-263 offers a promising therapeutic option for PF-ILDs.

Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here — using a mouse model of hypercapnia exposure, cell lineage tracing, spatial transcriptomics, and 3D cultures — we show that hypercapnia limits β-catenin signaling in alveolar type II (AT2) cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+ fibroblasts from those maintaining AT2 progenitor activity toward those that antagonize β-catenin signaling, thereby limiting progenitor function. Constitutive activation of β-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in patients with hypercapnia may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier and increasing lung flooding, ventilator dependency, and mortality.

Hypoxia is a sentinel feature of idiopathic pulmonary fibrosis (IPF). The IPF microenvironment contains high lactate levels, and hypoxia enhances cellular lactate production. Lactate, acting through the GPR81 lactate receptor, serves as a signal molecule regulating cellular processes. We previously identified intrinsically fibrogenic mesenchymal progenitor cells (MPCs) that drive fibrosis in the lungs of patients with IPF. However, whether hypoxia enhances IPF MPC fibrogenicity is unclear. We hypothesized that hypoxia increases IPF MPC fibrogenicity via lactate and its cognate receptor GPR81. Here we show that hypoxia promotes IPF MPC self-renewal. The mechanism involves hypoxia-mediated enhancement of LDHA function and lactate production and release. Hypoxia also increases HIF1α levels, and this increase in turn augments the expression of GPR81. Exogenous lactate operating through GPR81 promotes IPF MPC self-renewal. IHC analysis of IPF lung tissue demonstrates IPF MPCs expressing GPR81 and hypoxic markers on the periphery of the fibroblastic focus. We show that hypoxia enhances IPF MPC fibrogenicity in vivo. We demonstrate that knockdown of GPR81 inhibits hypoxia-induced IPF MPC self-renewal in vitro and attenuates hypoxia-induced IPF MPC fibrogenicity in vivo. Our data demonstrate that hypoxia creates a feed-forward loop that augments IPF MPC fibrogenicity via the lactate/GPR81/HIF1α pathway.

In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-Seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all aa and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest that elevated glucose and proline avidity underlie increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease. Recent findings have shown a marked metabolic reprogramming associated with changes in mitochondrial homeostasis and autophagy during pulmonary fibrosis. The microRNA-33 (miR-33) family of microRNAs (miRNAs) encoded within the introns of sterol regulatory element binding protein (SREBP) genes are master regulators of sterol and fatty acid (FA) metabolism. miR-33 controls macrophage immunometabolic response and enhances mitochondrial biogenesis, FA oxidation, and cholesterol efflux. Here, we show that miR-33 levels are increased in bronchoalveolar lavage (BAL) cells isolated from patients with IPF compared with healthy controls. We demonstrate that specific genetic ablation of miR-33 in macrophages protects against bleomycin-induced pulmonary fibrosis. The absence of miR-33 in macrophages improves mitochondrial homeostasis and increases autophagy while decreasing inflammatory response after bleomycin injury. Notably, pharmacological inhibition of miR-33 in macrophages via administration of anti–miR-33 peptide nucleic acids (PNA-33) attenuates fibrosis in different in vivo and ex vivo mice and human models of pulmonary fibrosis. These studies elucidate a major role of miR-33 in macrophages in the regulation of pulmonary fibrosis and uncover a potentially novel therapeutic approach to treat this disease.

