Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) in vitro in normal human lung fibroblasts; 2) in vivo in bleomycin and recombinant Ad-TGF-ß (adenovirus transforming growth factor-ß) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-ß-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-ß, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

Protein tyrosine phosphatase α mediates profibrotic signaling in lung fibroblasts through TGF-ß responsiveness.

Fibrotic lung diseases represent a diverse group of progressive and often fatal disorders with limited treatment options. Although the pathogenesis of these conditions remains incompletely understood, receptor type protein tyrosine phosphatase α (PTP-α encoded by PTPRA) has emerged as a key regulator of fibroblast signaling. We previously reported that PTP-α regulates cellular responses to cytokines and growth factors through integrin-mediated signaling and that PTP-α promotes fibroblast expression of matrix metalloproteinase 3, a matrix-degrading proteinase linked to pulmonary fibrosis. Here, we sought to determine more directly the role of PTP-α in pulmonary fibrosis. Mice genetically deficient in PTP-α (Ptpra(-/-)) were protected from pulmonary fibrosis induced by intratracheal bleomycin, with minimal alterations in the early inflammatory response or production of TGF-ß. Ptpra(-/-) mice were also protected from pulmonary fibrosis induced by adenoviral-mediated expression of active TGF-ß1. In reciprocal bone marrow chimera experiments, the protective phenotype tracked with lung parenchymal cells but not bone marrow-derived cells. Because fibroblasts are key contributors to tissue fibrosis, we compared profibrotic responses in wild-type and Ptpra(-/-) mouse embryonic and lung fibroblasts. Ptpra(-/-) fibroblasts exhibited hyporesponsiveness to TGF-ß, manifested by diminished expression of αSMA, EDA-fibronectin, collagen 1A, and CTGF. Ptpra(-/-) fibroblasts exhibited markedly attenuated TGF-ß-induced Smad2/3 transcriptional activity. We conclude that PTP-α promotes profibrotic signaling pathways in fibroblasts through control of cellular responsiveness to TGF-ß.

Neutrophil transmigration triggers repair of the lung epithelium via beta-catenin signaling.

Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated ß-catenin signaling, and this was verified by real-time PCR, nuclear translocation of ß-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of ß-catenin signaling in response to neutrophil transmigration. Knockdown of ß-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of ß-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of ß-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that ß-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

Role of ß-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury.

Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that ß-catenin signaling promotes epithelial repair after inflammatory injury, but the ß-catenin target genes that mediate this effect are unknown. Herein, we examined which ß-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of ß-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the ß-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that ß-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

Matrix metalloproteinase 3 is a mediator of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) may be triggered by epithelial injury that results in aberrant production of growth factors, cytokines, and proteinases, leading to proliferation of myofibroblasts, excess deposition of collagen, and destruction of the lung architecture. The precise mechanisms and key signaling mediators responsible for this aberrant repair process remain unclear. We assessed the importance of matrix metalloproteinase-3 (MMP-3) in the pathogenesis of IPF through i) determination of MMP-3 expression in patients with IPF, ii) in vivo experiments examining the relevance of MMP-3 in experimental models of fibrosis, and iii) in vitro experiments to elucidate possible mechanisms of action. Gene expression analysis, quantitative RT-PCR, and Western blot analysis of explanted human lungs revealed enhanced expression of MMP-3 in IPF, compared with control. Transient adenoviral vector-mediated expression of recombinant MMP-3 in rat lung resulted in accumulation of myofibroblasts and pulmonary fibrosis. Conversely, MMP-3-null mice were protected against bleomycin-induced pulmonary fibrosis. In vitro treatment of cultured lung epithelial cells with purified MMP-3 resulted in activation of the ß-catenin signaling pathway, via cleavage of E-cadherin, and induction of epithelial-mesenchymal transition. These processes were inhibited in bleomycin-treated MMP-3-null mice, as assessed by cytosolic translocation of ß-catenin and cyclin D1 expression. These observations support a novel role for MMP-3 in the pathogenesis of IPF, through activation of ß-catenin signaling and induction of epithelial-mesenchymal transition.

