Regulation of Pulmonary Bacterial Immunity by Follistatin-Like Protein 1.

Klebsiella pneumoniae is a common cause of antibiotic-resistant pneumonia. Follistatin-like protein 1 (FSTL-1) is highly expressed in the lung and is critical for lung homeostasis. The role of FSTL-1 in immunity to bacterial pneumonia is unknown. Wild-type (WT) and FSTL-1 hypomorphic (Hypo) mice were infected with Klebsiella pneumoniae to determine infectious burden, immune cell abundance, and cytokine production. FSTL-1 Hypo/TCRδ-/- and FSTL-1 Hypo/IL17ra-/- were also generated to assess the role of γδT17 cells in this model. FSTL-1 Hypo mice had reduced K. pneumoniae lung burden compared with that of WT controls. FSTL-1 Hypo mice had increased Il17a/interleukin-17A (IL-17A) and IL-17-dependent cytokine expression. FSTL-1 Hypo lungs also had increased IL-17A+ and TCRγδ+ cells. FSTL-1 Hypo/TCRδ-/- displayed a lung burden similar to that of FSTL-1 Hypo and reduced lung burden compared with the TCRδ-/- controls. However, FSTL-1 Hypo/TCRδ-/- mice had greater bacterial dissemination than FSTL-1 Hypo mice, suggesting that gamma delta T (γδT) cells are dispensable for FSTL-1 Hypo control of pulmonary infection but are required for dissemination control. Confusing these observations, FSTL-1 Hypo/TCRδ-/- lungs had an increased percentage of IL-17A-producing cells compared with that of TCRδ-/- mice. Removal of IL-17A signaling in the FSTL-1 Hypo mouse resulted in an increased lung burden. These findings identify a novel role for FSTL-1 in innate lung immunity to bacterial infection, suggesting that FSTL-1 influences type-17 pulmonary bacterial immunity.

MicroRNA-144-3p targets relaxin/insulin-like family peptide receptor 1 (RXFP1) expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis.

The hormone relaxin is considered a potential therapy for idiopathic pulmonary fibrosis (IPF). We have previously shown that a potential limitation to relaxin-based IPF therapy is decreased expression of a relaxin receptor, relaxin/insulin-like family peptide receptor 1 (RXFP1), in IPF fibroblasts. The mechanism that down-regulates RXFP1 in IPF remains unclear. To determine whether microRNAs (miRs) regulate RXFP1 gene expression, here we employed a bioinformatics approach to identify miRs predicted to target RXFP1 and identified a putative miR-144-3p target site in the RXFP1 mRNA. In situ hybridization of IPF lung biopsies revealed that miR-144-3p is expressed in fibroblastic foci. Furthermore, we found that miR-144-3p is up-regulated in IPF fibroblasts compared with lung fibroblasts from healthy donors. Transforming growth factor ß increased miR-144-3p expression in both healthy and IPF lung fibroblasts in a SMAD family 2/3 (SMAD2/3)-dependent manner, and Jun proto-oncogene AP-1 transcription factor subunit (AP-1) was required for constitutive miR-144-3p expression. Overexpression of an miR-144-3p mimic significantly reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in healthy lung fibroblasts. IPF lung fibroblasts transfected with anti-miR-144-3p had increased RXFP1 expression and reduced α-SMA expression. Of note, a lentiviral luciferase reporter carrying the WT 3' UTR of RXFP1 was significantly repressed in IPF lung fibroblasts, whereas a reporter carrying a mutated miR-144-3p-binding site exhibited less sensitivity toward endogenous miR-144-3p expression, indicating that miR-144-3p down-regulates RXFP1 in IPF lung fibroblasts by targeting its 3' UTR. We conclude that miR-144-3p directly represses RXFP1 mRNA and protein expression.

Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow-derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.

Expression of RXFP1 Is Decreased in Idiopathic Pulmonary Fibrosis. Implications for Relaxin-based Therapies.

RATIONALE: Relaxin is a hormone that has been considered as a potential therapy for patients with fibrotic diseases. OBJECTIVES: To gauge the potential efficacy of relaxin-based therapies in idiopathic pulmonary fibrosis (IPF), we studied gene expression for relaxin/insulin-like family peptide receptor 1 (RXFP1) in IPF lungs and controls. METHODS: We analyzed gene expression data obtained from the Lung Tissue Research Consortium and correlated RXFP1 gene expression data with cross-sectional clinical and demographic data. We also employed ex vivo donor and IPF lung fibroblasts to test RXFP1 expression in vitro. We tested CGEN25009, a relaxin-like peptide, in lung fibroblasts and in bleomycin injury. MEASUREMENTS AND MAIN RESULTS: We found that RXFP1 is significantly decreased in IPF. In patients with IPF, the magnitude of RXFP1 gene expression correlated directly with diffusing capacity of the lung for carbon monoxide (P < 0.0001). Significantly less RXFP1 was detected in vitro in IPF fibroblasts than in donor controls. Transforming growth factor-ß decreased RXFP1 in both donor and IPF lung fibroblasts. CGEN25009 was effective at decreasing bleomycin-induced, acid-soluble collagen deposition in vivo. The relaxin-like actions of CGEN25009 were abrogated by RXFP1 silencing in vitro, and, in comparison with donor lung fibroblasts, IPF lung fibroblasts exhibited decreased sensitivity to the relaxin-like effects of CGEN25009. CONCLUSIONS: IPF is characterized by the loss of RXFP1 expression. RXFP1 expression is directly associated with pulmonary function in patients with IPF. The relaxin-like effects of CGEN25009 in vitro are dependent on expression of RXFP1. Our data suggest that patients with IPF with the highest RXFP1 expression would be predicted to be most sensitive to relaxin-based therapies.