BLM-induced Lung Inflammation Research Articles

Fraxetin alleviates BLM-induced idiopathic pulmonary fibrosis by inhibiting NCOA4-mediated epithelial cell ferroptosis.

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung condition with few available treatments. The early driver of wound repair that contributes to IPF has been extensively identified as repetitive alveolar epithelial damage. According to recent reports, IPF is linked to ferroptosis, a unique type of cell death characterized by a fatal buildup of iron and lipid peroxidation. There is little information on epithelial cells that induce pulmonary fibrosis by going through ferroptosis. In this study, we used bleomycin (BLM) to examine the impact of ferroptosis on IPF in mouse lung epithelial cells (MLE-12). We discovered that BLM increases ferroptosis in MLE-12. Additionally, we found that NCOA4 is overexpressed and plays a key role in the ferroptosis of epithelial cells throughout the IPF process. Using Molecular docking, we found that Fraxetin, a natural component extracted from Fraxinus rhynchophylla, formed a stable binding to NCOA4. In vitro investigations showed that Fraxetin administration greatly decreased ferroptosis and NCOA4 expression, which in turn lowered the release of inflammatory cytokines. Fraxetin treatment significantly alleviated BLM-induced lung inflammation and fibrosis. Our findings imply that fraxetin possesses inhibitory roles in ferroptosis and can be a potential drug against IPF.

Dickkopf1 Promotes Pulmonary Fibrosis upon Bleomycin-Induced Lung Injury

Orchestration of inflammation and tissue repair processes is critical to maintaining homeostasis upon tissue injury. Tissue fibrosis is a pathological process characterized by aberrant accumulation of extracellular matrix proteins, such as collagen, upon injury. Dickkopf1 (DKK1) is a quintessential Wnt antagonist. The role of DKK1 in bleomycin (BLM)-induced lung injury and fibrosis model remains elusive. This study shows that BLM-induced lung injury markedly elevated DKK1 protein expressions in the lungs in mice, consistent with human pulmonary fibrosis patient lung tissues. The elevated DKK1 levels coincided with immune cell infiltration and collagen deposition. Notably, the reduced expression of DKK1 in Dkk1 hypomorphic doubleridge (Dkk1d/d) mice abrogated BLM-induced lung inflammation and fibrosis. Immune cell infiltration, collagen deposition, expression of profibrotic cytokine transforming growth factor β1 (TGF-β1), and extracellular matrix protein-producing myofibroblast marker α-smooth muscle actin (α-SMA) were reduced in Dkk1d/d mice. Consistent with these results, local DKK1 antibody administration after BLM-induced lung injury substantially decreased lung inflammation and fibrosis phenotypes. Taken together, these results demonstrate that DKK1 is a proinflammatory and profibrotic ligand that promotes inflammation and fibrosis upon BLM-induced lung injury, placing it as an attractive molecular target for dysregulated pulmonary inflammation and tissue repair.

Cangrelor alleviates bleomycin-induced pulmonary fibrosis by inhibiting platelet activation in mice

Pulmonary fibrosis is a progressive chronic inflammatory lung disease whose pathogenesis is complicated. Platelets and neutrophils play important roles in the progression of pulmonary inflammation. We have reported that cangrelor, a non-sepesific GPR17 antagonist, alleviates pulmonary fibrosis partly by inhibiting macrophage inflammation in mice. Cangrelor is also a well-known anti-platelet agent. To test whether cangrelor mitigated pulmonary fibrosis partly through the inhibition of platelets, bleomycin (BLM) was used to induce pulmonary fibrosis in C57BL/6 J mice. We found that cangrelor (10 mg/kg) not only significantly decreased BLM-induced release of inflammatory cytokines (PF4, CD40 L and MPO), but also decreased the increment of platelets, neutrophils and platelet-neutrophil aggregates in the fibrotic lung and in the peripheral blood of BLM-treated mice. In addition, cangrelor decreased the number of CD40 and MPO double positive neutrophils and the expression level of CD40 in BLM-treated mouse lungs. Based on these results we conclude that cangrelor alleviates BLM-induced lung inflammation and pulmonary fibrosis in mice, partly through inhibition of platelet activation, therefore reducing the infiltration of neutrophils due to the adhesion of platelets and neutrophils mediated by CD40 - CD40 L interaction. Cangrelor could be a potential therapeutic medicine for pulmonary fibrosis.

Serelaxin enhances the therapeutic effects of human amnion epithelial cell-derived exosomes in experimental models of lung disease.