KRAS is one of the most frequently activated oncogenes in human cancers. While the role of KRAS mutation in tumorigenesis and tumor maintenance has been extensively studied, the relationship between KRAS and the tumor immune microenvironment is not fully understood. Herein, we identified a novel role of KRAS in driving tumor evasion from innate immune surveillance. In lung adenocarcinoma patient samples and Kras-driven genetic mouse models of lung cancer, mutant KRAS activated the expression of cluster of differentiation 47 (CD47), an antiphagocytic signal in cancer cells, leading to decreased phagocytosis of cancer cells by macrophages. Mechanistically, mutant KRAS activated PI3K-STAT3 signaling, which restrained miR-34a expression and relieved the post-transcriptional repression of miR-34a on CD47. In three independent lung cancer patient cohorts, KRAS mutation status positively correlated with CD47 expression. Therapeutically, disruption of the KRAS-CD47 signaling axis with KRAS siRNA, the KRASG12C inhibitor AMG 510 or miR-34a mimic suppressed CD47 expression, enhanced the phagocytic capacity of macrophages and restored innate immune surveillance. Our results revealed a direct mechanistic link between active KRAS and innate immune evasion and identified CD47 as a major effector underlying KRAS-mediated immunosuppressive tumor microenvironment. [ABSTRACT FROM AUTHOR]

The impact of diet-induced maternal obesity on offspring airway hyperresponsiveness was studied in a diversity outbred mouse model that mirrors human genetic diversity. Female mice were started on high-fat or regular diet 8 weeks before breeding and throughout pregnancy and lactation. After weaning, all offspring were fed a regular diet. By 12 weeks, body weight and fat were increased in offspring of high-fat diet-fed dams, which was accompanied by metabolic dysfunction and hyperinsulinemia. This was followed by increased epithelial sensory innervation and increased bronchoconstriction to inhaled 5-hydroxytryptamine at 16 weeks. Bronchoconstriction was nerve mediated and blocked by vagotomy or atropine. A high-fat diet before pregnancy exerted the most influence on offspring airway physiology. Maternal obesity induced metabolic dysfunction and hyperinsulinemia, resulting in hyperinnervation and subsequent increased reflex-mediated hyperresponsiveness in their offspring. This is relevant to our understanding of asthma inheritance, considering the genetic diversity of humans.

Both CO2 retention, or hypercapnia, and skeletal muscle dysfunction predict higher mortality in critically ill patients. Mechanistically, muscle injury and reduced myogenesis contribute to critical illness myopathy, and while hypercapnia causes muscle wasting, no research has been conducted on hypercapnia-driven dysfunctional myogenesis in vivo. Autophagy flux regulates myogenesis by supporting skeletal muscle stem cell - satellite cell - activation, and previous data suggest that hypercapnia inhibits autophagy. We tested whether hypercapnia worsens satellite cell autophagy flux and myogenic potential and if autophagy induction reverses these deficits. Satellite cell transplantation and lineage-tracing experiments showed that hypercapnia undermined satellite cells' activation, replication, and myogenic capacity. Bulk and single-cell sequencing analyses indicated that hypercapnia disrupts autophagy, senescence, and other satellite cell programs. Autophagy activation was reduced in hypercapnic cultured myoblasts, and autophagy genetic knockdown phenocopied these changes in vitro. Rapamycin stimulation led to AMPK activation and downregulation of the mTOR pathway, which are both associated with accelerated autophagy flux and cell replication. Moreover, hypercapnic mice receiving rapamycin showed improved satellite cell autophagy flux, activation, replication rate, and posttransplantation myogenic capacity. In conclusion, we have shown that hypercapnia interferes with satellite cell activation, autophagy flux, and myogenesis, and systemic rapamycin administration improves these outcomes.

Determining how alveoli are formed and maintained is critical to understanding lung organogenesis and regeneration after injury. To study the cellular dynamics of this critical stage of lung development, we have used scanned oblique-plane illumination microscopy of living lung slices to observe alveologenesis in real time at high resolution over several days. Contrary to the prevailing notion that alveologenesis occurs by airspace subdivision via ingrowing septa, we find that alveoli form by ballooning epithelial outgrowth supported by contracting mesenchymal ring structures. Systematic analysis has produced a computational model of finely timed cellular structural changes that drive normal alveologenesis. With this model, we can now quantify how perturbing known regulatory intercellular signaling pathways and cell migration processes effects alveologenesis. In the future, this new paradigm and platform can be leveraged for mechanistic studies and screening for therapies to promote lung regeneration.

