CXCL14 is a candidate biomarker for Hedgehog signalling in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is associated with aberrant expression of developmental pathways, including Hedgehog (Hh). As Hh signalling contributes to multiple pro-fibrotic processes, Hh inhibition may represent a therapeutic option for IPF. However, no non-invasive biomarkers are available to monitor lung Hh activity. METHODS: We assessed gene and protein expression in IPF and control lung biopsies, mouse lung, fibroblasts stimulated in vitro with sonic hedgehog (SHh), and plasma in IPF patients versus controls, and cancer patients before and after treatment with vismodegib, a Hh inhibitor. RESULTS: Lung tissue from IPF patients exhibited significantly greater expression of Hh-related genes versus controls. The gene most significantly upregulated in both IPF lung biopsies and fibroblasts stimulated in vitro with SHh was CXCL14, which encodes a soluble secreted chemokine whose expression is inhibited in vitro by the addition of vismodegib. CXCL14 expression was induced by SHh overexpression in mouse lung. Circulating CXCL14 protein levels were significantly higher in plasma from IPF patients than controls. In cancer patients, circulating CXCL14 levels were significantly reduced upon vismodegib treatment. CONCLUSIONS: CXCL14 is a systemic biomarker that could be used to identify IPF patients with increased Hh pathway activity and monitor the pharmacodynamic effects of Hh antagonist therapy in IPF. TRIAL REGISTRATION NUMBER: Post-results, NCT00968981.

Endogenously expressed IL-13Rα2 attenuates IL-13-mediated responses but does not activate signaling in human lung fibroblasts.

IL-13 can bind to two distinct receptors: a heterodimer of IL-13Rα1/IL-4Rα and IL-13Rα2. Whereas IL-13Rα1/IL-4Rα engagement by IL-13 leads to the activation of STAT6, the molecular events triggered by IL-13 binding to IL-13Rα2 remain incompletely understood. IL-4 can bind to and signal through the IL-13Rα1/IL-4Rα complex but does not interact with IL-13Rα2. Idiopathic pulmonary fibrosis is a progressive and generally fatal parenchymal lung disease of unknown etiology with no current pharmacologic treatment options that substantially prolong survival. Preclinical models of fibrotic diseases have implicated IL-13 activity on multiple cell types, including macrophages and fibroblasts, in initiating and perpetuating pathological fibrosis. In this study, we show that IL-13, IL-4, IL-13Rα2, and IL-13-inducible target genes are expressed at significantly elevated levels in lung tissue from patients with idiopathic pulmonary fibrosis compared with control lung tissue. IL-4 and IL-13 induce virtually identical transcriptional responses in human monocytes, macrophages, and lung fibroblasts. IL-13Rα2 expression can be induced in lung fibroblasts by IL-4 or IL-13 via a STAT6-dependent mechanism, or by TNF-α via a STAT6-independent mechanism. Endogenously expressed IL-13Rα2 decreases, but does not abolish, sensitivity of lung fibroblasts to IL-13 and does not affect sensitivity to IL-4. Genome-wide transcriptional analyses of lung fibroblasts stimulated with IL-13 in the presence of Abs that selectively block interactions of IL-13 with IL-13Rα1/IL-4Rα or IL-13Rα2 show that endogenously expressed IL-13Rα2 does not activate any unique IL-13-mediated gene expression patterns, confirming its role as a decoy receptor for IL-13 signaling.

Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis.

BACKGROUND: There is microscopic spatial and temporal heterogeneity of pathological changes in idiopathic pulmonary fibrosis (IPF) lung tissue, which may relate to heterogeneity in pathophysiological mediators of disease and clinical progression. We assessed relationships between gene expression patterns, pathological features, and systemic biomarkers to identify biomarkers that reflect the aggregate disease burden in patients with IPF. METHODS: Gene expression microarrays (N=40 IPF; 8 controls) and immunohistochemical analyses (N=22 IPF; 8 controls) of lung biopsies. Clinical characterisation and blood biomarker levels of MMP3 and CXCL13 in a separate cohort of patients with IPF (N=80). RESULTS: 2940 genes were significantly differentially expressed between IPF and control samples (|fold change| >1.5, p<0.05). Two clusters of co-regulated genes related to bronchiolar epithelium or lymphoid aggregates exhibited substantial heterogeneity within the IPF population. Gene expression in bronchiolar and lymphoid clusters corresponded to the extent of bronchiolisation and lymphoid aggregates determined by immunohistochemistry in adjacent tissue sections. Elevated serum levels of MMP3, encoded in the bronchiolar cluster, and CXCL13, encoded in the lymphoid cluster, corresponded to disease severity and shortened survival time (p<10(-7) for MMP3 and p<10(-5) for CXCL13; Cox proportional hazards model). CONCLUSIONS: Microscopic pathological heterogeneity in IPF lung tissue corresponds to specific gene expression patterns related to bronchiolisation and lymphoid aggregates. MMP3 and CXCL13 are systemic biomarkers that reflect the aggregate burden of these pathological features across total lung tissue. These biomarkers may have clinical utility as prognostic and/or surrogate biomarkers of disease activity in interventional studies in IPF.

A WISP1 antibody inhibits MRTF signaling to prevent the progression of established liver fibrosis.

Fibrosis is the major risk factor associated with morbidity and mortality in patients with non-alcoholic steatohepatitis (NASH)-driven chronic liver disease. Although numerous efforts have been made to identify the mediators of the initiation of liver fibrosis, the molecular underpinnings of fibrosis progression remain poorly understood, and therapies to arrest liver fibrosis progression are elusive. Here, we identify a pathway involving WNT1-inducible signaling pathway protein 1 (WISP1) and myocardin-related transcription factor (MRTF) as a central mechanism driving liver fibrosis progression through the integrin-dependent transcriptional reprogramming of myofibroblast cytoskeleton and motility. In mice, WISP1 deficiency protects against fibrosis progression, but not fibrosis onset. Moreover, the therapeutic administration of a novel antibody blocking WISP1 halted the progression of existing liver fibrosis in NASH models. These findings implicate the WISP1-MRTF axis as a crucial determinant of liver fibrosis progression and support targeting this pathway by antibody-based therapy for the treatment of NASH fibrosis.