In vivo interferon-gamma induced changes in gene expression dramatically alter neutrophil phenotype.

The cytokine Interferon-γ (IFN-γ) exerts powerful immunoregulatory effects on the adaptive immune system and also enhances functions of the neutrophil (PMN). The clinical use of IFN-γ has been driven by the finding that its administration to patients with chronic granulomatous disease (CGD) results in decreased incidence and severity of infections. However, IFN-γ has no effect on the characteristic defect of CGD, the inability to convert oxygen to microbicidal metabolites including superoxide anion (O2-) during the phagocytosis associated oxidative burst. We administered varying doses of IFN-γ to adult volunteers and studied the effects on plasma drug levels and response molecules and PMNs isolated from blood drawn at intervals over a 96- hour period. Plasma concentrations of IFN-γ, IP-10 and neopterin, and stimulated release of O2- from PMNs exhibited dose- and time-dependent increases after IFN-γ administration. Gene expression in PMNs was altered for 2775 genes; changes occurred rapidly after administration and returned to baseline in 24-36 hours. Several genes involved with neutrophil host defense were upregulated including those for components of the O2- generating NADPH oxidase; innate-immune and Fc receptors; proteins involved in MHCI and II; a regulator of circulating PMN number; guanylate binding proteins; and a key enzyme in synthesis of an essential NOS cofactor. Coordinate changes were detected in protein levels of representative products from several of these genes. Lysates from isolated neutrophils also demonstrated a spike in NO following IFN-γ administration. IFN-γ appears to increase non-oxygen dependent microbicidal functions of PMNs which could provide strategies to compensate for deficiencies, explain its clinical benefit for CGD patients and expand therapeutic applications of IFN-γ to other disorders. Trial registration: Protocol registered in ClinicalTrials.gov, NCT02609932, Effect of IFN-γ on Innate Immune Cells.

Metabolic abnormalities in G6PC3-deficient human neutrophils result in severe functional defects.

Severe congenital neutropenia type 4 (SCN-4) is an autosomal recessive condition in which mutations in the G6PC3 gene encoding for the catalytic 3 subunit of glucose-6-phosphatase-ß result in neutropenia, neutrophil dysfunction, and other syndromic features. We report a child with SCN-4 caused by compound heterozygous mutations in G6PC3, a previously identified missense mutation in exon 6 (c.758G>A[p.R235H]), and a novel missense mutation in exon 2 (c.325G>A[p.G109S]). The patient had recurrent bacterial infections, inflammatory bowel disease, neutropenia, and intermittent thrombocytopenia. Administration of granulocyte colony-stimulating factor (G-CSF) resolved the neutropenia and allowed for detailed evaluation of human neutrophil function. Random and directed migration by the patient's neutrophils was severely diminished. Associated with this were defects in CD11b expression and F-actin assembly. Bactericidal activity at bacteria/neutrophil ratios >1:1 was also diminished and was associated with attenuated ingestion. Superoxide anion generation was <25% of control values, but phox proteins appeared quantitatively normal. Extensive metabolomics analysis at steady state and upon incubation with stable isotope-labeled tracers (U-13C-glucose, 13C,15N-glutamine, and U-13C-fructose) demonstrated dramatic impairments in early glycolysis (hexose phosphate levels), hexosemonophosphate shunt (required for the generation of the NADPH), and the total adenylate pool, which could explain the dramatic cell dysfunction displayed by the patient's neutrophils. Preliminary experiments with fructose supplementation to bypass the enzyme block demonstrated that the metabolic profile could be reversed, but was not sustained long enough for functional improvement. In human deficiency of G6PC3, metabolic defects resulting from the enzyme deficiency account for diverse neutrophil functional defects and present a major risk of infection.

Leukocyte elastase induces lung epithelial apoptosis via a PAR-1-, NF-kappaB-, and p53-dependent pathway.