Pulmonary surfactant is a lipoprotein complex lining the alveolar surface to decrease the surface tension and facilitate inspiration. Surfactant deficiency is often seen in premature infants and in children and adults with respiratory distress syndrome. Mechanical stretch of alveolar type 2 epithelial (AT2) cells during lung expansion is the primary physiological factor that stimulates surfactant secretion; however, it is unclear whether there is a mechanosensor dedicated to this process. Here, we show that loss of the mechanosensitive channels TMEM63A and TMEM63B (TMEM63A/B) resulted in atelectasis and respiratory failure in mice due to a deficit of surfactant secretion. TMEM63A/B were predominantly localized at the limiting membrane of the lamellar body (LB), a lysosome-related organelle that stores pulmonary surfactant and ATP in AT2 cells. Activation of TMEM63A/B channels during cell stretch facilitated the release of surfactant and ATP from LBs fused with the plasma membrane. The released ATP evoked Ca2+ signaling in AT2 cells and potentiated exocytic fusion of more LBs. Our study uncovered a vital physiological function of TMEM63 mechanosensitive channels in preparing the lungs for the first breath at birth and maintaining respiration throughout life.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder of endosomal protein trafficking associated with pulmonary fibrosis in specific subtypes, including HPS-1 and HPS-2. Single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity while double mutant HPS1/2 mice exhibit spontaneous fibrosis with aging, which has been attributed to HPS mutations in alveolar epithelial type II (AT2) cells. We utilized HPS mouse models and human lung tissue to investigate mechanisms of AT2 cell dysfunction driving fibrotic remodeling in HPS. Starting at 8 weeks of age, HPS mice exhibited progressive loss of AT2 cell numbers. HPS AT2 cell function was impaired ex vivo and in vivo. Incorporating AT2 cell lineage tracing in HPS mice, we observed aberrant differentiation with increased AT2-derived alveolar epithelial type I cells. Transcriptomic analysis of HPS AT2 cells revealed elevated expression of genes associated with aberrant differentiation and p53 activation. Lineage tracing and organoid modeling studies demonstrated that HPS AT2 cells were primed to persist in a Krt8+ reprogrammed transitional state, mediated by p53 activity. Intrinsic AT2 progenitor cell dysfunction and p53 pathway dysregulation are novel mechanisms of disease in HPS-related pulmonary fibrosis, with the potential for early targeted intervention before the onset of fibrotic lung disease.

Red blood cells (RBCs), traditionally recognized for their role in transporting oxygen, play a pivotal role in the body's immune response by expressing TLR9 and scavenging excess host cell-free DNA. DNA capture by RBCs leads to accelerated RBC clearance and triggers inflammation. Whether RBCs can also acquire microbial DNA during infections is unknown. Murine RBCs acquire microbial DNA in vitro and bacterial-DNA-induced macrophage activation was augmented by WT but not Tlr9-deleted RBCs. In a mouse model of polymicrobial sepsis, RBC-bound bacterial DNA was elevated in WT but not in erythroid Tlr9-deleted mice. Plasma cytokine analysis in these mice revealed distinct sepsis clusters characterized by persistent hypothermia and hyperinflammation in the most severely affected subjects. RBC-Tlr9 deletion attenuated plasma and tissue IL-6 production in the most severe group. Parallel findings in human subjects confirmed that RBCs from septic patients harbored more bacterial DNA compared to healthy individuals. Further analysis through 16S sequencing of RBC-bound DNA illustrated distinct microbial communities, with RBC-bound DNA composition correlating with plasma IL-6 in patients with sepsis. Collectively, these findings unveil RBCs as overlooked reservoirs and couriers of microbial DNA, capable of influencing host inflammatory responses in sepsis.