There is growing interest in stem cell-derived exosomes for their therapeutic and regenerative benefits given their manufacturing and regulatory advantages over cell-based therapies. As existing fibrosis impedes the viability and efficacy of stem cell/exosome-based strategies for treating chronic diseases, here we tested the effects of the anti-fibrotic drug, serelaxin, on the therapeutic efficacy of human amnion epithelial cell (AEC)-derived exosomes in experimental lung disease. Female Balb/c mice were subjected to either the 9.5-week model of ovalbumin and naphthalene (OVA/NA)-induced chronic allergic airway disease (AAD) or 3-week model of bleomycin (BLM)-induced pulmonary fibrosis; then administered increasing concentrations of AEC-exosomes (5μg or 25μg), with or without serelaxin (0.5mg/kg/day) for 7-days. 1x106 AECs co-administered with serelaxin over the corresponding time-period were included for comparison in both models, as was pirfenidone-treatment of the BLM model. Control groups received saline/corn oil or saline, respectively. Both experimental models presented with significant tissue inflammation, remodelling, fibrosis and airway/lung dysfunction at the time-points studied. While AEC-exosome (5μg or 25μg)-administration alone demonstrated some benefits in each model, serelaxin was required for AEC-exosomes (25μg) to rapidly normalise chronic AAD-induced airway fibrosis and airway reactivity, and BLM-induced lung inflammation, epithelial damage and subepithelial/basement membrane fibrosis. Combining serelaxin with AEC-exosomes (25μg) also demonstrated broader protection compared to co-administration of serelaxin with 1x106 AECs or pirfenidone. Serelaxin enhanced the therapeutic efficacy of AEC-exosomes in treating basement membrane-induced fibrosis and related airway dysfunction.

Involvement of Alveolar Epithelial Cell Necroptosis in Idiopathic Pulmonary Fibrosis Pathogenesis.

Alveolar epithelial cell (AEC) injury leading to cell death is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF). Among regulated/programmed cell death, the excessive apoptosis of AECs has been widely implicated in IPF pathogenesis. Necroptosis is a type of regulated/programmed necrosis. A multiprotein complex composed of receptor-interacting protein kinase (RIPK)-1 and -3 plays a key regulatory role in initiating necroptosis. Although necroptosis participates in disease pathogeneses through the release of damage-associated molecular patterns, its association with IPF progression remains elusive. In this study, we attempted to illuminate the involvement of RIPK3-regulated necroptosis in IPF pathogenesis. IPF lung tissues were used to detect necroptosis, and the role of RIPK3 was determined using cell culturing models of AECs. Lung fibrosis models of bleomycin (BLM) treatment were also used. RIPK3 expression levels were increased in IPF lungs, and both apoptosis and necroptosis were detected mainly in AECs. Necrostatin-1 and RIPK3 knockout experiments in AECs revealed the participation of necroptosis in BLM and hydrogen peroxide-induced cell death. BLM treatment induced RIPK3 expression in AECs and increased high-mobility group box 1 and IL-1β levels in mouse lungs. The efficient attenuation of BLM-induced lung inflammation and fibrosis was determined in RIPK3 knockout mice and by necrostatin-1 with a concomitant reduction in high-mobility group box 1 and IL-1β. RIPK3-regulated necroptosis in AECs is involved in the mechanism of lung fibrosis development through the release of damage-associated molecular patterns as part of the pathogenic sequence of IPF.

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation.

B-cell activating factor regulates IL-1β- and IL-17A-mediated pulmonary fibrosis in mice

Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive devastating, yet untreatable fibrotic disease of unknown origin. We investigated the contribution of the B-cell activating factor (BAFF), a TNF family member recently implicated in the regulation of pathogenic IL-17-producing cells in autoimmune diseases. Methods The contribution of BAFF was assessed in a murine model of lung fibrosis induced by airway administered bleomycin, an intercalating agent which causes DNA strand breaks. Lung fibrosis was evaluated in Baff deficient mice and in wild-type mice after neutralization of BAFF with BAFF-R-Ig. Lung cells from bleomycin-treated mice were collected at day 7, labeled with anti-CD115, anti-CD3, anti-CD19 and anti-Gr1 antibodies and sorted by FACS for evaluation of BAFF mRNA expression by qPCR. BAFF was quantified in bronchoalveolar lavage fluid (BALF) of controls and patients with IPF. Results We show that murine BAFF levels were strongly increased in the bronchoalveolar space and lungs after bleomycin exposure. We identified Gr1 + neutrophils as an important source of BAFF upon BLM induced lung inflammation and fibrosis. Genetic ablation of BAFF or BAFF neutralization by a soluble receptor significantly attenuated pulmonary fibrosis and IL-1β levels. We further demonstrate that bleomycin-induced BAFF expression and lung fibrosis were IL-1β- and IL-17A-dependent. BAFF was required for rIL-17A-induced lung fibrosis and augmented IL-17A production by CD3 + T cells from murine fibrotic lungs ex vivo . Finally, we report elevated levels of BAFF in bronchoalveolar lavages from IPF patients. Conclusion The present data, demonstrating a critical role of BAFF by amplifying the IL-17A effect in experimental lung fibrosis, together with increased BAFF levels in the airways of patients with IPF, suggest that BAFF might be a therapeutic target of IPF.

