Probes for INS

Variants of NHL repeat-containing protein 2 (NHLRC2) have been associated with severe fibrotic interstitial lung disease in early childhood and NHLRC2 has been listed as a differentially expressed gene between rapidly and slowly progressing idiopathic pulmonary fibrosis (IPF) patients. However, its cell type-specific localization in human lung tissue is unknown. The aim of this study was to evaluate NHLRC2 mRNA and protein expression in different cell types of lung tissue samples and to investigate the effect of transforming growth factor (TGF)-β1 exposure on NHLRC2 expression in vitro.The NHLRC2 expression in lung tissue samples was studied by immunohistochemistry (50 IPF, 10 controls) and mRNA in situ hybridization (8 IPF, 3 controls). The immunohistochemical NHLRC2 expression was quantified with image analysis software and associated with the clinical and smoking data of the patients. NHLRC2 expression levels in primary stromal and small airway epithelial cell lines after exposure to TGF-β1 was measured by quantitative reverse transcription polymerase chain reaction and Western blot analysis.NHLRC2 expression was detected especially in bronchiolar epithelial cells, type II pneumocytes and macrophages in normal lung. In the lungs of IPF patients, NHLRC2 was mainly expressed in hyperplastic alveolar epithelial cells lining fibroblast foci and honeycombs. NHLRC2 expression assessed by image analysis was higher in IPF compared to controls (p < 0.001). Ever-smokers had more prominent NHLRC2 staining than non-smokers (p = 0.037) among IPF patients. TGF-β1 exposure did not influence NHLRC2 levels in lung cell lines.NHLRC2 expression was higher in IPF compared to controls being widely expressed in type II pneumocytes, macrophages, bronchiolar epithelium, and hyperplastic alveolar epithelium. Additionally, its expression was not regulated by the exposure to TGF-β1 in vitro. Further studies are needed to clarify the role of NHLRC2 in IPF.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive lung scarring. IPF-related pulmonary vascular remodeling and pulmonary hypertension (PH) result in particularly poor prognosis.To study the pathogenesis of vascular remodeling in fibrotic lungs and its contribution to progression of fibrosis.We used an experimental model of lung fibrosis associated with PH by transient overexpression of active TGF-β1. Samples from patients with fibrotic lung diseases were analyzed in-depth by immunostaining, gene expression, and gene mutations.We found a reduction in endothelial cells (ECs) and activation of vascular smooth muscle cells (VSMCs) in fibrotic lungs. Co-culturing fibroblasts with VSMCs or ECs from fibrotic lungs induced fibrotic phenotypes in fibroblasts. IPF fibroblasts induced EC death and activation of VSMCs in co-culture systems. Decreased levels of BMPR2 and its signaling were observed in ECs and VSMCs from fibrotic lungs in both rats and humans. On fibroblasts treated with media from VSMCs, BMPR2 suppression in VSMCs led to fibrogenic effects. Tacrolimus activated BMPR2 signaling and attenuated fibrosis and PH in rodent lungs. Whole exome sequencing revealed rare mutations in PH-related genes, including BMPR2, in IPF patients undergoing transplantation. A unique missense BMPR2 mutation (p.Q721R) was discovered to have dysfunctional effects on BMPR2 signaling.Endothelial dysfunction and vascular remodeling in PH secondary to pulmonary fibrosis enhance fibrogenesis through impaired BMPR2 signaling. Tacrolimus may have value as treatment of advanced IPF and concomitant PH. Genetic abnormalities may determine the development of PH in advanced IPF.