Airway remodeling is a critical factor determining the pathogenesis and treatment sensitivity of severe asthma (SA) or uncontrolled asthma (UA). The activation of epithelial-mesenchymal trophic units (EMTUs) regulated by airway epithelial cells (AECs) has been proven to induce airway remodeling directly. However, the triggers for EMTU activation and the underlying mechanism of airway remodeling are not fully elucidated. Here, we screened the differentially expressed gene cathepsin C (CTSC; also known as dipeptidyl peptidase 1 [DPP-1]) in epithelia of patients with SA and UA using RNA-sequencing data and further verified the increased expression of CTSC in induced sputum of patients with asthma, which was positively correlated with severity and airway remodeling. Moreover, direct instillation of exogenous CTSC induced airway remodeling. Genetic inhibition of CTSC suppressed EMTU activation and airway remodeling in two asthma models with airway remodeling. Mechanistically, increased secretion of CTSC from AECs induced EMTU activation through the p38-mediated pathway, further inducing airway remodeling. Meanwhile, inhibition of CTSC also reduced the infiltration of inflammatory cells and the production of inflammatory factors in the lungs of asthmatic mice. Consequently, targeting CTSC with compound AZD7986 protected against airway inflammation, EMTU activation, and remodeling in the asthma model. Based on the dual effects of CTSC on airway inflammation and remodeling, CTSC is a potential biomarker and therapeutic target for SA or UA.

Hermansky-Pudlak syndrome (HPS), particularly types 1 and 4, is characterized by progressive pulmonary fibrosis, a major cause of morbidity and mortality. However, the precise mechanisms driving pulmonary fibrosis in HPS are not fully elucidated. Our previous studies suggested that CHI3L1-driven fibroproliferation may be a notable factor in HPS-associated fibrosis. This study aimed to explore the role of CHI3L1-CRTH2 interaction on type 2 innate lymphoid cells (ILC2s) and explored the potential contribution of ILC2-fibroblast crosstalk in the development of pulmonary fibrosis in HPS. We identified ILC2s in lung tissues from patients with idiopathic pulmonary fibrosis and HPS. Using bleomycin-challenged WT and Hps1-/- mice, we observed that ILC2s were recruited and appeared to contribute to fibrosis development in the Hps1-/- mice, with CRTH2 playing a notable role in ILC2 accumulation. We sorted ILC2s, profiled fibrosis-related genes and mediators, and conducted coculture experiments with primary lung ILC2s and fibroblasts. Our findings suggest that ILC2s may directly stimulate the proliferation and differentiation of primary lung fibroblasts partially through amphiregulin-EGFR-dependent mechanisms. Additionally, specific overexpression of CHI3L1 in the ILC2 population using the IL-7Rcre driver, which was associated with increased fibroproliferation, indicates that ILC2-mediated, CRTH2-dependent mechanisms might contribute to optimal CHI3L1-induced fibroproliferative repair in HPS-associated pulmonary fibrosis.

Radiation-induced lung injury (RILI) initiates radiation pneumonitis and progresses to fibrosis as the main side effect experienced by patients with lung cancer treated with radiotherapy. There is no effective drug for RILI. Sustained vascular activation is a major contributor to the establishment of chronic disease. Here, using a whole thoracic irradiation (WTI) mouse model, we investigated the mechanisms and effectiveness of thrombopoietin mimetic (TPOm) for preventing RILI. We demonstrated that administering TPOm 24 hours before irradiation decreased histologic lung injury score, apoptosis, vascular permeability, expression of proinflammatory cytokines, and neutrophil infiltration in the lungs of mice 2 weeks after WTI. We described the expression of c-MPL, a TPO receptor, in mouse primary pulmonary microvascular endothelial cells, showing that TPOm reduced endothelial cell-neutrophil adhesion by inhibiting ICAM-1 expression. Seven months after WTI, TPOm-treated lung exhibited less collagen deposition and expression of MMP-9, TIMP-1, IL-6, TGF-β, and p21. Moreover, TPOm improved lung vascular structure, lung density, and respiration rate, leading to a prolonged survival time after WTI. Single-cell RNA sequencing analysis of lungs 2 weeks after WTI revealed that TPOm shifted populations of capillary endothelial cells toward a less activated and more homeostatic phenotype. Taken together, TPOm is protective for RILI by inhibiting endothelial cell activation.

