RESUMEN
Systemic sclerosis (SSc) is a progressive, multiorgan disease with limited treatment options. Although a recent proof-of-concept study using romilkimab or SAR156597, a bispecific IL-4/IL-13 antibody, suggests a direct role of these cytokines in the pathophysiology of SSc, their contributions to the balance between inflammation and fibrosis are unclear. Here, we determine the roles of type 2 inflammation in fibrogenesis using FRA2-Tg (Fos-related antigen 2-overexpressing transgenic) mice, which develop spontaneous, age-dependent progressive lung fibrosis. We defined the molecular signatures of inflammation and fibrosis at three key stages in disease progression, corresponding to preonset, inflammatory dominant, and fibrosis dominant biology, and revealed an early increase in cytokine-cytokine receptor interactions and antigen-processing and presentation pathways followed by enhanced Th2- and M2 macrophage-driven type 2 responses. This type 2 inflammation progressed to extensive fibrotic pathology by 14-18 weeks of age, with these gene signatures overlapping significantly with those seen in the lungs of patients with SSc with interstitial lung disease (ILD). These changes were also evident in the histopathology, which showed perivascular and peribronchiolar inflammation with prominent eosinophilia and accumulation of profibrotic M2-like macrophages followed by rapid progression to fibrosis with thickened alveolar walls with multifocal fibrotic bands and signs of interstitial pneumonia. Critically, treatment with a bispecific antibody targeting IL-4 and IL-13 during the inflammatory phase abrogated the Th2 and M2 responses and led to near-complete abrogation of lung fibrosis. These data recapitulate important features of fibrotic progression in the lungs of patients with SSc-ILD and enhance our understanding of the progressive pathobiology of SSc. This study also further establishes FRA2-Tg mice as a valuable tool for testing future therapeutic agents in SSc-ILD.
Asunto(s)
Enfermedades Pulmonares Intersticiales , Fibrosis Pulmonar , Esclerodermia Sistémica , Humanos , Ratones , Animales , Fibrosis Pulmonar/patología , Interleucina-13 , Interleucina-4 , Enfermedades Pulmonares Intersticiales/patología , Fibrosis , Pulmón/patología , Inflamación/patología , Ratones Transgénicos , CitocinasRESUMEN
The composition of extracellular matrix (ECM) is altered during pathologic scarring in damaged organs including the lung. One major change in the ECM involves the cross-linking of collagen, which promotes fibroblast to myofibroblast differentiation. We examined the role of lysyl oxidase (LOX)-like 2 in lung progenitors and fibroblasts cultured from normal or IPF lung samples and in a humanized mouse model of IPF using a monoclonal antibody (Simtuzumab). Primary lung fibroblasts from normal donor lungs and IPF lung explants were examined for expression of LOXL2. Targeting LOXL2 with Simtuzumab on normal and IPF fibroblasts was examined both in vitro and in vivo for synthetic, functional, and profibrotic properties. LOXL2 was increased at transcript and protein level in IPF compared with normal lung samples. In a dose-dependent manner, Simtuzumab enhanced differentiation of fibroblasts into myofibroblasts. Inhibition of LOXL2 also enhanced fibroblast invasion and accelerated the outgrowth of fibroblasts from dissociated human lung cell preparations. Finally, preventative or delayed delivery of Simtuzumab enhanced lung fibrosis in a humanized mouse model of pulmonary fibrosis. Consistent with its failure in a Phase 2 clinical trial, Simtuzumab exhibited no therapeutic efficacy in translational in vitro and in vivo assays.