Background: Orai1 is a plasma membrane Ca2+ channel that is involved in store-operated calcium entry (SOCE). In pulmonary cells, SOCE regulates gene expression and stimulates cytokine, mucin, and protease secretion. Activation of Orai1/SOCE results in recruitment of neutrophils to the lungs. Orai1 activation is also upstream of transcription factors such as nuclear factor of activated T cells, which facilitate onset of inflammation. In cystic fibrosis (CF), the immune response is dysregulated, and the lung is chronically inflamed, but Orai1 expression in the CF lung is poorly understood. Orai1 is a promising target for drug development, so we tested the hypothesis that Orai1 was upregulated in CF lungs. Methods: We used LungMAP to analyze single-cell ribonucleic acid (RNA) sequencing data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, CF, and asthma (disease control) donors (4 male/4 female per group). We imaged sections by confocal and super resolution microscopy and analyzed Orai1 and STIM1 expression, colocalization, and particle size in different pulmonary cell types. Results: Orai1 was broadly expressed throughout the lung, but expression was greatest in immune cells. At messenger RNA and protein levels, there were no consistent trends in expression levels between the three groups. Orai1 must interact with STIM1 to activate SOCE, so we used STIM1/Orai1 colocalization as a marker of Orai1 activity. Using this approach, we found significantly greater colocalization between these proteins in CF and asthma lung epithelia (CF 50%, asthma 54%, normal 15%), interstitia (CF 57%, asthma 49%, normal 16%), and luminal immune cells (CF 66%, asthma 70%, normal 38%). Orai1 also aggregates as part of its interaction process. Using super-resolution microscopy, we found significantly more Orai1 and STIM1 aggregation in immune cells from CF and asthmatic lungs (average Orai1 particle size: CF 52 nm, asthma 63 nm, normal 28 nm; average STIM1 particle size: CF 77 nm, asthma 59 nm, normal 14 nm). We also looked at Orai1 in peripheral blood neutrophils from normal and CF donors (5 per group). All CF subjects took elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA), but under baseline conditions, there were significantly bigger puncta in CF neutrophils (CF 10 nm, normal 6 nm), suggesting that these patients continued to have significant inflammation despite taking ELX/TEZ/IVA, and mean percentage predicted forced expiratory volume in 1 second in our CF cohort was 55 ± 22%, indicating that these patients had persistent lung disease. Conclusions: This is the first comprehensive analysis of Orai1 and STIM1 expression in lungs from normal and CF donors. We found evidence that Orai1 was more active in CF than normal lungs. This novel application of super-resolution microscopy has the potential to be used in clinical settings for analysis of ex vivo patient samples and to evaluate inflammation in people with CF. Although traditional biomarkers of inflammation such as serum cytokine levels are useful for rapid detection of systemic inflammation, our technique allows for precise localization of upstream inflammatory signaling biomarkers at the cellular level and in fixed samples. Therefore, these data suggest that Orai1 has a key role in CF lung inflammation and attest to the potential of anti-inflammatory therapeutics that target Orai1.We used LungMAP to analyze single-cell ribonucleic acid (RNA) sequencing data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, CF, and asthma (disease control) donors (4 male/4 female per group). W

In diseases such as asthma and cystic fibrosis (CF), the immune response is dysregulated and the lung is chronically inflamed. Orai1 activation is required for the initiation and persistence of inflammation. However, Orai1 expression in the lung is poorly understood. We therefore tested the hypothesis that Orai1 expression was upregulated in asthmatic and CF lungs.We used LungMAP to analyze single-cell RNAseq data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, asthma, and CF donors. We imaged sections by confocal and super resolution microscopy, and analyzed Orai1 and STIM1 expression in different pulmonary cell types.Orai1 was broadly-expressed, but expression was greatest in immune cells. At mRNA and protein levels, there were no consistent trends in expression levels between the three phenotypes. Orai1 must interact with STIM1 in order to activate and conduct Ca2+. We therefore used STIM1/Orai1 co-localization as a marker of Orai1 activity. Using this approach, we found significantly increased co-localization between these proteins in epithelia, interstitial and luminal immune cells, but not alveoli, from asthma and CF lungs. Orai1 also aggregates as part of its activation process. Using super resolution microscopy, we also found significantly increased Orai1 aggregation in immune cells from asthmatic and CF lungs.We found evidence that Orai1 was more active in asthma and CF than normal lungs. These data suggest that Orai1 is a relevant target for reducing pulmonary inflammation.

