IL-4 drastically decreases deuterosomal and multiciliated cells via alteration in progenitor cell differentiation.

BACKGROUND: Allergic inflammation affects the epithelial cell populations resulting in goblet cell hyperplasia and decreased ciliated cells. Recent advances in single-cell RNA sequencing (scRNAseq) have enabled the identification of new cell subtypes and genomic features of single cells. In this study, we aimed to investigate the effect of allergic inflammation in nasal epithelial cell transcriptomes at the single-cell level. METHODS: We performed scRNAseq in cultured primary human nasal epithelial (HNE) cells and in vivo nasal epithelium. The transcriptomic features and epithelial cell subtypes were determined under IL-4 stimulation, and cell-specific marker genes and proteins were identified. RESULTS: We confirmed that cultured HNE cells were similar to in vivo epithelial cells through scRNAseq. Cell-specific marker genes were utilized to cluster the cell subtypes, and FOXJ1+ -ciliated cells were sub-classified into multiciliated and deuterosomal cells. PLK4 and CDC20B were specific for deuterosomal cells, and SNTN, CPASL, and GSTA2 were specific for multiciliated cells. IL-4 altered the proportions of cell subtypes, resulting in a decrease in multiciliated cells and loss of deuterosomal cells. The trajectory analysis revealed deuterosomal cells as precursor cells of multiciliated cells and deuterosomal cells function as a bridge between club and multiciliated cells. A decrease in deuterosomal cell marker genes was observed in nasal tissue samples with type 2 inflammation. CONCLUSION: The effects of IL-4 appear to be mediated through the loss of the deuterosomal population, resulting in the reduction in multiciliated cells. This study also newly suggests cell-specific markers that might be pivotal for investigating respiratory inflammatory diseases.

Serum proteomic profiling of rheumatoid arthritis-interstitial lung disease with a comparison to idiopathic pulmonary fibrosis.

Although interstitial lung disease (ILD) causes significant morbidity and mortality in rheumatoid arthritis (RA), it is difficult to predict the development or progression of ILD, emphasising the need for improved discovery through minimally invasive diagnostic tests. Aptamer-based proteomic profiling was used to assess 1321 proteins from 159 patients with rheumatoid arthritis with interstitial lung disease (RA-ILD), RA without ILD, idiopathic pulmonary fibrosis and healthy controls. Differential expression and gene set enrichment analyses revealed molecular signatures that are strongly associated with the presence and severity of RA-ILD and provided insight into unexplored pathways of disease. These warrant further study as non-invasive diagnostic tools and future therapeutic targets.

Cell-Type-Specific Expression of Hyaluronan Synthases HAS2 and HAS3 Promotes Goblet Cell Hyperplasia in Allergic Airway Inflammation.

Allergic rhinitis (AR) is a multifactorial airway disease characterized by basal and goblet cell hyperplasia. Hyaluronic acid (HA) is a major component of extracellular matrix and a critical contributor to tissue repair and remodeling after injury. We previously demonstrated that the intermediate progenitor cell (IPC) surface marker CD44v3 is upregulated in the basal and suprabasal layers of well-differentiated primary human nasal epithelial (HNE) cells after stimulation with the Th2 (T-helper cell type 2) cytokine IL-4, and an antibody blocking the CD44v3-HA interaction suppressed IL-4-induced goblet cell hyperplasia. We now show that the expression of HA and two HA synthases, HAS2 and HAS3, was upregulated in both the nasal surface epithelium of subjects with AR and IL-4-stimulated HNE cells. Inhibition of HA synthesis by 4-methylumbelliferone suppressed IL-4-induced goblet cell hyperplasia. Moreover, HAS2 and HAS3 were expressed in IPCs depending on the differentiation events, as follows: the rapid, transient upregulation of HAS2 induced basal IPC proliferation and basal-to-suprabasal transition, whereas the delayed upregulation of HAS3 promoted the transition of suprabasal IPCs to a goblet cell fate. 4-methylumbelliferone treatment in a house dust mite-induced murine AR model attenuated goblet cell metaplasia. Last, HA concentrations in nasal epithelial lining fluids from patients with AR positively correlated with the concentrations of mediators causing allergic inflammation. These data suggest that HA produced after the sequential upregulation of HAS2 and HAS3 contributes to goblet cell hyperplasia in allergic airway inflammation and modulates disease progression.

Lung lymphatic thrombosis and dysfunction caused by cigarette smoke exposure precedes emphysema in mice.