RESUMEN
Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant repair that diminishes lung function via mechanisms that remain poorly understood. CC chemokine receptor (CCR10) and its ligand CCL28 were both elevated in IPF compared with normal donors. CCR10 was highly expressed by various cells from IPF lungs, most notably stage-specific embryonic antigen-4-positive mesenchymal progenitor cells (MPCs). In vitro, CCL28 promoted the proliferation of CCR10+ MPCs while CRISPR/Cas9-mediated targeting of CCR10 resulted in the death of MPCs. Following the intravenous injection of various cells from IPF lungs into immunodeficient (NOD/SCID-γ, NSG) mice, human CCR10+ cells initiated and maintained fibrosis in NSG mice. Eph receptor A3 (EphA3) was among the highest expressed receptor tyrosine kinases detected on IPF CCR10+ cells. Ifabotuzumab-targeted killing of EphA3+ cells significantly reduced the numbers of CCR10+ cells and ameliorated pulmonary fibrosis in humanized NSG mice. Thus, human CCR10+ cells promote pulmonary fibrosis, and EphA3 mAb-directed elimination of these cells inhibits lung fibrosis.
Asunto(s)
Fibrosis Pulmonar Idiopática/metabolismo , Células Madre Mesenquimatosas/metabolismo , Receptor EphA3/metabolismo , Receptores CCR10/metabolismo , Células Epiteliales Alveolares/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Sistemas CRISPR-Cas , Quimiocinas CC/metabolismo , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Humanos , Fibrosis Pulmonar Idiopática/patología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Rationale: Aberrant lung remodeling in idiopathic pulmonary fibrosis (IPF) is characterized by elevated MMP9 (matrix metalloproteinase 9) expression, but the precise role of this matrix metalloproteinase in this disease has yet to be fully elucidated.Objectives: To evaluate antifibrotic effects of MMP9 inhibition on IPF.Methods: Quantitative genomic, proteomic, and functional analyses both in vitro and in vivo were used to determine MMP9 expression in IPF cells and the effects of MMP9 inhibition on profibrotic mechanisms.Measurements and Main Results: In the present study, we demonstrate that MMP9 expression was increased in airway basal cell (ABC)-like cells from IPF lungs compared with ABC cells from normal lungs. The inhibition of MMP9 activity with an anti-MMP9 antibody, andecaliximab, blocked TGF-ß1 (transforming growth factor ß1)-induced Smad2 phosphorylation. However, in a subset of cells from patients with IPF, TGF-ß1 activation in their ABC-like cells was unaffected or enhanced by MMP9 blockade (i.e., nonresponders). Further analysis of nonresponder ABC-like cells treated with andecaliximab revealed an association with type 1 IFN expression, and the addition of IFNα to these cells modulated both MMP9 expression and TGF-ß1 activation. Finally, the inhibition of MMP9 ameliorated pulmonary fibrosis induced by responder lung cells but not a nonresponder in a humanized immunodeficient mouse model of IPF.Conclusions: Together, these data demonstrate that MMP9 regulates the activation of ABC-like cells in IPF and that targeting this MMP might be beneficial to a subset of patients with IPF who show sufficient expression of type 1 IFNs.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/fisiopatología , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , California/epidemiología , Femenino , Humanos , Fibrosis Pulmonar Idiopática/epidemiología , Fibrosis Pulmonar Idiopática/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones , Michigan/epidemiología , Modelos Animales , Proteómica , Estados UnidosRESUMEN
Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.
Asunto(s)
Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Isoformas de Proteínas/metabolismo , Factor de Células Madre/metabolismo , Animales , Bleomicina/farmacología , Recuento de Células/métodos , Células Cultivadas , Técnicas de Cocultivo/métodos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis/tratamiento farmacológico , Fibrosis/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Pulmón/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Through the progressive accumulation of genetic and epigenetic alterations in cellular physiology, non-small-cell lung cancer (NSCLC) evolves in distinct steps involving mutually exclusive oncogenic mutations in K-Ras or EGFR along with inactivating mutations in the p53 tumor suppressor. Herein, we show two independent in vivo lung cancer models in which CHUK/IKK-α acts as a major NSCLC tumor suppressor. In a novel transgenic mouse strain, wherein IKKα ablation is induced by tamoxifen (Tmx) solely in alveolar type II (AT-II) lung epithelial cells, IKKα loss increases the number and size of lung adenomas in response to the chemical carcinogen urethane, whereas IKK-ß instead acts as a tumor promoter in this same context. IKKα knockdown in three independent human NSCLC lines (independent of K-Ras or p53 status) enhances their growth as tumor xenografts in immune-compromised mice. Bioinformatics analysis of whole transcriptome profiling followed by quantitative protein and targeted gene expression validation experiments reveals that IKKα loss can result in the up-regulation of activated HIF-1-α protein to enhance NSCLC tumor growth under hypoxic conditions in vivo.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Quinasa I-kappa B/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proliferación Celular/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/genética , Femenino , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Quinasa I-kappa B/deficiencia , Neoplasias Pulmonares/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regulación hacia Arriba , Proteínas ras/genéticaRESUMEN
Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-ß pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.
