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The article presents the discussion on Blots were imaged on a Li-Cor Odyssey instrument and intensity being quantified relative to loading control (ACTB) using ImageStudio Lite software. Topics include nucleus RNA sequencing (snRNA-seq) for human lung tissue being performed as described and reported previously; and running a regularized negative binomial regression against sequencing depth for any given barcode/cell in the dataset. more...

Previously we have shown that a gain-of-function MUC5B promoter variant (rs35705950) is the strongest risk factor for the development of idiopathic pulmonary fibrosis. We have also found that Muc5b overexpression reduces mucociliary clearance in mice, potentially leading to recurrent injury to the bronchoalveolar epithelia. Hypersensitivity pneumonitis (HP) is induced by inhalation of numerous causative antigens that may be affected by mucociliary clearance. We conducted this study to determine the role of Muc5b in a mouse model of HP induced by Saccharopolyspora rectivirgula (SR) antigen. We used Muc5b-deficient and wild-type (WT) mice to determine whether Muc5b plays a role in inflammation and fibrosis at 3 and 6 wk in an SR model of HP. We measured cell concentrations and MUC5B expression in whole lung lavage (WLL) and quantified fibrosis using hydroxyproline assay and second harmonic generation. Muc5b expression in WLL fluid was significantly increased in SR-exposed WT mice compared with saline controls. WT mice challenged with SR developed more inflammation and lung fibrosis at 6 wk compared with 3 wk postexposure. Moreover, we found that 6 wk following challenge with SR, Muc5b-deficient mice had less lung inflammation and less lung fibrosis than Muc5b WT mice. Furthermore, Muc5b-deficient mice had significantly lower concentrations of TGF-β1 in the WLL compared with Muc5b WT mice at 6 wk of exposure. Muc5b appears to play a role in fibrosis in the animal model of HP and this may have implications for HP in humans. [ABSTRACT FROM AUTHOR] more...

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B, and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of ciliumgenes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF. [ABSTRACT FROM AUTHOR] more...

The common gain-of-function MUC5B promoter variant (rs35705950) is the strongest risk factor for the development of idiopathic pulmonary fibrosis (IPF). While the role of complement in IPF is controversial, both MUC5B and the complement system play a role in lung host defense. The aim of this study was to evaluate the relationship between complement component 3 (C3) and MUC5B in patients with IPF and in bleomycin-induced lung injury in mice. To do this, we evaluated C3 gene expression in whole lung tissue from 300 subjects with IPF and 175 healthy controls. Expression of C3 was higher in IPF than healthy controls {1.40-fold increase [95% confidence interval (CI) 1.31-1.50]; P < 0.0001} and even greater among IPF subjects with the highest-risk IPF MUC5B promoter genotype [TT vs. GG = 1.59-fold (95% CI 1.15-2.20); P < 0.05; TT vs. GT = 1.66- fold (95% CI 1.20 -2.30); P < 0.05]. Among subjects with IPF, C3 expression was significantly higher in the lung tissue without microscopic honeycombing than in the lung tissue with microscopic honeycombing [1.40-fold increase (95% CI 1.23- 1.59); P < 0.01]. In mice, while bleomycin exposure increased Muc5b protein expression, C3-deficient mice were protected from bleomycin-induced lung injury. In aggregate, our findings indicate that the MUC5B promoter variant is associated with higher C3 expression and suggest that the complement system may contribute to the pathogenesis of IPF. [ABSTRACT FROM AUTHOR] more...

Background: Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs. Results: Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and% predicted DLCO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples. Conclusions: We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity. [ABSTRACT FROM AUTHOR] more...