Transcriptomic analyses have advanced the understanding of complex disease pathophysiology including chronic obstructive pulmonary disease (COPD). However, identifying relevant biologic causative factors has been limited by the integration of high dimensionality data. COPD is characterized by lung destruction and inflammation, with smoke exposure being a major risk factor. To define previously unknown biological mechanisms in COPD, we utilized unsupervised and supervised interpretable machine learning analyses of single-cell RNA-Seq data from the mouse smoke-exposure model to identify significant latent factors (context-specific coexpression modules) impacting pathophysiology. The machine learning transcriptomic signatures coupled to protein networks uncovered a reduction in network complexity and new biological alterations in actin-associated gelsolin (GSN), which was transcriptionally linked to disease state. GSN was altered in airway epithelial cells in the mouse model and in human COPD. GSN was increased in plasma from patients with COPD, and smoke exposure resulted in enhanced GSN release from airway cells from patients with COPD. This method provides insights into rewiring of transcriptional networks that are associated with COPD pathogenesis and provides a translational analytical platform for other diseases.

Pulmonary fibrosis is characterized by stiffening of the extracellular matrix. Fibroblasts migrate in the direction of greater stiffness, a phenomenon termed durotaxis. The mechanically guided fibroblast migration could be a crucial step in the progression of lung fibrosis. In this study, we found primary human lung fibroblasts sense increasing matrix stiffness with a change of mitochondrial dynamics in favor of mitochondrial fission and increased production of ATP. Mitochondria polarize in the direction of a physiologically relevant stiffness gradient, with conspicuous localization to the leading edge, primarily lamellipodia and filopodia, of migrating lung fibroblasts. Matrix stiffness–regulated mitochondrial fission and durotactic lung fibroblast migration are mediated by a dynamin-related protein 1/mitochondrial fission factor–dependent (DRP1/MFF-dependent) pathway. Importantly, we found that the DRP1/MFF pathway is activated in fibrotic lung myofibroblasts in both human IPF and bleomycin-induced mouse lung fibrosis. These findings suggest that energy-producing mitochondria need to be sectioned via fission and repositioned in durotactic lung fibroblasts to meet the higher energy demand. This represents a potentially new mechanism through which mitochondria may contribute to the progression of fibrotic lung diseases. Inhibition of durotactic migration of lung fibroblasts may play an important role in preventing the progression of human idiopathic pulmonary fibrosis.

Dysfunction of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, is implicated in pulmonary disease pathogenesis, highlighting the importance of human in vitro models. However, AEC2-like cells in culture have yet to be directly compared to their in vivo counterparts at single-cell resolution. Here, we performed head-to-head comparisons among the transcriptomes of primary (1°) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell–derived AEC2s (iAEC2s). We found each population occupied a distinct transcriptomic space with cultured AEC2s (1° and iAEC2s) exhibiting similarities to and differences from freshly purified 1° cells. Across each cell type, we found an inverse relationship between proliferative and maturation states, with preculture 1° AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2s did not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s cocultured with fibroblasts acquired a transitional cell state described in mice and humans to arise during fibrosis or following injury. Hence, we provide direct comparisons of the transcriptomic programs of 1° and engineered AEC2s, 2 in vitro models that can be harnessed to study human lung health and disease.

Keratin expression dynamically changes in airway basal cells (BCs) after acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In bronchiolitis obliterans (BO) after lung transplantation, BC clonogenicity declines, which is associated with a switch from keratin15 (Krt15) to keratin14 (Krt14). We investigated these keratins’ roles using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs did not differentiate into club and ciliated cells but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo after injury. Krt14, but not Krt15, bound the tumor suppressor stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, whereas dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts with Krt14-KO phenotype and resembles the phenotype in BO with decreased clonogenicity, increased Krt14, and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior, which is relevant in chronic disease states like BO.

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.

Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.