Osteopontin Potentiates Pulmonary Inflammation and Fibrosis by Modulating IL-17/IFN-γ-secreting T-cell Ratios in Bleomycin-treated Mice

Lung fibrosis is a life-threatening disease caused by overt or insidious inflammatory responses. However, the mechanism of tissue injury-induced inflammation and subsequent fibrogenesis remains unclear. Recently, we and other groups reported that Th17 responses play a role in amplification of the inflammatory phase in a murine model induced by bleomycin (BLM). Osteopontin (OPN) is a cytokine and extracellular-matrix-associated signaling molecule. However, whether tissue injury causes inflammation and consequent fibrosis through OPN should be determined. In this study, we observed that BLM-induced lung inflammation and subsequent fibrosis was ameliorated in OPN-deficient mice. OPN was expressed ubiquitously in the lung parenchymal and bone-marrow-derived components and OPN from both components contributed to pathogenesis following BLM intratracheal instillation. Th17 differentiation of CD4+ αβ T cells and IL-17-producing γδ T cells was significantly reduced in OPN-deficient mice compared to WT mice. In addition, Th1 differentiation of CD4+ αβ T cells and the percentage of IFN-γ-producing γδ T cells increased. T helper cell differentiation in vitro revealed that OPN was preferentially upregulated in CD4+ T cells under Th17 differentiation conditions. OPN expressed in both parenchymal and bone marrow cell components and contributed to BLM-induced lung inflammation and fibrosis by affecting the ratio of pathogenic IL-17/protective IFN-γ T cells.

B cell activating factor is central to bleomycin- and IL-17-mediated experimental pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive devastating, yet untreatable fibrotic disease of unknown origin. We investigated the contribution of the B-cell activating factor (BAFF), a TNF family member recently implicated in the regulation of pathogenic IL-17-producing cells in autoimmune diseases. The contribution of BAFF was assessed in a murine model of lung fibrosis induced by airway administered bleomycin. We show that murine BAFF levels were strongly increased in the bronchoalveolar space and lungs after bleomycin exposure. We identified Gr1+ neutrophils as an important source of BAFF upon BLM-induced lung inflammation and fibrosis. Genetic ablation of BAFF or BAFF neutralization by a soluble receptor significantly attenuated pulmonary fibrosis and IL-1β levels. We further demonstrate that bleomycin-induced BAFF expression and lung fibrosis were IL-1β and IL-17A dependent. BAFF was required for rIL-17A-induced lung fibrosis and augmented IL-17A production by CD3+ T cells from murine fibrotic lungs ex vivo. Finally we report elevated levels of BAFF in bronchoalveolar lavages from IPF patients. Our data therefore support a role for BAFF in the establishment of pulmonary fibrosis and a crosstalk between IL-1β, BAFF and IL-17A.