The lymphatic vasculature is critical for lung function, but defects in lymphatic function in the pathogenesis of lung disease is understudied. In mice, lymphatic dysfunction alone is sufficient to cause lung injury that resembles human emphysema. Whether lymphatic function is disrupted in cigarette smoke (CS)-induced emphysema is unknown. In this study, we investigated the effect of CS on lung lymphatic function. Analysis of human lung tissue revealed significant lung lymphatic thrombosis in patients with emphysema compared to control smokers that increased with disease severity. In a mouse model, CS exposure led to lung lymphatic thrombosis, decreased lymphatic drainage, and impaired leukocyte trafficking that all preceded the development of emphysema. Proteomic analysis demonstrated an increased abundance of coagulation factors in the lymph draining from the lungs of CS-exposed mice compared to control mice. In addition, in vitro assays demonstrated a direct effect of CS on lymphatic endothelial cell integrity. These data show that CS exposure results in lung lymphatic dysfunction and a shift in thoracic lymph towards a prothrombic state. Furthermore, our data suggest that lymphatic dysfunction is due to effects of CS on the lymphatic vasculature that precede emphysema. These studies demonstrate a novel component of CS-induced lung injury that occurs early in the pathogenesis of emphysema.

Association of circulating cell-free double-stranded DNA and metabolic derangements in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with unclear aetiology and poorly understood pathophysiology. Although plasma levels of circulating cell-free DNA (ccf-DNA) and metabolomic changes have been reported in IPF, the associations between ccf-DNA levels and metabolic derangements in lung fibrosis are unclear. Here, we demonstrate that ccf-double-stranded DNA (dsDNA) is increased in patients with IPF with rapid progression of disease compared with slow progressors and healthy controls and that ccf-dsDNA associates with amino acid metabolism, energy metabolism and lipid metabolism pathways in patients with IPF.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Reversal of emphysema by restoration of pulmonary endothelial cells.

Chronic obstructive pulmonary disease (COPD) is marked by airway inflammation and airspace enlargement (emphysema) leading to airflow obstruction and eventual respiratory failure. Microvasculature dysfunction is associated with COPD/emphysema. However, it is not known if abnormal endothelium drives COPD/emphysema pathology and/or if correcting endothelial dysfunction has therapeutic potential. Here, we show the centrality of endothelial cells to the pathogenesis of COPD/emphysema in human tissue and using an elastase-induced murine model of emphysema. Airspace disease showed significant endothelial cell loss, and transcriptional profiling suggested an apoptotic, angiogenic, and inflammatory state. This alveolar destruction was rescued by intravenous delivery of healthy lung endothelial cells. Leucine-rich α-2-glycoprotein-1 (LRG1) was a driver of emphysema, and deletion of Lrg1 from endothelial cells rescued vascular rarefaction and alveolar regression. Hence, targeting endothelial cell biology through regenerative methods and/or inhibition of the LRG1 pathway may represent strategies of immense potential for the treatment of COPD/emphysema.

Association of plasma mitochondrial DNA with COPD severity and progression in the SPIROMICS cohort.

BACKGROUND: There is a lack of mechanism-driven, clinically relevant biomarkers in chronic obstructive pulmonary disease (COPD). Mitochondrial dysfunction, a proposed disease mechanism in COPD, is associated with the release of mitochondrial DNA (mtDNA), but plasma cell-free mtDNA has not been previously examined prospectively for associations with clinical COPD measures. METHODS: P-mtDNA, defined as copy number of mitochondrially-encoded NADH dehydrogenase-1 (MT-ND1) gene, was measured by real-time quantitative PCR in 700 plasma samples from participants enrolled in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort. Associations between p-mtDNA and clinical disease parameters were examined, adjusting for age, sex, smoking status, and for informative loss to follow-up. RESULTS: P-mtDNA levels were higher in participants with mild or moderate COPD, compared to smokers without airflow obstruction, and to participants with severe COPD. Baseline increased p-mtDNA levels were associated with better CAT scores in female smokers without airflow obstruction and female participants with mild or moderate COPD on 1-year follow-up, but worse 6MWD in females with severe COPD. Higher p-mtDNA levels were associated with better 6MWD in male participants with severe COPD. These associations were no longer significant after adjusting for informative loss to follow-up. CONCLUSION: In this study, p-mtDNA levels associated with baseline COPD status but not future changes in clinical COPD measures after accounting for informative loss to follow-up. To better characterize mitochondrial dysfunction as a potential COPD endotype, these results should be confirmed and validated in future studies. TRIAL REGISTRATION:  ClinicalTrials.gov NCT01969344 (SPIROMICS).