The incidence and sites of mucus accumulation, and molecular regulation of mucin gene expression, in COVID-19 lung disease have not been reported.Characterize incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease.Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by AB-PAS and immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. SARS-CoV-2-infected human bronchial epithelial (HBE) cultures were utilized to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures.MUC5B and variably MUC5AC RNA levels were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the sub-acute/chronic disease phase following SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of COVID-19 subjects in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days post inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days post inoculation. SARS-CoV-2 infection of HBE cultures induced expression of EGFR ligands and inflammatory cytokines (e.g., IL-1α/β) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways, or dexamethasone administration, reduced SARS-CoV-2-induced mucin expression.SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation post SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The potential pathogenetic mechanisms underlying the varied morphology of congenital pulmonary airway malformations (CPAMs) have not been molecularly determined, but a subset have been shown to contain clusters of mucinous cells (MCC). These clusters are believed to serve as precursors for potential invasive mucinous adenocarcinoma, and they are associated with KRAS codon 12 mutations. To assess the universality of KRAS mutations in MCCs, we sequenced exon 2 of KRAS in 61 MCCs from 18 patients, and we found a KRAS codon 12 mutation in all 61 MCCs. Furthermore, all MCCs from a single patient always had the same KRAS mutation, and the same KRAS mutation was also found in non-mucinous lesional tissue. Next generation sequencing of seven MCCs showed no other mutations or copy number variations. Sequencing of 46 additional CPAMs with MCCs revealed KRAS mutations in non-mucinous lesional tissue in all cases. RNA in situ hybridization confirmed widespread distribution of cells with mutant KRAS RNA, even extending outside of the bronchiolar type epithelium. We identified 25 additional CPAMs with overall histologic architecture similar to CPAMs with KRAS mutations but without identifiable MCCs, and we found KRAS mutations in 17 (68%). The histologic features of these KRAS mutated CPAMs included type 1 and type 3 morphology, as well as lesions with an intermediate histologic appearance, and analysis revealed a strong correlation between the specific amino acid substitution and histomorphology. These findings, together with previously published model organism data, suggests that the formation of type 1 and 3 CPAMs is driven by mosaic KRAS mutations arising in the lung epithelium early in development and places them within the growing field of mosaic RASopathies. The presence of widespread epithelial mutation explains late metastatic disease in incompletely resected patients and reinforces the recommendation for complete resection of these lesions.

Interstitial lung disease is frequently comorbid with dermatomyositis and has a poor prognosis, especially in patients with the anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody. However, the pathogenesis of dermatomyositis-related interstitial lung disease remains unclear.We examined 18 and 19 patients with dermatomyositis-related interstitial lung disease and idiopathic pulmonary fibrosis (control), respectively. Lung tissues obtained from these patients were semi-quantitatively evaluated by immunohistochemical staining with in-house anti-human MDA5 monoclonal antibodies, as well as anti-human immunoglobulin (Ig) G, IgM, IgA, and complement component 3(C3) antibodies. We established human MDA5 transgenic mice and treated them with rabbit anti-human MDA5 polyclonal antibodies, and evaluated lung injury and Ig and C3 expression.MDA5 was moderately or strongly expressed in the lungs of patients in both groups, with no significant differences between the groups. However, patients with dermatomyositis-related interstitial lung disease showed significantly stronger expression of C3 (p < 0.001), IgG (p < 0.001), and IgM (p = 0.001) in the lungs than control. Moreover, lung C3, but IgG, IgA, nor IgM expression was significantly stronger in MDA5 autoantibody-positive dermatomyositis-related interstitial lung disease (n = 9) than in MDA5 autoantibody-negative dermatomyositis-related interstitial lung disease (n = 9; p = 0.022). Treatment with anti-MDA5 antibodies induced lung injury in MDA5 transgenic mice, and strong immunoglobulin and C3 expression was observed in the lungs of the mice.Strong immunoglobulin and C3 expression in the lungs involve lung injury related to dermatomyositis-related interstitial lung disease. Enhanced immune complex formation in the lungs may contribute to the poor prognosis of MDA5 autoantibody-positive dermatomyositis-related interstitial lung disease.