Oncostatin M expression induced by bacterial triggers drives airway inflammatory and mucus secretion in severe asthma.

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population.

Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis-associated interstitial lung disease (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells and interrogated these signatures in a single-cell RNA-Seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.

Molecular programs of fibrotic change in aging human lung.

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging.

TGFß2 and TGFß3 isoforms drive fibrotic disease pathogenesis.

Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.

Osteopontin Links Myeloid Activation and Disease Progression in Systemic Sclerosis.

Progressive lung fibrosis is a major cause of mortality in systemic sclerosis (SSc) patients, but the underlying mechanisms remain unclear. We demonstrate that immune complexes (ICs) activate human monocytes to promote lung fibroblast migration partly via osteopontin (OPN) secretion, which is amplified by autocrine monocyte colony stimulating factor (MCSF) and interleukin-6 (IL-6) activity. Bulk and single-cell RNA sequencing demonstrate that elevated OPN expression in SSc lung tissue is enriched in macrophages, partially overlapping with CCL18 expression. Serum OPN is elevated in SSc patients with interstitial lung disease (ILD) and prognosticates future lung function deterioration in SSc cohorts. Serum OPN levels decrease following tocilizumab (monoclonal anti-IL-6 receptor) treatment, confirming the connection between IL-6 and OPN in SSc patients. Collectively, these data suggest a plausible link between autoantibodies and lung fibrosis progression, where circulating OPN serves as a systemic proxy for IC-driven profibrotic macrophage activity, highlighting its potential as a promising biomarker in SSc ILD.

Keratin-dependent regulation of Aire and gene expression in skin tumor keratinocytes.

Expression of the intermediate filament protein keratin 17 (K17) is robustly upregulated in inflammatory skin diseases and in many tumors originating in stratified and pseudostratified epithelia. We report that autoimmune regulator (Aire), a transcriptional regulator, is inducibly expressed in human and mouse tumor keratinocytes in a K17-dependent manner and is required for timely onset of Gli2-induced skin tumorigenesis in mice. The induction of Aire mRNA in keratinocytes depends on a functional interaction between K17 and the heterogeneous nuclear ribonucleoprotein hnRNP K. Further, K17 colocalizes with Aire protein in the nucleus of tumor-prone keratinocytes, and each factor is bound to a specific promoter region featuring an NF-κB consensus sequence in a relevant subset of K17- and Aire-dependent proinflammatory genes. These findings provide radically new insight into keratin intermediate filament and Aire function, along with a molecular basis for the K17-dependent amplification of inflammatory and immune responses in diseased epithelia.

Isolation and characterization of the human CP49 gene promoter.

PURPOSE: This study focuses on the identification of regulatory elements that contribute to lens-specific expression of the human CP49 gene within the 5'-flanking DNA sequences. METHODS: The DNA sequence upstream of the human CP49 coding region was subcloned as a set of 5' and 3' deletion series. The constructs were transfected into lens (N/N1003A) and nonlens (NIH3T3) cell lines and chicken primary lens cultures, to test for promoter activity and specificity. To further test the specificity, a portion of the 5' flanking DNA sequence was used to drive transgene expression in mice. The flanking DNA sequence was analyzed for potential transcription factor-binding sites. RESULTS: The 5'-flanking DNA preferentially activated reporter gene expression in a lens-preferred manner when transfected into cultured cells. Transgene expression driven by the CP49 promoter region was lens specific. Analysis of the proximal promoter sequence revealed the presence of potential binding sites for the AP-1, AP-2, and OCT-1 transcription factors and the absence of TATA and CAAT boxes. CONCLUSIONS: The sequence upstream of the CP49 gene possesses promoter activity and is able to drive lens-preferred expression in both transfection and transgenic experiments. Promoter activity is dependent on the presence of the proximal 300 bp directly upstream of the coding region.

Analysis of non-crystallin lens fiber cell gene expression in c-Maf -/- mice.

PURPOSE: To examine the role of the c-Maf transcription factor in the regulation of non-crystallin lens fiber cell specific gene expression. METHODS: Expression of lens fiber cell genes in c-Maf null mice was analyzed by immunohistochemical and RT-PCR methods. In addition, the effect of c-Maf on fiber cell gene promoter fragments was examined in co-transfection experiments. RESULTS: Examination of protein expression in c-Maf homozygous mutant mice revealed undetectable levels of CP49, CP115 and MIP protein. mRNA transcripts for these non-crystallin genes were observed at reduced levels in subsequent RT-PCR experiments. The co-transfection of a c-Maf expression construct with reporter constructs containing the proximal promoters of the CP49 and CP115 genes, the structural components of the lens fiber cell beaded filament, did not result in activation of reporter gene expression, as was observed with a crystallin promoter. CONCLUSIONS: While c-Maf has been shown to play a critical role in lens specific expression of the numerous crystallin genes, it does not appear to regulate some non-crystallin fiber cell genes in the same manner. In contrast to most crystallin genes, detectable levels of CP49, CP115, and MIP transcripts are achieved in the absence of c-Maf. In co-transfection experiments, the CP49 and CP115 proximal promoter sequences were not activated by c-Maf. This suggests that increases in transcription of non-crystallin lens fiber cell genes can be enacted in a c-Maf independent fashion.