Decreased asbestos-induced lung inflammation and fibrosis after radiation and bone marrow transplant.
ABSTRACT: The effect of lung irradiation on subsequent inflammatory or fibrotic lung injuries remains poorly understood. We postulated that irradiation and bone marrow transplantation might impact the development and progression of lung remodeling resulting from asbestos inhalation. Our objective was to determine whether irradiation and bone marrow transplantation affected inflammation and fibrosis associated with inhaled asbestos exposure. Inflammation, cytokine production, and fibrosis were assessed in lungs of naïve and sex-mismatched chimeric mice exposed to asbestos for 3, 9, or 40 days. Potential engraftment of donor-derived cells in recipient lungs was examined by fluorescence in situ hybridization and immunohistochemistry. Compared with asbestos-exposed naïve (nonchimeric) mice, chimeric mice exposed to asbestos for 3, 9, or 40 days demonstrated significant abrogation of acute increases in asbestos-associated inflammatory mediators and fibrosis. Donor-derived cells trafficked to lung but did not significantly engraft as phenotypic lung cells. Irradiation and bone marrow transplantation alters inflammatory and fibrotic responses to asbestos, likely through modulation of soluble inflammatory mediators.
Project description:Lung exposure to radiation induces an injury response that includes the release of cytokines and chemotactic mediators; these signals recruit immune cells to execute inflammatory and wound-healing processes. However, radiation alters the pulmonary microenvironment, dysregulating the immune responses and preventing a return to homeostasis. Importantly, dysregulation is observed as a chronic inflammation, which can progress into pneumonitis and promote pulmonary fibrosis; inflammatory monocytes, which are bone marrow derived and express CCR2, have been shown to migrate into the lung after radiation exposure. Although the extent to which recruited inflammatory monocytes contribute to radiation-induced pulmonary fibrosis has not been fully investigated, we hypothesize that its pathogenesis is reliant on this population. The CC chemokine ligand, CCL2, is a chemotactic mediator responsible for trafficking of CCR2+ inflammatory cells into the lung. Therefore, the contribution of this mediator to fibrosis development was analyzed. Interleukin (IL)-1?, a potent pro-inflammatory cytokine expressed during the radiation response, and its receptor, IL-1R1, were also evaluated. To this end, CCR2-/-, IL-1?-/- and IL-1R1-/- chimeric mice were generated and exposed to 12.5 Gy thoracic radiation, and their response was compared to wild-type (C57BL/6) syngeneic controls. Fibrotic foci were observed in the periphery of the lungs of C57 syngeneic mice and CCR2-/- recipient mice that received C57 bone marrow (C57 > CCR2-/-) by 16 and 12 weeks after irradiation, respectively. In contrast, in the mice that had received bone marrow lacking CCR2 (CCR2-/- > C57 and CCR2-/- syngeneic mice), no pulmonary fibrosis was observed at 22 weeks postirradiation. This observation correlated with decreased numbers of infiltrating and interstitial macrophages compared to controls, as well as reduced proportions of pro-inflammatory Ly6C+ macrophages observed at 12-18 weeks postirradiation, suggesting that CCR2+ macrophages contribute to radiation-induced pulmonary fibrosis. Interestingly, reduced proportions of CD206+ lung macrophages were also present at these time points in CCR2-/- chimeric mice, regardless of donor bone marrow type, suggesting that the phenotype of resident subsets may be influenced by CCR2. Furthermore, chimeras, in which either IL-1? was ablated from infiltrating cells or IL-1R1 from lung tissues, were also protected from fibrosis development, correlating with attenuated CCL2 production; these data suggest that IL-1? may influence chemotactic signaling after irradiation. Overall, our data suggest that CCR2+ infiltrating monocyte-derived macrophages may play a critical role in the development of radiation-induced pulmonary fibrosis.