CD44v3-Positive Intermediate Progenitor Cells Contribute to Airway Goblet Cell Hyperplasia.

In allergic airway diseases, intermediate progenitor cells (IPCs) increase in number in the surface epithelium. IPCs arise from basal cells, the origin of hallmark pathological changes, including goblet cell hyperplasia and mucus hypersecretion. Thus, targeting IPCs will benefit future treatment of allergic airway diseases. However, the lack of adequate cell surface markers for IPCs limits their identification and characterization. We now show that CD44 containing exon v3 (CD44v3) is a surface marker for IPCs that are capable of both proliferating and generating differentiated goblet cells in allergic human nasal epithelium. In primary human nasal epithelial cells that had differentiated at an air-liquid interface, IL-4 upregulated mRNA expression of three CD44v variants that include exon v3 (CD44v3-v6, CD44v3,v8-v10, and CD44v3-v10), and it induced expression of CD44v3 protein in the basal and suprabasal layers of the culture. FACS analysis revealed two subpopulations differing in CD44v3 concentrations, as follows: CD44v3low cells expressed high amounts of proliferative and basal cell markers (Ki-67 and TP63), whereas CD44v3high cells strongly expressed progenitor and immature and mature goblet cell markers (SOX2, CA2, and SPDEF). Importantly, a blocking anti-CD44 antibody suppressed IL-4-induced mucin production by human nasal epithelial cells. Furthermore, CD44v3 was coexpressed with TP63, KRT5, or SOX2 and was upregulated in the basal and suprabasal layers of the nasal surface epithelium of subjects with allergic rhinitis. Taken together, these data demonstrate that high CD44v3 expression contributes to goblet cell hyperplasia in inflammation of the allergic airway.

MTOR suppresses autophagy-mediated production of IL25 in allergic airway inflammation.

INTRODUCTION: Airway epithelial cells are recognised as an essential controller for the initiation and perpetuation of asthmatic inflammation, yet the detailed mechanisms remain largely unknown. This study aims to investigate the roles and mechanisms of the mechanistic target of rapamycin (MTOR)-autophagy axis in airway epithelial injury in asthma. METHODS: We examined the MTOR-autophagy signalling in airway epithelium from asthmatic patients or allergic mice induced by ovalbumin or house dust mites, or in human bronchial epithelial (HBE) cells. Furthermore, mice with specific MTOR knockdown in airway epithelium and autophagy-related lc3b-/- mice were used for allergic models. RESULTS: MTOR activity was decreased, while autophagy was elevated, in airway epithelium from asthmatic patients or allergic mice, or in HBE cells treated with IL33 or IL13. These changes were associated with upstream tuberous sclerosis protein 2 signalling. Specific MTOR knockdown in mouse bronchial epithelium augmented, while LC3B deletion diminished allergen-induced airway inflammation and mucus hyperproduction. The worsened inflammation caused by MTOR deficiency was also ameliorated in lc3b-/- mice. Mechanistically, autophagy was induced later than the emergence of allergen-initiated inflammation, particularly IL33 expression. MTOR deficiency increased, while knocking out of LC3B abolished the production of IL25 and the eventual airway inflammation on allergen challenge. Blocking IL25 markedly attenuated the exacerbated airway inflammation in MTOR-deficiency mice. CONCLUSION: Collectively, these results demonstrate that allergen-initiated inflammation suppresses MTOR and induces autophagy in airway epithelial cells, which results in the production of certain proallergic cytokines such as IL25, further promoting the type 2 response and eventually perpetuating airway inflammation in asthma.

Autophagy in chronic lung disease.

The development of chronic lung disease occurs as a consequence of multiple cellular events that involve an initial insult which often leads to the development of chronic inflammation, and the dysregulation of cellular proliferation and cell death mechanisms. Multiple cell types in the lung are key to the respiratory and protective/barrier functions necessary to manage the chronic exposures to environmental, mechanical, and oxidative stressors. Autophagy is essential to lung development and homeostasis, as well as the prevention and development of disease. The cellular process involves the collection and removal of unwanted organelles and proteins through lysosomal degradation. In recent years, investigations have addressed the roles of autophagy and selective autophagy in numerous chronic lung diseases. Here, we highlight recent advances on the role of autophagy in the pathogenesis of asthma, chronic obstructive pulmonary disease and emphysema, pulmonary arterial hypertension, and idiopathic pulmonary fibrosis.