Mutations in the FOXF1 gene, encoding the mesenchymal Forkhead Box (FOX) transcription factor, are linked to Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV), a severe congenital disorder associated with the loss of alveolar capillaries and lung hypoplasia. While proangiogenic functions of FOXF1 have been extensively studied, the role of FOXF1 in mesenchymal-epithelial signaling during lung development remains uncharacterized. Herein, we utilized murine lung organoids to demonstrate that the S52F FOXF1 mutation (found in ACDMPV patients) stimulates canonical WNT/β-catenin signaling in type 2 alveolar epithelial cells (AEC2s), leading to increased proliferation of AEC2s and decreased differentiation of AEC2s into AEC1s. Alveolar organoids containing Foxf1WT/S52F lung fibroblasts and wild-type epithelial cells grew faster on Matrigel and exhibited AEC2 hyperplasia. AEC2 hyperplasia and loss of AEC1s were found in the lungs of Foxf1WT/S52F embryos, a mouse model of ACDMPV. Activation of canonical WNT/β-catenin signaling in AEC2s of lung organoids and Foxf1WT/S52F mice was associated with decreased expression of non-canonical WNT5A ligand in lung fibroblasts. Mechanistically, FOXF1 directly activates the Wnt5a gene transcription through an evolutionarily conserved +6320/+6326 region located in the first intron of the Wnt5a gene. Site-directed mutagenesis of the +6320/+6326 region prevented the transcriptional activation of the Wnt5a enhancer by FOXF1. Treatment with exogenous WNT5A ligand inhibited the effects of the S52F FOXF1 mutation on canonical WNT/β-catenin signaling in alveolar organoids, preventing aberrant AEC2 cell expansion and restoring differentiation of AEC1s. Activation of either FOXF1 or WNT5A may provide an attractive strategy to improve lung function in ACDMPV patients.

The gain-of-function minor allele of the MUC5B promoter (rs35705950) is the strongest risk factor for idiopathic pulmonary fibrosis (IPF), a devastating fibrotic lung disease that leads to progressive respiratory failure in adults. We have previously demonstrated that Muc5b overexpression in mice worsens lung fibrosis following bleomycin exposure and have hypothesized that excess Muc5b promotes endoplasmic reticulum (ER) stress and apoptosis, stimulating fibrotic lung injury. Here, we report that ER stress pathway members ATF4 and ATF6 co-express with MUC5B in epithelia of the distal IPF airway and honeycomb cyst, and this is more pronounced in carriers of the gain-of-function MUC5B promoter variant. Similarly, in mice exposed to bleomycin, Muc5b expression is temporally associated with markers of ER stress. Using bulk and single cell RNA sequencing (scRNA-seq) in bleomycin-exposed mice, we found that pathologic ER-stress associated transcripts Atf4 and Ddit3 were elevated in alveolar epithelia of SFTPC-Muc5b transgenic (SFTPC-Muc5bTg) mice relative to wild type mice. Activation of the ER stress response inhibits protein translation for most genes by phosphorylation of Eif2α, which prevents guanine exchange by Eif2B, and facilitates translation of Atf4. The integrated stress response inhibitor (ISRIB), facilitates interaction of phosphorylated Eif2α with Eif2B, overcoming translation inhibition associated with ER stress and reducing Atf4 translation. We found that a single dose of ISRIB diminished Atf4 translation in SFTPC-Muc5bTg mice following bleomycin injury. Moreover, ISRIB resolved the exaggerated fibrotic response of SFTPC-Muc5bTg mice to bleomycin. In summary, we demonstrate that MUC5B/Muc5b expression is associated with pathologic ER stress and that restoration of normal translation with a single dose of ISRIB promotes lung repair in bleomycin-injured Muc5b-overexpressing mice.

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.

Pulmonary hypertension (PH) can occur as a complication of schistosomiasis. In humans, schistosomiasis-PH persists despite antihelminthic therapy and parasite eradication. We hypothesized that persistent disease arises as a consequence of exposure repetition.Following intraperitoneal sensitization, mice were experimentally exposed to Schistosoma eggs by intravenous injection, either once or three times repeatedly. The phenotype was characterized by right heart catheterization and tissue analysis.Following intraperitoneal sensitization, a single intravenous Schistosoma egg exposure resulted in a PH phenotype that peaked at 7-14 days, followed by spontaneous resolution. Three sequential exposures resulted in a persistent PH phenotype. Inflammatory cytokines were not significantly different between mice exposed to one or three egg doses, but there was an increase in perivascular fibrosis in those who received three egg doses. Significant perivascular fibrosis was also observed in autopsy specimens from patients who died of this condition.Repeatedly exposing mice to schistosomiasis causes a persistent PH phenotype, accompanied by perivascular fibrosis. Perivascular fibrosis may contribute to the persistent schistosomiasis-PH observed in humans with this disease.

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.