Project description:Pulmonary exposure to multiwalled carbon nanotubes (MWCNT) induces an inflammatory and rapid fibrotic response, although the long-term signaling mechanisms are unknown. The aim of this study was to examine the effects of 1, 10, 40, or 80 ?g MWCNT administered by pharyngeal aspiration on bronchoalveolar lavage (BAL) fluid for polymorphonuclear cell (PMN) infiltration, lactate dehydrogenase (LDH) activity, and lung histopathology for inflammatory and fibrotic responses in mouse lungs 1 mo, 6 mo, and 1 yr postexposure. Further, a 120-?g crocidolite asbestos group was incorporated as a positive control for comparative purposes. Results showed that MWCNT increased BAL fluid LDH activity and PMN infiltration in a dose-dependent manner at all three postexposure times. Asbestos exposure elevated LDH activity at all 3 postexposure times and PMN infiltration at 1 mo and 6 mo postexposure. Pathological changes in the lung, the presence of MWCNT or asbestos, and fibrosis were noted at 40 and 80 ?g MWCNT and in asbestos-exposed mice at 1 yr postexposure. To determine potential signaling pathways involved with MWCNT-associated pathological changes in comparison to asbestos, up- and down-regulated gene expression was determined in lung tissue at 1 yr postexposure. Exposure to MWCNT tended to favor those pathways involved in immune responses, specifically T-cell responses, whereas exposure to asbestos tended to favor pathways involved in oxygen species production, electron transport, and cancer. Data indicate that MWCNT are biopersistent in the lung and induce inflammatory and fibrotic pathological alterations similar to those of crocidolite asbestos, but may reach these endpoints by different mechanisms.
Project description:Members of the transforming growth factor (TGF)-beta superfamily, including TGF-betas and bone morphogenetic proteins (BMPs), are essential for the maintenance of tissue homeostasis and regeneration after injury. We have observed that the BMP antagonist, gremlin, is highly up-regulated in idiopathic pulmonary fibrosis (IPF).To investigate the role of gremlin in the regulation of BMP signaling in pulmonary fibrosis.Progressive asbestos-induced fibrosis in the mouse was used as a model of human IPF. TGF-beta and BMP expression and signaling activities were measured from murine and human fibrotic lungs. The mechanism of gremlin induction was analyzed in cultured lung epithelial cells. In addition, the possible therapeutic role of gremlin inhibition was tested by administration of BMP-7 to mice after asbestos exposure.Gremlin mRNA levels were up-regulated in the asbestos-exposed mouse lungs, which is in agreement with the human IPF biopsy data. Down-regulation of BMP signaling was demonstrated by reduced levels of Smad1/5/8 and enhanced Smad2 phosphorylation in asbestos-treated lungs. Accordingly, analyses of cultured human bronchial epithelial cells indicated that asbestos-induced gremlin expression could be prevented by inhibitors of the TGF-beta receptor and also by inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathways. BMP-7 treatment significantly reduced hydroxyproline contents in the asbestos-treated mice.The TGF-beta and BMP signaling balance is important for lung regenerative events and is significantly perturbed in pulmonary fibrosis. Rescue of BMP signaling activity may represent a potential beneficial strategy for treating human pulmonary fibrosis.
Project description:Lung fibrosis is characterized by vascular leakage and myofibroblast recruitment, and both phenomena are mediated by lysophosphatidic acid (LPA) via its type-1 receptor (LPA1). Following lung damage, the accumulated myofibroblasts activate and secrete excessive extracellular matrix (ECM), and form fibrotic foci. Studies have shown that bone marrow-derived cells are an important source of myofibroblasts in the fibrotic organ. However, the type of cells in the bone marrow contributing predominantly to the myofibroblasts and the involvement of LPA-LPA1 signalling in this is yet unclear. Using a bleomycin-induced mouse lung-fibrosis model with an enhanced green fluorescent protein (EGFP) transgenic mouse bone marrow replacement, we first demonstrated that bone marrow derived-mesenchymal stem cells (BMSCs) migrated markedly to the bleomycin-injured lung. The migrated BMSC contributed significantly to ?-smooth muscle actin (?-SMA)-positive myofibroblasts. By transplantation of GFP-labelled human BMSC (hBMSC) or EGFP transgenic mouse BMSC (mBMSC), we further showed that BMSC might be involved in lung fibrosis in severe combined immune deficiency (SCID)/Beige mice induced by bleomycin. In addition, using quantitative-RT-PCR, western blot, Sircol collagen assay and migration assay, we determined the underlying mechanism was LPA-induced BMSC differentiation into myofibroblast and the secretion of ECM via LPA1. By employing a novel LPA1 antagonist, Antalpa1, we then showed that Antalpa1 could attenuate lung fibrosis by inhibiting both BMSC differentiation into myofibroblast and the secretion of ECM. Collectively, the above findings not only further validate LPA1 as a drug target in the treatment of pulmonary fibrosis but also elucidate a novel pathway in which BMSCs contribute to the pathologic process.