Cigarette smoke-initiated autoimmunity facilitates sensitisation to elastin-induced COPD-like pathologies in mice.

It is currently not understood whether cigarette smoke exposure facilitates sensitisation to self-antigens and whether ensuing auto-reactive T cells drive chronic obstructive pulmonary disease (COPD)-associated pathologies.To address this question, mice were exposed to cigarette smoke for 2 weeks. Following a 2-week period of rest, mice were challenged intratracheally with elastin for 3 days or 1 month. Rag1-/- , Mmp12-/- , and Il17a-/- mice and neutralising antibodies against active elastin fragments were used for mechanistic investigations. Human GVAPGVGVAPGV/HLA-A*02:01 tetramer was synthesised to assess the presence of elastin-specific T cells in patients with COPD.We observed that 2 weeks of cigarette smoke exposure induced an elastin-specific T cell response that led to neutrophilic airway inflammation and mucus hyperproduction following elastin recall challenge. Repeated elastin challenge for 1 month resulted in airway remodelling, lung function decline and airspace enlargement. Elastin-specific T cell recall responses were dose dependent and memory lasted for over 6 months. Adoptive T cell transfer and studies in T cells deficient Rag1-/- mice conclusively implicated T cells in these processes. Mechanistically, cigarette smoke exposure-induced elastin-specific T cell responses were matrix metalloproteinase (MMP)12-dependent, while the ensuing immune inflammatory processes were interleukin 17A-driven. Anti-elastin antibodies and T cells specific for elastin peptides were increased in patients with COPD.These data demonstrate that MMP12-generated elastin fragments serve as a self-antigen and drive the cigarette smoke-induced autoimmune processes in mice that result in a bronchitis-like phenotype and airspace enlargement. The study provides proof of concept of cigarette smoke-induced autoimmune processes and may serve as a novel mouse model of COPD.

N-Glycosylation Regulates Chitinase 3-like-1 and IL-13 Ligand Binding to IL-13 Receptor α2.

Chitinase 3-like-1 (Chi3l1) and IL-13 are both ligands of IL-13 receptor α2 (IL-13Rα2). The binding of the former activates mitogen-activated protein kinase, AKT, and Wnt/ß-catenin signaling, and plays important roles in innate and adaptive immunity, cellular apoptosis, oxidative injury, allergic inflammation, tumor metastasis and wound healing, fibrosis, and repair in the lung. In contrast, the latter binding is largely a decoy event that diminishes the effects of IL-13. Here, we demonstrate that IL-13Rα2 N-glycosylation is a critical determinant of which ligand binds. Structure-function evaluations demonstrated that Chi3l1-IL-13Rα2 binding was increased when sites of N-glycosylation are mutated, and studies with tunicamycin and Peptide:N-glycosidase F (PNGase F) demonstrated that Chi3l1-IL-13Rα2 binding and signaling were increased when N-glycosylation was diminished. In contrast, structure-function experiments demonstrated that IL-13 binding to IL-13Rα2 was dependent on each of the four sites of N-glycosylation in IL-13Rα2, and experiments with tunicamycin and PNGase F demonstrated that IL-13-IL-13Rα2 binding was decreased when IL-13Rα2 N-glycosylation was diminished. Studies with primary lung epithelial cells also demonstrated that Chi3l1 inhibited, whereas IL-13 stimulated, N-glycosylation as evidenced by the ability of Chi3l1 to inhibit and IL-13 to stimulate the subunits of the oligosaccharide complex A and B (STT3A and STT3B). These studies demonstrate that N-glycosylation is a critical determinant of Chi3l1 and IL-13 binding to IL-13Rα2, and highlight the ability of Chi3l1 and IL-13 to alter key elements of the N-glycosylation apparatus in a manner that would augment their respective binding.

DNA Methylation Is Predictive of Mortality in Current and Former Smokers.