Leukocyte elastase induces apoptosis of lung epithelial cells via alterations in mitochondrial permeability, but the signaling pathways regulating this response remain uncertain. Here we investigated the involvement of proteinase-activated receptor-1 (PAR-1), the transcription factor NF-kappaB, and the protooncogene p53 in this pathway. Elastase-induced apoptosis of lung epithelial cells correlated temporally with activation of NF-kappaB, phosphorylation, and nuclear translocation of p53, increased p53 up-regulated modulator of apoptosis (PUMA) expression, and mitochondrial translocation of Bax resulting in enhanced permeability. Elastase-induced apoptosis was also prevented by pharmacologic inhibitors of NF-kappaB and p53 and by short interfering RNA knockdown of PAR-1, p53, or PUMA. These inhibitors prevented elastase-induced PUMA expression, mitochondrial translocation of Bax, increased mitochondrial permeability, and attenuated apoptosis. NF-kappaB inhibitors also reduced p53 phosphorylation. We conclude that elastase-induced apoptosis of lung epithelial cells is mediated by a PAR-1-triggered pathway involving activation of NF-kappaB and p53, and a PUMA- and Bax-dependent increase in mitochondrial permeability leading to activation of distal caspases. Further, p53 contributes to elastase-induced apoptosis by both transcriptional and post-transcriptional mechanisms.

A novel method for long term bone marrow culture and genetic modification of murine neutrophils via retroviral transduction.

Neutrophils are a critical component of the innate immune response to invading microbial pathogens. However, an excessive and/or prolonged neutrophil response can result in tissue injury that is thought to underlie the pathogenesis of various inflammatory diseases. The development of novel therapeutic strategies for inflammatory diseases depends on an improved understanding of regulation of neutrophil function. However, investigations into neutrophil function have been constrained in part by the difficulty of genetically modifying neutrophils using current techniques. To overcome this, we have developed a novel method for the genetic modification of murine bone marrow derived progenitor cells using retroviral transduction followed by long term bone marrow culture to generate mature neutrophils. These neutrophils are functionally mature as determined by morphology, surface marker (Gr1, CD11b, CD62L and CXCR2) expression, and functional attributes including the ability to generate superoxide, exocytose granule contents, chemotax, and phagocytose and kill bacteria. Further, the in vitro matured neutrophils are capable of migrating to an inflammatory site in vivo. We utilized this system to express the Bcl-2 transgene in mature neutrophils using the retroviral vectors pMIG and pMIT. Bcl-2 overexpression conferred a substantial delay in spontaneous apoptosis of neutrophils as assessed by annexin V and 7-amino-actinomycin D (7AAD) staining. Moreover, Bcl-2 overexpression did not alter granulopoiesis, as assessed by morphology and surface marker expression. This system enables the genetic manipulation of progenitor cells that can be differentiated in vitro to mature neutrophils that are functional in vitro and in vivo.

Ndfip1 protein promotes the function of itch ubiquitin ligase to prevent T cell activation and T helper 2 cell-mediated inflammation.

Nedd4 family interacting protein-1 (Ndfip1) is a protein whose only known function is that it binds Nedd4, a HECT-type E3 ubiquitin ligase. Here we show that mice lacking Ndfip1 developed severe inflammation of the skin and lung and died prematurely. This condition was due to a defect in Ndfip1(-/-) T cells. Ndfip1(-/-) T cells were activated, and they proliferated and adopted a T helper 2 (Th2) phenotype more readily than did their Ndfip1(+/+) counterparts. This phenotype resembled that of Itchy mutant mice, suggesting that Ndfip1 might affect the function of Itch, an E3 ubiquitin ligase. We show that T cell activation promoted both Ndfip1 expression and its association with Itch. In the absence of Ndfip1, JunB half-life was prolonged after T cell activation. Thus, in the absence of Ndfip1, Itch is inactive and JunB accumulates. As a result, T cells produce Th2 cytokines and promote Th2-mediated inflammatory disease.