Project description:To determine whether genetic polymorphisms in several candidate genes related to innate immunity and protease-antiprotease balance modify individual susceptibility to develop asbestos-related fibrotic pleuropulmonary changes.Sixteen polymorphisms from nine genes (NLRP3, CARD8, TNF, TGFB1, GC, MMP1, MMP9, MMP12 and TIMP2) were genotyped from 951 Finnish asbestos-exposed workers. The genotype/haplotype data were compared to signs of fibrosis and pleural thickenings using linear and logistic regression analysis adjusted for potential confounders.A functional polymorphism (Q705K; rs35829419) in the NLRP3 gene was associated with interstitial lung fibrosis (p=0.013), and the TGFB1 rs2241718 SNP with visceral pleural fibrosis (VPF) (p=0.044). In stratified analysis, the carriage of at least one NLRP3 variant allele conferred a 2.5-fold increased risk for pathological interstitial lung fibrosis (OR 2.44, 95% CI 0.97 to 6.14). Conversely, the carriage of at least one TGFB1 rs2241718 variant allele protected against VPF (OR 0.62, 95% CI 0.39 to 0.98). The TIMP2 rs2277698 SNP and a haplotype consisting of the TGFB1 rs1800469 and rs1800470 SNPs were associated with the degree of pleural thickening calcification (p=0.037 and p=0.035), and the CARD8 rs2043211 SNP with the greatest thickness of pleural plaques (p=0.015).Our results support the hypothesis that the NLRP3 inflammasome is important in the development of fibrotic lung disease by associating the NLRP3 rs35829419 variant allele with increased risk of asbestos-related interstitial lung fibrosis, and the TGFB1 rs2241718 variant allele with decreased risk of asbestos-related VPF. Polymorphisms in CARD8 and TIMP2 are proposed to modify the development and/or calcification of pleural thickenings.
Project description:Pulmonary exposure to multiwalled carbon nanotubes (MWCNT) induces an inflammatory and rapid fibrotic response, although the long-term signaling mechanisms are unknown. The aim of this study was to examine the effects of 1, 10, 40, or 80 µg MWCNT administered by pharyngeal aspiration on bronchoalveolar lavage (BAL) fluid for polymorphonuclear cell (PMN) infiltration, lactate dehydrogenase (LDH) activity, and lung histopathology for inflammatory and fibrotic responses in mouse lungs 1 mo, 6 mo, and 1 yr postexposure. Further, a 120-µg crocidolite asbestos group was incorporated as a positive control for comparative purposes. Results showed that MWCNT increased BAL fluid LDH activity and PMN infiltration in a dose-dependent manner at all three postexposure times. Asbestos exposure elevated LDH activity at all 3 postexposure times and PMN infiltration at 1 mo and 6 mo postexposure. Pathological changes in the lung, the presence of MWCNT or asbestos, and fibrosis were noted at 40 and 80 µg MWCNT and in asbestos-exposed mice at 1 yr postexposure. To determine potential signaling pathways involved with MWCNT-associated pathological changes in comparison to asbestos, up- and down-regulated gene expression was determined in lung tissue at 1 yr postexposure. Exposure to MWCNT tended to favor those pathways involved in immune responses, specifically T-cell responses, whereas exposure to asbestos tended to favor pathways involved in oxygen species production, electron transport, and cancer. Data indicate that MWCNT are biopersistent in the lung and induce inflammatory and fibrotic pathological alterations similar to those of crocidolite asbestos, but may reach these endpoints by different mechanisms. Overall design: We evaluated bronchoalveolar lavage (BAL) fluid for polymorphonuclear cell (PMN) infiltration and lactate dehydrogenase (LDH) activity, and lung histopathology for inflammatory and fibrotic responses in mouse lungs after 1 year post-exposure to a dispersion media (DM) control (Porter et al., 2008); 1, 10, 40, or 80 g MWCNT; or 120 g crocidolite asbestos by pharyngeal aspiration. Additionally, we determined gene expression changes in mouse lung tissue with increasing doses of MWCNT at 1 year post-exposure to determine potential underlying mechanisms of fibrotic signaling after exposure to MWCNT.