Asunto(s)
Fibrosis Pulmonar Idiopática/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Miofibroblastos/metabolismo , Animales , Biopsia , Diferenciación Celular , Femenino , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/patología , Ratones , Ratones SCID , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Tomografía Computarizada por Rayos X , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Recent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types. METHODS: Surgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441. RESULTS: DNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells. CONCLUSIONS: Together, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.
Asunto(s)
Senescencia Celular/genética , Cromonas/farmacología , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Células Madre Mesenquimatosas/citología , Morfolinas/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Daño del ADN , Reparación del ADN , Proteína Quinasa Activada por ADN/deficiencia , Proteínas de Unión al ADN/deficiencia , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Pulmón/patología , Ratones , Ratones SCIDRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease. A maladaptive epithelium due to chronic injury is a prominent feature and contributor to pathogenic cellular communication in IPF. Recent data highlight the concept of a "reprogrammed" lung epithelium as critical in the development of lung fibrosis. Extracellular vesicles (EVs) are potent mediator of cellular crosstalk, and recent evidence supports their role in lung pathologies such as IPF. Here, we demonstrate that syndecan-1 is overexpressed by the epithelium in the lungs of IPF patients and in murine models after bleomycin injury. Moreover, we find that syndecan-1 is a pro-fibrotic signal that alters alveolar type II (ATII) cell phenotypes by augmenting TGFß and Wnt signaling among other pro-fibrotic pathways. Importantly, we demonstrate that syndecan-1 controls the packaging of several anti-fibrotic microRNAs into EVs that have broad effects over several fibrogenic signaling networks as a mechanism of regulating epithelial plasticity and pulmonary fibrosis. Collectively, our work reveals new insight into how EVs orchestrate cellular signals that promote lung fibrosis and demonstrate the importance of syndecan-1 in coordinating these programs.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Vesículas Extracelulares/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Sindecano-1/metabolismo , Células Epiteliales Alveolares/patología , Animales , Bleomicina/efectos adversos , Línea Celular , Modelos Animales de Enfermedad , Vesículas Extracelulares/patología , Femenino , Humanos , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Lesión Pulmonar/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Transducción de Señal , Sindecano-1/genética , Transcriptoma , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unremitting extracellular matrix deposition, leading to a distortion of pulmonary architecture and impaired gas exchange. Fibroblasts from IPF patients acquire an invasive phenotype that is essential for progressive fibrosis. Here, we performed RNA sequencing analysis on invasive and noninvasive fibroblasts and found that the immune checkpoint ligand CD274 (also known as PD-L1) was upregulated on invasive lung fibroblasts and was required for the invasive phenotype of lung fibroblasts, is regulated by p53 and FAK, and drives lung fibrosis in a humanized IPF model in mice. Activating CD274 in IPF fibroblasts promoted invasion in vitro and pulmonary fibrosis in vivo. CD274 knockout in IPF fibroblasts and targeting CD274 by FAK inhibition or CD274-neutralizing antibodies blunted invasion and attenuated fibrosis, suggesting that CD274 may be a novel therapeutic target in IPF.
