Background: Idiopathic pulmonary fibrosis (IPF) bears high mortality due to unclear pathogenesis and limited therapeutic options. Therefore, identifying novel regulators is required to develop alternative therapeutic strategies. Methods: The lung fibroblasts from IPF patients and Reticulocalbin 3 (RCN3) fibroblast-selective knockdown mouse model were used to determine the importance of Rcn3 in IPF; the epigenetic analysis and protein interaction assays, including BioID, were used for mechanistic studies. Results: Reticulocalbin 3 (RCN3) upregulation is associated with the fibrotic activation of lung fibroblasts from IPF patients and Rcn3 overexpression blunts the antifibrotic effects of pirfenidone and nintedanib. Moreover, repressing Rcn3 expression in mouse fibroblasts ameliorates bleomycin-induced lung fibrosis and pulmonary dysfunction in vivo. Mechanistically, RCN3 promotes fibroblast activation by maintaining persistent activation of TGFβ1 signalling via the TGFβ1-RCN3-TGFBR1 positive feedback loop, in which RCN3 upregulated by TGFβ1 exposure detains EZH2 (an epigenetic methyltransferase) in the cytoplasm through RCN3-EZH2 interaction, leading to the release of the EZH2-H3K27me3 epigenetic repression of TGFBR1 and the persistent expression of TGFBR1. Conclusions: These findings introduce a novel regulating mechanism of TGFβ1 signalling in fibroblasts and uncover a critical role of the RCN3-mediated loop in lung fibrosis. RCN3 upregulation may cause resistance to IPF treatment and targeting RCN3 could be a novel approach to ameliorate pulmonary fibrosis. Graphical

Background: Septic-acute respiratory distress syndrome (ARDS), characterized by the acute lung injury (ALI) secondary to aberrant systemic inflammatory response, has high morbidity and mortality. Despite increased understanding of ALI pathogenesis, the therapies to prevent lung dysfunction underlying systemic inflammatory disorder remain elusive. The high density lipoprotein (HDL) has critical protective effects in sepsis and its dysfunction has a manifested contribution to septic organ failure. However, the adverse changes in HDL composition and function in septic-ARDS patients are large unknown.Methods: To investigate HDL remodeling in septic-ARDS, we analyzed the changes of HDL composition from 40 patients with septic-ARDS (A-HDL) and 40 matched normal controls (N-HDL). To determine the deleterious functional remodeling of HDL, A-HDL or N-HDL was administrated to C57BL/6 and apoA-I knock-out (KO) mice after cecal ligation and puncture (CLP) procedure. Mouse lung microvascular endothelial cells (MLECs) were further treated by these HDLs to investigate whether the adverse effects of A-HDL were associated with endothelial dysfunction.Results: Septic-ARDS patients showed significant changes of HDL composition, accompanied with significantly decreased HDL-C. We further indicated that A-HDL treatment aggravated CLP induced ALI. Intriguingly, these deleterious effects of A-HDL were associated with pulmonary endothelial dysfunction, rather than the increased plasma lipopolysaccharide (LPS). Further in vitro results demonstrated the direct effects of A-HDL on MLECs, including increased endothelial permeability, enhanced expressions of adhesion proteins and pro-inflammatory cytokines via activating NF-κB signaling and decreased junction protein expression.Conclusions: Our results depicted the remodeling of HDL composition in sepsis, which predisposes lung to ARDS via inducing ECs dysfunction. These results also demonstrated the importance of circulating HDL in regulating alveolar homeostasis.