Rationale: Smoking results in at least a decade lower life expectancy. Mortality among current smokers is two to three times as high as never smokers. DNA methylation is an epigenetic modification of the human genome that has been associated with both cigarette smoking and mortality.Objectives: We sought to identify DNA methylation marks in blood that are predictive of mortality in a subset of the COPDGene (Genetic Epidemiology of COPD) study, representing 101 deaths among 667 current and former smokers.Methods: We assayed genome-wide DNA methylation in non-Hispanic white smokers with and without chronic obstructive pulmonary disease (COPD) using blood samples from the COPDGene enrollment visit. We tested whether DNA methylation was associated with mortality in models adjusted for COPD status, age, sex, current smoking status, and pack-years of cigarette smoking. Replication was performed in a subset of 231 individuals from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study.Measurements and Main Results: We identified seven CpG sites associated with mortality (false discovery rate < 20%) that replicated in the ECLIPSE cohort (P < 0.05). None of these marks were associated with longitudinal lung function decline in survivors, smoking history, or current smoking status. However, differential methylation of two replicated PIK3CD (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta) sites were associated with lung function at enrollment (P < 0.05). We also observed associations between DNA methylation and gene expression for the PIK3CD sites.Conclusions: This study is the first to identify variable DNA methylation associated with all-cause mortality in smokers with and without COPD. Evaluating predictive epigenomic marks of smokers in peripheral blood may allow for targeted risk stratification and aid in delivery of future tailored therapeutic interventions.

Apolipoprotein E and Periostin Are Potential Biomarkers of Nasal Mucosal Inflammation. A Parallel Approach of In Vitro and In Vivo Secretomes.

No previously suggested biomarkers of nasal mucosal inflammation have been practically applied in clinical fields, and nasal epithelium-derived secreted proteins as biomarkers have not specifically been investigated. The goal of this study was to identify secreted proteins that dynamically change during the differentiation from basal cells to fully differentiated cells and examine whether nasal epithelium-derived proteins can be used as biomarkers of nasal mucosal inflammation, such as chronic rhinosinusitis. To achieve this goal, we analyzed two secretomes using the isobaric tag for relative and absolute quantification technique. From in vitro secretomes, we identified the proteins altered in apical secretions of primary human nasal epithelial cells according to the degree of differentiation; from in vivo secretomes, we identified the increased proteins in nasal lavage fluids obtained from patients 2 weeks after endoscopic sinus surgery for chronic sinusitis. We then used a parallel approach to identify specific biomarkers of nasal mucosal inflammation; first, we selected apolipoprotein E as a nasal epithelial cell-derived biomarker through screening proteins that were upregulated in both in vitro and in vivo secretomes, and verified highly secreted apolipoprotein E in nasal lavage fluids of the patients by Western blotting. Next, we selected periostin as an inflammatory mediator-inducible biomarker from in vivo secretomes, the secretion of which was not induced under in vitro culture conditions. We demonstrated that those two nasal epithelium-derived proteins are possible biomarkers of nasal mucosal inflammation.

GLUT1-dependent glycolysis regulates exacerbation of fibrosis via AIM2 inflammasome activation.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive, fatal lung disease that affects older adults. One of the detrimental natural histories of IPF is acute exacerbation of IPF (AE-IPF), of which bacterial infection is reported to play an important role. However, the mechanism by which bacterial infection modulates the fibrotic response remains unclear. OBJECTIVES: Altered glucose metabolism has been implicated in the pathogenesis of fibrotic lung diseases. We have previously demonstrated that glucose transporter 1 (GLUT1)-dependent glycolysis regulates fibrogenesis in a murine fibrosis model. To expand on these findings, we hypothesised that GLUT1-dependent glycolysis regulates acute exacerbation of lung fibrogenesis during bacterial infection via AIM2 inflammasome activation. RESULTS: In our current study, using a murine model of Streptococcus pneumoniae (S. pneumoniae) infection, we investigated the potential role of GLUT1 on mediating fibrotic responses to an acute exacerbation during bleomycin-induced fibrosis. The results of our current study illustrate that GLUT1 deficiency ameliorates S. pneumoniae-mediated exacerbation of lung fibrosis (wild type (WT)/phosphate buffered saline (PBS), n=3; WT/S. pneumoniae, n=3; WT/Bleomycin, n=5 ; WT/Bleomycin+S. pneumoniae, n=7; LysM-Cre-Glut1fl/f /PBS, n=3; LysM-Cre-Glut1fl/fl /S. pneumoniae, n=3; LysM-Cre-Glut1fl/fl /Bleomycin, n=6; LysM-Cre-Glut1fl/fl /Bleomycin+S. pneumoniae, n=9, p=0.041). Further, the AIM2 inflammasome, a multiprotein complex essential for sensing cytosolic bacterial DNA as a danger signal, is an important regulator of this GLUT1-mediated fibrosis and genetic deficiency of AIM2 reduced bleomycin-induced fibrosis after S. pneumoniae infection (WT/PBS, n=6; WT/Bleomycin+S. pneumoniae, n=15; Aim2-/-/PBS, n=6, Aim2-/-/Bleomycin+S. pneumoniae, n=11, p=0.034). GLUT1 deficiency reduced expression and function of the AIM2 inflammasome, and AIM2-deficient mice showed substantial reduction of lung fibrosis after S. pneumoniae infection. CONCLUSION: Our results demonstrate that GLUT1-dependent glycolysis promotes exacerbation of lung fibrogenesis during S. pneumoniae infection via AIM2 inflammasome activation.