Asunto(s)
Antígeno B7-H1/metabolismo , Fibroblastos/metabolismo , Fibrosis/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Animales , Antígeno B7-H1/genética , Adhesión Celular , Femenino , Fibroblastos/patología , Fibrosis/patología , Humanos , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/terapia , Pulmón/patología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Fenotipo , TranscriptomaRESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/metabolismo , Fibrosis Pulmonar Idiopática/inmunología , Pulmón/patología , Receptor de Muerte Celular Programada 1/metabolismo , Subgrupos de Linfocitos T/inmunología , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4/inmunología , Separación Celular , Células Cultivadas , Citometría de Flujo , Humanos , Inmunofenotipificación , Ratones , Ratones SCID , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
Although cellular senescence may be a protective mechanism in modulating proliferative capacity, fibroblast senescence is now recognized as a key pathogenic mechanism in idiopathic pulmonary fibrosis (IPF). In aged mice, abundance and persistence of apoptosis-resistant senescent fibroblasts play a central role in nonresolving lung fibrosis after bleomycin challenge. Therefore, we investigated whether quercetin can restore the susceptibility of senescent IPF fibroblasts to proapoptotic stimuli and mitigate bleomycin-induced pulmonary fibrosis in aged mice. Unlike senescent normal lung fibroblasts, IPF lung fibroblasts from patients with stable and rapidly progressing disease were highly resistant to Fas ligand (FasL)-induced and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Senescent IPF fibroblasts exhibited decreased expression of FasL and TRAIL receptors and caveolin-1, as well as increased AKT activation, compared with senescent normal lung fibroblasts. Although quercetin alone was not proapoptotic, it abolished the resistance to FasL- or TRAIL-induced apoptosis in IPF fibroblasts. Mechanistically, quercetin upregulated FasL receptor and caveolin-1 expression and modulated AKT activation. In vivo quercetin reversed bleomycin-induced pulmonary fibrosis and attenuated lethality, weight loss, and the expression of pulmonary senescence markers p21 and p19-ARF and senescence-associated secretory phenotype in aged mice. Collectively, these data indicate that quercetin reverses the resistance to death ligand-induced apoptosis by promoting FasL receptor and caveolin-1 expression and inhibiting AKT activation, thus mitigating the progression of established pulmonary fibrosis in aged mice. Therefore, quercetin may be a viable therapeutic option for IPF and other age-related diseases that progress with the accumulation of senescent fibroblasts.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Fibroblastos/patología , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Quercetina/farmacología , Animales , Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Femenino , Fibroblastos/efectos de los fármacos , Humanos , Fibrosis Pulmonar Idiopática/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Células Epiteliales/inmunología , Fibrosis Pulmonar Idiopática/inmunología , Pulmón/inmunología , Mucosa Respiratoria/inmunología , Animales , Línea Celular , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/trasplante , Femenino , Fibrosis , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/citología , Pulmón/patología , Ratones , Ratones Endogámicos NOD , Receptores CCR10/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/patología , Organismos Libres de Patógenos Específicos , Quimera por TrasplanteRESUMEN
BACKGROUND: Recruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. We tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. METHODS: Blood monocytes from advanced and stable PCa patients were cultured, and the conditioned media (CM) were collected and analyzed using standard invasion and wound closure assays to measure effects on invasion and motility of PCa tumor cells. We then identified the proteome profile of these monocytes using proteome array and ELISA. RESULTS: Conditioned media from circulating monocytes in patients with metastatic prostate cancer (PCa-M) increased invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic castration-resistant (mCRPC) patients presented high levels of chitinase-3-like 1 (CHI3L1, YKL-40) when compared to patients with stable disease (PCa-N) and healthy control individuals (HC). The only described receptor for CHI3L1, interleukin-13 receptor α2 (IL-13Rα2), was significantly up-regulated in the human metastatic PCa cell line, ARCaPM . Accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPM cells while siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa-M patients. CONCLUSIONS: Thus, we show that circulating monocytes from metastatic PCa patients exert a tumor-promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa.
