Milena S. Espindola

Microbes are associated with host innate immune response in idiopathic pulmonary fibrosis

&NA; Rationale: Differences in the lung microbial community influence idiopathic pulmonary fibrosis (IPF) progression. Whether the lung microbiome influences IPF host defense remains unknown. Objectives: To explore the host immune response and microbial interaction in IPF as they relate to progression‐free survival (PFS), fibroblast function, and leukocyte phenotypes. Methods: Paired microarray gene expression data derived from peripheral blood mononuclear cells as well as 16S ribosomal RNA sequencing data from bronchoalveolar lavage obtained as part of the COMET‐IPF (Correlating Outcomes with Biochemical Markers to Estimate Time‐Progression in Idiopathic Pulmonary Fibrosis) study were used to conduct association pathway analyses. The responsiveness of paired lung fibroblasts to Toll‐like receptor 9 (TLR9) stimulation by CpG‐oligodeoxynucleotide (CpG‐ODN) was integrated into microbiome‐gene expression association analyses for a subset of individuals. The relationship between associated pathways and circulating leukocyte phenotypes was explored by flow cytometry. Measurements and Main Results: Down‐regulation of immune response pathways, including nucleotide‐binding oligomerization domain (NOD)‐, Toll‐, and RIG1‐like receptor pathways, was associated with worse PFS. Ten of the 11 PFS‐associated pathways correlated with microbial diversity and individual genus, with species accumulation curve richness as a hub. Higher species accumulation curve richness was significantly associated with inhibition of NODs and TLRs, whereas increased abundance of Streptococcus correlated with increased NOD‐like receptor signaling. In a network analysis, expression of up‐regulated signaling pathways was strongly associated with decreased abundance of operational taxonomic unit 1341 (OTU1341; Prevotella) among individuals with fibroblasts responsive to CpG‐ODN stimulation. The expression of TLR signaling pathways was also linked to CpG‐ODN responsive fibroblasts, OTU1341 (Prevotella), and Shannon index of microbial diversity in a network analysis. Lymphocytes expressing C‐X‐C chemokine receptor 3 CD8 significantly correlated with OTU1348 (Staphylococcus). Conclusions: These findings suggest that host‐microbiome interactions influence PFS and fibroblast responsiveness.

Targeting of TAM Receptors Ameliorates Fibrotic Mechanisms in Idiopathic Pulmonary Fibrosis

Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling, which progressively abolishes lung function in an RTK (receptor tyrosine kinase)‐dependent manner. Gas6 (growth arrest‐specific 6) ligand, Tyro3 (TYRO3 protein tyrosine kinase 3), and Axl (anexelekto) RTK expression and activity are increased in IPF. Objectives: To determine if targeting these RTK pathways would inhibit fibroblast activation and the development of pulmonary fibrosis. Methods: Quantitative genomic, proteomic, and functional analyses were used to determine Gas6/TAM (Tyro3, Axl, and Mertk [MER proto‐oncogene, tyrosine kinase]) RTK expression and activation in tissues and fibroblasts from normal and IPF lungs. The profibrotic impact of these RTK pathways were also examined in bleomycin‐induced pulmonary fibrosis and in SCID/Bg mice that developed pulmonary fibrosis after the intravenous administration of primary IPF fibroblasts. Measurements and Main Results: Gas6, Axl, and Tyro3 were increased in both rapidly and slowly progressive IPF compared with normal lung samples and fibroblasts. Targeting these pathways with either specific antibodies directed at Gas6 or Axl, or with small‐molecule TAM inhibitors indicated that the small molecule‐mediated targeting approach was more efficacious in both in vitro and in vivo studies. Specifically, the TAM receptor inhibitor R428 (also known as BGB324) significantly inhibited the synthetic, migratory, and proliferative properties of IPF fibroblasts compared with the other Gas6/TAM receptor targeting agents. Finally, loss of Gas6 expression decreased lung fibrotic responses to bleomycin and treatment with R428 inhibited pulmonary fibrosis in humanized SCID/Bg mice. Conclusions: Gas6/TAM receptor activity contributes to the activation of pulmonary fibroblasts in IPF, suggesting that targeting this RTK pathway might be an effective antifibrotic strategy in this disease.

Modeling Idiopathic Pulmonary Fibrosis in Humanized Severe Combined Immunodeficient Mice

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease of unknown etiopathogenesis with limited therapeutic options. IPF is characterized by an abundance of fibroblasts and loss of epithelial progenitors, which cumulates in unrelenting fibrotic lung remodeling and loss of normal oxygenation. IPF has been challenging to model in rodents; nonetheless, mouse models of lung fibrosis provide clues as to the natural progression of lung injury and remodeling, but many have not been useful in predicting efficacy of therapeutics in clinical IPF. We provide a detailed methodologic description of various iterations of humanized mouse models, initiated by the i.v. injection of cells from IPF lung biopsy or explants specimens into severe combined immunodeficiency (SCID)/beige or nonobese diabetic SCID γ mice. Unlike cells from normal lung samples, IPF cells promote persistent, nonresolving lung remodeling in SCID mice. Finally, we provide examples and discuss potential advantages and pitfalls of human-specific targeting approaches in a humanized SCID model of pulmonary fibrosis.