Low Dose Carbon Monoxide Exposure in Idiopathic Pulmonary Fibrosis Produces a CO Signature Comprised of Oxidative Phosphorylation Genes.

Compelling preclinical studies indicate that low-dose carbon monoxide (CO) abrogates experimental lung fibrosis. We recently reported the results of a multicenter, double-blinded, clinical trial of inhaled CO in patients with idiopathic pulmonary fibrosis (IPF). Identifying no significantly changes in metalloproteinase-7 (MMP7) serum concentration, or secondary endpoints of physiologic measurements, hospitalization, death, or patient-reported outcomes. In the present study, we evaluated the effect of low dose CO exposure (100-200 ppm) for 12 weeks on genome-wide gene expression in peripheral blood mononuclear cells (PBMC) derived from these IPF study subjects. We conducted transcriptome profiling on 38 IPF subjects with time points available at 0, 12, and 24 weeks. Total RNA isolated from PBMCs was hybridized onto the Affymetrix Human Gene 2.0 ST Array. We identified 621 genes significantly upregulated in the 24-week CO exposed group compared with the 12-week. Pathway analysis demonstrated association with Oxidative Phosphorylation (adjusted P < 0.05). We identified a clear CO signature dominated with 23 oxidative phosphorylation-related genes (FDR <10%). We confirmed the expression of nine selected gene products using Nanostring's nCounter analysis system. These findings suggest this signature may serve as a potential genomic biomarker for CO exposure and for potential titration of dosage to allow precision testing of therapies in future low dose CO therapeutic studies in IPF.

DROSHA-Dependent AIM2 Inflammasome Activation Contributes to Lung Inflammation during Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) has been linked to chronic lung inflammation. Drosha ribonuclease III (DROSHA), a class 2 ribonuclease III enzyme, plays a key role in microRNA (miRNA) biogenesis. However, the mechanisms by which DROSHA affects the lung inflammation during idiopathic pulmonary fibrosis (IPF) remain unclear. Here, we demonstrate that DROSHA regulates the absent in melanoma 2 (AIM2) inflammasome activation during idiopathic pulmonary fibrosis (IPF). Both DROSHA and AIM2 protein expression were elevated in alveolar macrophages of patients with IPF. We also found that DROSHA and AIM2 protein expression were increased in alveolar macrophages of lung tissues in a mouse model of bleomycin-induced pulmonary fibrosis. DROSHA deficiency suppressed AIM2 inflammasome-dependent caspase-1 activation and interleukin (IL)-1ß and IL-18 secretion in primary mouse alveolar macrophages and bone marrow-derived macrophages (BMDMs). Transduction of microRNA (miRNA) increased the formation of the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks, which is required for AIM2 inflammasome activation in BMDMs. Our results suggest that DROSHA promotes AIM2 inflammasome activation-dependent lung inflammation during IPF.

Necroptosis: a crucial pathogenic mediator of human disease.

Necroptosis is a genetically regulated form of necrotic cell death that has emerged as an important pathway in human disease. The necroptosis pathway is induced by a variety of signals, including death receptor ligands, and regulated by receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL), which form a regulatory necrosome complex. RIPK3-mediated phosphorylation of MLKL executes necroptosis. Recent studies, using animal models of tissue injury, have revealed that RIPK3 and MLKL are key effectors of injury propagation. This Review explores the functional roles of RIPK3 and MLKL as crucial pathogenic determinants and markers of disease progression and severity in experimental models of human disease, including acute and chronic pulmonary diseases; renal, hepatic, cardiovascular, and neurodegenerative diseases; cancer; and critical illness.