Project description:BACKGROUND: Retroperitoneal fibrosis (RPF) is a rare fibroinflammatory disease that leads to hydronephrosis and renal failure. In a case-control study, we have recently shown that asbestos exposure was the most important risk factor for RPF in the Finnish population. The aim of this study was to evaluate the relation of asbestos exposure to radiologically confirmed lung and pleural fibrosis among patients with RPF. METHODS: Chest high-resolution computed tomography (HRCT) was performed on 16 unexposed and 22 asbestos-exposed RPF patients and 18 asbestos-exposed controls. Parietal pleural plaques (PPP), diffuse pleural thickening (DPT) and parenchymal fibrosis were scored separately. RESULTS: Most of the asbestos-exposed RPF patients and half of the asbestos-exposed controls had bilateral PPP, but only a few had lung fibrosis. Minor bilateral plaques were detected in two of the unexposed RPF patients, and none had lung fibrosis. DPT was most frequent and thickest in the asbestos-exposed RPF-patients. In three asbestos-exposed patients with RPF we observed exceptionally large pleural masses that were located anteriorly in the pleural space and continued into the anterior mediastinum.Asbestos exposure was associated with DPT in comparisons between RPF patients and controls (case-control analysis) as well as among RPF patients (case-case analysis). CONCLUSION: The most distinctive feature of the asbestos-exposed RPF patients was a thick DPT. An asbestos-related pleural finding was common in the asbestos-exposed RPF patients, but only a few of these patients had parenchymal lung fibrosis. RPF without asbestos exposure was not associated with pleural or lung fibrosis. The findings suggest a shared etiology for RPF and pleural fibrosis and furthermore possibly a similar pathogenetic mechanisms.
Project description:Hermansky-Pudlak syndrome (HPS) is a family of recessive disorders of intracellular trafficking defects that are associated with highly penetrant pulmonary fibrosis. Naturally occurring HPS mice reliably model important features of the human disease, including constitutive alveolar macrophage activation and susceptibility to profibrotic stimuli.To decipher which cell lineage(s) in the alveolar compartment is the predominant driver of fibrotic susceptibility in HPS.We used five different HPS and Chediak-Higashi mouse models to evaluate genotype-specific fibrotic susceptibility. To determine whether intrinsic defects in HPS alveolar macrophages cause fibrotic susceptibility, we generated bone marrow chimeras in HPS and wild-type mice. To directly test the contribution of the pulmonary epithelium, we developed a transgenic model with epithelial-specific correction of the HPS2 defect in an HPS mouse model.Bone marrow transplantation experiments demonstrated that both constitutive alveolar macrophage activation and increased susceptibility to bleomycin-induced fibrosis were conferred by the genotype of the lung epithelium, rather than that of the bone marrow-derived, cellular compartment. Furthermore, transgenic epithelial-specific correction of the HPS defect significantly attenuated bleomycin-induced alveolar epithelial apoptosis, fibrotic susceptibility, and macrophage activation. Type II cell apoptosis was genotype specific, caspase dependent, and correlated with the degree of fibrotic susceptibility.We conclude that pulmonary fibrosis in naturally occurring HPS mice is driven by intracellular trafficking defects that lower the threshold for pulmonary epithelial apoptosis. Our findings demonstrate a pivotal role for the alveolar epithelium in the maintenance of alveolar homeostasis and regulation of alveolar macrophage activation.