Acute respiratory distress syndrome (ARDS) is characterized by acute lung injury (ALI) secondary to an excessive alveolar inflammatory response. Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum (ER) lumen protein in the secretory pathway. We previously reported the indispensable role of Rcn3 in type II alveolar epithelial cells (AECIIs) during lung development and the lung injury repair process. In the present study, we further observed a marked induction of Rcn3 in the alveolar epithelium during LPS-induced ALI. In vitro alveolar epithelial (MLE-12) cells consistently exhibited a significant induction of Rcn3 accompanied with NF-κB activation in response to LPS exposure. We examined the role of Rcn3 in the alveolar inflammatory response by using mice with a selective deletion of Rcn3 in alveolar epithelial cells upon doxycycline administration. The Rcn3 deficiency significantly blunted the ALI and alveolar inflammation induced by intratracheal LPS instillation but not that induced by an intraperitoneal LPS injection (secondary insult); the alleviated ALI was accompanied by decreases in NF-κB activation and NLRP3 levels but not in GRP78 and cleaved caspase-3 levels. The studies conducted in MLE-12 cells consistently showed that Rcn3 knockdown blunted the activations of NF-κB signaling and NLRP3-dependent inflammasome upon LPS exposure. Collectively, these findings suggest a novel role for Rcn3 in regulating the alveolar inflammatory response to pulmonary infection via the NF-κB/NLRP3/inflammasome axis and shed additional light on the mechanism of ARDS/ALI.

Objectives. To investigate the association of serum autoantibodies against M2-muscarinic acetylcholine receptor (anti-M2-R) with atrial fibrosis in long-standing persistent atrial fibrillation (AF) patients. Methods. Twenty-four long-standing persistent AF patients, scheduled to undergo hybrid ablation surgery, were enrolled in the study. Twenty-six patients with sinus rhythm, scheduled to undergo coronary artery bypass grafting surgery, were enrolled into the non-AF group. We detected serum anti-M2-R levels. Left atrial appendages were subjected to histological and molecular biological assays. Patients in the AF group received follow-up for two years. Results. The AF group showed significantly higher serum anti-M2-R levels compared to the non-AF group (496.2 ± 232.5 vs. 86.3 ± 25.7 pmol/L, p<0.001). The AF group exhibited severe fibrosis in the left atrial appendages, as indicated by increased collagen volume fraction (45.2 ± 4.7% vs. 27.6 ± 8.3%, p<0.001), and higher levels of collagen I (0.52 ± 0.04 vs. 0.24 ± 0.06, p<0.001) and collagen III (0.51 ± 0.07 vs. 0.36 ± 0.09, p<0.001). TGF-β1 and CTGF were also upregulated in the AF group. A positive correlation between serum anti-M2-R levels and fibrosis of the left atrial appendage and fibrogenic indexes was observed. Conclusions. Serum anti-M2-R levels are higher in AF patients and are associated with the severity of atrial fibrosis. In addition, serum anti-M2-R levels are positively correlated to TGF-β1 and CTGF expression in the left atrial appendage.

Background: Although reticulocalbin 3 (Rcn3) has a critical role in alveolar epithelial function as well as in pathogenesis of pulmonary fibrosis, no study has yet examined its diagnostic and prognostic values for interstitial lung disease (ILD). This study aimed to evaluate Rcn3 as a potential marker in differential diagnosis of idiopathic pulmonary fibrosis (IPF) and connective tissue disease-associated interstitial lung disease (CTD-ILD) and in reflecting the severity of disease. Methods: This was a retrospective observational pilot study included 71 ILD patients and 39 healthy controls. These patients were stratified into IPF group (39) and CTD-ILD group (32). The severity of ILD was evaluated through pulmonary function test. Results: Serum Rcn3 level was statistically higher in CTD-ILD patients than that in IPF patients (p = 0.017) and healthy controls (p = 0.010). Serum Rcn3 further showed statistically negative correlation with pulmonary function indexes (TLC% pred and DLCO% pred) and positive correlation with inflammatory indexes (CRP and ESR) (r = − 0.367, p = 0.039; r = − 0.370, p = 0.037; r = 0.355, p = 0.046; r = 0.392, p = 0.026, respectively) in CTD-ILD patients rather than IPF patients. ROC analysis demonstrated that serum Rcn3 had superior diagnostic value for CTD-ILD and a cutoff value of 2.73 ng/mL had a sensitivity of 69%, a specificity of 69% and an accuracy of 45% for diagnose of CTD-ILD. Conclusions: Serum Rcn3 levels might be a clinically useful biomarker in screening and evaluating CTD-ILD.