Divergent roles for Clusterin in Lung Injury and Repair

Lung fibrosis is an unabated wound healing response characterized by the loss and aberrant function of lung epithelial cells. Herein, we report that extracellular Clusterin promoted epithelial cell apoptosis whereas intracellular Clusterin maintained epithelium viability during lung repair. Unlike normal and COPD lungs, IPF lungs were characterized by significantly increased extracellular Clusterin whereas the inverse was evident for intracellular Clusterin. In vitro and in vivo studies demonstrated that extracellular Clusterin promoted epithelial cell apoptosis while intercellular Clusterin modulated the expression of the DNA repair proteins, MSH2, MSH6, OGG1 and BRCA1. The fibrotic response in Clusterin deficient (CLU−/−) mice persisted after bleomycin and it was associated with increased DNA damage, reduced DNA repair responses, and elevated cellular senescence. Remarkably, this pattern mirrored that observed in IPF lung tissues. Together, our results show that cellular localization of Clusterin leads to divergent effects on epithelial cell regeneration and lung repair during fibrosis.

Characterization of CD28 null T cells in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease, with unknown etiopathogenesis and suboptimal therapeutic options. Previous reports have shown that increased T-cell numbers and CD28null phenotype is predictive of prognosis in IPF, suggesting that these cells might have a role in this disease. Flow cytometric analysis of explanted lung cellular suspensions showed a significant increase in CD8+ CD28null T cells in IPF relative to normal lung explants. Transcriptomic analysis of CD3+ T cells isolated from IPF lung explants revealed a loss of CD28-transcript expression and elevation of pro-inflammatory cytokine expression in IPF relative to normal T cells. IPF lung explant-derived T cells (enriched with CD28null T cells), but not normal donor lung CD28+ T cells induced dexamethasone-resistant lung remodeling in humanized NSG mice. Finally, CD28null T cells expressed similar CTLA4 and significantly higher levels of PD-1 proteins relative to CD28+ T cells and blockade of either proteins in humanized NSG mice, using anti-CTLA4, or anti-PD1, mAb treatment-accelerated lung fibrosis. Together, these results demonstrate that IPF CD28null T cells may promote lung fibrosis but the immune checkpoint proteins, CTLA-4 and PD-1, appears to limit this effect.

CCR10+ epithelial cells from idiopathic pulmonary fibrosis lungs drive remodeling

Idiopathic pulmonary fibrosis (IPF) is a devastating fibrotic lung disease of unknown etiology and limited therapeutic options. In this report, we characterize what we believe is a novel CCR10+ epithelial cell population in IPF lungs. There was a significant increase in the percentage of CCR10+ epithelial cells in IPF relative to normal lung explants and their numbers significantly correlated to lung remodeling in humanized NSG mice. Cultured CCR10-enriched IPF epithelial cells promoted IPF lung fibroblast invasion and collagen 1 secretion. Single-cell RNA sequencing analysis showed distinct CCR10+ epithelial cell populations enriched for inflammatory and profibrotic transcripts. Consistently, cultured IPF but not normal epithelial cells induced lung remodeling in humanized NSG mice, where the number of CCR10+ IPF, but not normal, epithelial cells correlated with hydroxyproline concentration in the remodeled NSG lungs. A subset of IPF CCR10hi epithelial cells coexpress EphA3 and ephrin A signaling induces the expression of CCR10 by these cells. Finally, EphA3+CCR10hi epithelial cells induce more consistent lung remodeling in NSG mice relative to EphA3-CCR10lo epithelial cells. Our results suggest that targeting epithelial cells, highly expressing CCR10, may be beneficial in IPF.

Role of Immune Checkpoint Proteins in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease, with unknown etiopathogenesis and suboptimal therapeutic options. Due to the lack of clinical efficacy of standard immuno-suppressants in IPF, the role of the immune response in this disease remains elusive. Nevertheless, previous reports have shown that increased T cell numbers and phenotype is predictive of prognosis in IPF, suggesting that these cells might have a role in this disease. Transcriptomic analysis of CD3+ T cells isolated from IPF lungs removed prior to lung transplant (i.e. explant lung) revealed a loss of CD28 expression and both elevated checkpoint and lymphocyte activation pathways. Flow cytometric analysis of a mixture of immune and non-immune cells isolated from explanted IPF lungs showed elevated PD-1 and CTLA4 protein expression on CD4- lymphocytes and PD-L1 expression on EpCAM+ and CD45- EpCAM- cells. Lung remodeling and loss of BAL surfactant protein C were observed in NOD SCID IL-2Rγ-/- (NSG) mice that received an intravenous injection of a mixture of IPF cells, including purified IPF T cells. Finally, in humanized NSG mice, anti-CTLA4, but not anti-PD1, mAb treatment induced an expansion of CD3+ T cells and accelerated lung fibrosis. Together, these results demonstrate that IPF T cells are profibrotic but the immune checkpoint protein, CTLA-4, appears to limit this effect in IPF.