Project description:Pulmonary fibrosis may result from abnormal alveolar wound repair after injury. Hepatocyte growth factor (HGF) improves alveolar epithelial wound repair in the lung. Stem cells were shown to play a major role in lung injury, repair and fibrosis. We studied the presence, origin and antifibrotic properties of HGF-expressing stem cells in usual interstitial pneumonia.Immunohistochemistry was performed in lung tissue sections and primary alveolar epithelial cells obtained from patients with usual interstitial pneumonia (UIP, n?=?7). Bone marrow derived stromal cells (BMSC) from adult male rats were transfected with HGF, instilled intratracheally into bleomycin injured rat lungs and analyzed 7 and 14 days later.In UIP, HGF was expressed in specific cells mainly located in fibrotic areas close to the hyperplastic alveolar epithelium. HGF-positive cells showed strong co-staining for the mesenchymal stem cell markers CD44, CD29, CD105 and CD90, indicating stem cell origin. HGF-positive cells also co-stained for CXCR4 (HGF+/CXCR4+) indicating that they originate from the bone marrow. The stem cell characteristics were confirmed in HGF secreting cells isolated from UIP lung biopsies. In vivo experiments showed that HGF-expressing BMSC attenuated bleomycin induced pulmonary fibrosis in the rat, indicating a beneficial role of bone marrow derived, HGF secreting stem cells in lung fibrosis.HGF-positive stem cells are present in human fibrotic lung tissue (UIP) and originate from the bone marrow. Since HGF-transfected BMSC reduce bleomycin induced lung fibrosis in the bleomycin lung injury and fibrosis model, we assume that HGF-expressing, bone-marrow derived stem cells in UIP have antifibrotic properties.
Project description:In radiotherapy, radiation (IR)-induced lung fibrosis has severe and dose-limiting side effects. To elucidate the molecular effects of IR fibrosis, we examined the fibrosis process in irradiated mouse lung tissues. High focal IR (90 Gy) was exposed to a 3-mm volume of the left lung in C57BL6 mice. In the diffused irradiation, 20 Gy dose delivered with a 7-mm collimator almost covered the entire left lung. Histological examination for lung tissues of both irradiated and neighboring regions was done for 4 weeks after irradiation. Long-term effects (12 months) of 20Gy IR were compared on a diffuse region of the left lung and non-irradiated right lung. Fibrosis was initiated as early as 2 weeks after IR in the irradiated lung region and neighboring region. Upregulation of gtse1 in both 90Gy-irradiated and neighboring regions was observed. Upregulation of fgl1 in both 20Gy diffused irradiated and non-irradiated lungs was identified. When gtse1 or flg1 was knock-downed, TGFβ or IR-induced epithelial-mesenchymal transition was inhibited, accompanied with the inhibition of cellular migration, suggesting fibrosis responsible genes. Immunofluorescence analysis using mouse fibrotic lung tissues suggested that fibrotic regions showed increased expressions of Gtse1 and Fgl1, indicating novel molecular signatures of gtse1and fgl1 for IR-induced lung fibrosis. Even though their molecular mechanisms and IR doses or irradiated volumes for lung fibrosis may be different, these genes may be novel targets for understanding IR-induced lung fibrosis and in treatment strategies. KEY MESSAGES: Upregulation of gtse1 by 90Gy focal irradiation and upregulation of fgl1 by 20Gy diffused irradiation are identified in mouse lung fibrosis model. Gtse1 and Fgl1 are involved in radiation or TGFβ-induced epithelial-mesenchymal transition. Radiation-induced fibrotic regions of mouse lungs showed increased expressions of Gtse1 and Fgl1. Gtse1 and Fgl1 are suggested to be novel targets for radiation-induced lung fibrosis.