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Comunicación Celular , Movimiento Celular , Células Epiteliales/metabolismo , Monocitos/metabolismo , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Proteína 1 Similar a Quitinasa-3/metabolismo , Medios de Cultivo Condicionados/farmacología , Humanos , Interleucina-1beta/metabolismo , Masculino , Neoplasias de la Próstata/patologíaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease characterized by the persistence of activated myofibroblasts resulting in excessive deposition of extracellular matrix proteins and profound tissue remodeling. In the present study, the expression of tumor necrosis factor- (TNF-) related apoptosis-inducing ligand (TRAIL) was key to the resolution of bleomycin-induced pulmonary fibrosis. Both in vivo and in vitro studies demonstrated that Gr-1+TRAIL+ bone marrow-derived myeloid cells blocked the activation of lung myofibroblasts. Although soluble TRAIL was increased in plasma from IPF patients, the presence of TRAIL+ myeloid cells was markedly reduced in IPF lung biopsies, and primary lung fibroblasts from this patient group expressed little of the TRAIL receptor-2 (DR5) when compared with appropriate normal samples. IL-13 was a potent inhibitor of DR5 expression in normal fibroblasts. Together, these results identified TRAIL+ myeloid cells as a critical mechanism in the resolution of pulmonary fibrosis, and strategies directed at promoting its function might have therapeutic potential in IPF.
Asunto(s)
Fibrosis Pulmonar/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/inmunología , Fibroblastos/metabolismo , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Células Mieloides/metabolismo , Fibrosis Pulmonar/inmunología , Transducción de Señal/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/efectos de los fármacos , Antibióticos Antineoplásicos/uso terapéutico , Bleomicina/uso terapéutico , Fibroblastos/efectos de los fármacos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/genética , Proteínas de la Membrana/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular/efectos de los fármacos , Humanos , Fibrosis Pulmonar Idiopática/fisiopatología , Proteínas de la Membrana/genética , Persona de Mediana Edad , Proto-Oncogenes Mas , Transducción de Señal/genéticaRESUMEN
The respiratory tract is faced daily with 10,000 L of inhaled air. While the majority of air contains harmless environmental components, the pulmonary immune system also has to cope with harmful microbial or sterile threats and react rapidly to protect the host at this intimate barrier zone. The airways are endowed with a broad armamentarium of cellular and humoral host defense mechanisms, most of which belong to the innate arm of the immune system. The complex interplay between resident and infiltrating immune cells and secreted innate immune proteins shapes the outcome of host-pathogen, host-allergen, and host-particle interactions within the mucosal airway compartment. Here, we summarize and discuss recent findings on pulmonary innate immunity and highlight key pathways relevant for biomarker and therapeutic targeting strategies for acute and chronic diseases of the respiratory tract.
Asunto(s)
Biomarcadores/metabolismo , Inmunidad Innata , Enfermedades Pulmonares/inmunología , Pulmón/inmunología , Mucosa Respiratoria/inmunología , Animales , Humanos , Enfermedades Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Transducción de Señal , Investigación Biomédica TraslacionalRESUMEN
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.
Asunto(s)
Fibrosis Pulmonar Idiopática/patología , Animales , Anticuerpos Neutralizantes/farmacología , Bencilaminas , Ciclamas , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/uso terapéutico , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/metabolismo , Interleucina-13/metabolismo , Pulmón/patología , Ratones SCID , Fenotipo , Receptores CXCR4/metabolismo , Receptores de Interleucina-4/metabolismoRESUMEN
Syndecan-1 is a transmembrane proteoglycan expressed prominently by lung epithelium and has pleiotropic functions such as regulating cell migration, proliferation, and survival. Loss of syndecan-1 expression by lung cancer cells is associated with higher-grade cancers and worse clinical prognosis. We evaluated the effects of syndecan-1 in various cell-based and animal models of lung cancer and found that lung tumorigenesis was moderated by syndecan-1. We also demonstrate that syndecan-1 (or lack thereof) alters the miRNA cargo carried within exosomes exported from lung cancer cells. Analysis of the changes in miRNA expression identified a distinct shift toward augmented procancer signaling consistent with the changes found in lung adenocarcinoma. Collectively, our work identifies syndecan-1 as an important factor in lung cancer cells that shapes the tumor microenvironment through alterations in miRNA packaging within exosomes.