Glucagon like peptide-1 attenuates bleomycin-induced pulmonary fibrosis, involving the inactivation of NF-κB in mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with high mortality and poor prognosis. Previous studies confirmed that NF-κB plays a critical role in the pathogenesis of pulmonary fibrosis and glucagon like peptide-1 (GLP-1) has a property of anti-inflammation by inactivation of NF-κB. Furthermore, the GLP-1 receptor was detected in the lung tissues. Our aim was to investigate the potential value and mechanisms of GLP-1 on BLM-induced pulmonary fibrosis in mice. Mice with BLM-induced pulmonary fibrosis were treated with or without GLP-1 administration. 28 days after BLM infusion, the number of total cells, macrophages, neutrophils, lymphocytes, and the content of TGF-β1 in BALF were measured. Hematoxylin-eosin (HE) staining and Masson's trichrome (MT) staining were performed. The Ashcroft score and hydroxyproline content were analyzed. RT-qPCR and western blot were used to evaluate the expression of α-SMA and VCAM-1. The phosphorylation of NF-κB p65 was also assessed by western blot. DNA binding of NF-κB p65 was measured through Trans(AM) p65 transcription factor ELISA kit. GLP-1 reduced inflammatory cell infiltration and the content of TGF-β1 in BLAF in mice with BLM injection. The Ashcroft score and hydroxyproline content were decreased by GLP-1 administration. Meanwhile, BLM-induced overexpression of α-SMA and VCAM-1 were blocked by GLP-1 treatment in mice. GLP-1 also reduced the ratio of phosphor-NF-κB p65/total-NF-κB p65 and NF-κB p65 DNA binding activity in BLM-induced pulmonary fibrosis in mice. Our data found that BLM-induced lung inflammation and pulmonary fibrosis were significantly alleviated by GLP-1 treatment in mice, possibly through inactivation of NF-κB.

Metformin Reduces Inflammation and Lung Fibrosis in a Bleomycin‐Induced Lung Injury Model (LB505)

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder characterized by the destruction of lung parenchyma together with deposition of extracellular matrix (ECM) in the interstitial and alveolar spaces. Metformin is developed as an oral anti‐diabetic drug in the biguanide class. Recently, it has been suggested to have anti‐inflammatory and anti‐tissue remodeling effects. Therefore, in this study, we investigated the roles of metformin in bleomycin‐induced lung fibrosis. C57BL/6 mice were intratracheally injected with bleomycin (BLM, 0.2 U/100 μl) on day 0. Metformin was administered orally to the mice once a day from day 1 to day 21. On day 21, the mice were sacrificed. We performed H&E and Masson’s trichrome (MT) staining to evaluate lung inflammation and collagen deposition and analyzed the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) by differential cell counts. From total RNA of lung tissue, the expression level of pro‐collagen, collagen‐1, fibronectin, and tumor growth factor‐β (TGF‐β) was determined by real‐time PCR. Metformin suppressed lung tissue inflammation in BLM‐instilled mice on day 21. Consistent with this, the number of inflammatory cells in BALF was diminished by metformin treatment. BLM increased the level of pro‐collagen, collagen‐1, fibronectin, and TGF‐β mRNA expression on day 21, which was suppressed in metformin‐treated mice. Metformin reduced peribronchial collagen deposition induced by BLM on day 21. Metformin has protective effects on BLM‐induced lung inflammation and remodeling.

On the Therapeutical Action of Digitalis

<b>Rationale:</b> Idiopathic interstitial pneumonias are characterized by variable degrees of lung inflammation and fibrosis, and the mechanisms leading to these responses remain unclear. Protease activated receptor (PAR)-2, a G-protein-coupled receptor has been recognized as a key molecule in inflammation and fibrosis. In a previous study, we found that activation of PAR-2 induces transforming growth factor (TGF)-β expression and epithelial-to-mesenchymal transition (EMT) associated with lung fibrosis in vitro. We hypothesized PAR-2 inhibiting peptide (IP) might reduce bleomycin (BLM)-induced lung injury and fibrosis in vivo. <b>Methods:</b> C57BL/6 mice were used in all experiments. BLM was administered by intratracheal injection under anesthesia. PAR-2 IP was intranasally instilled half an hour before BLM injection and once daily until the end of the experiment. Blood sampling and bronchoalveolar lavage (BAL) were performed 7, 14 and 21 days after BLM instillation. The lungs were excised and the samples were used for histological analysis, RT-PCR and ELISA. <b>Results:</b> PAR-2 IP reduced BLM-induced lymphocytes in BAL fluid (BALF). Lung fibrosis was reduced in PAR-2 IP-treated BLM mice. PAR-2 IP downregulated the expression of monocyte chemotactic protein (MCP)-1 and TGF-β, and inhibited profibrotic responses, as seen by reduced collagen induction by BLM. Immunohistochemistry showed PAR-2 IP reduced BLM-induced EMT. Induction of high-mobility group box (HMGB)-1 by BLM which may mediate EMT was reduced by PAR-2 IP treatment. <b>Conclusion:</b> PAR-2 inhibition reduces BLM-induced lung inflammation and fibrosis, suggesting that PAR-2 might play a pivotal role in BLM-induced lung injury. The results could lead to new therapeutic strategies for lung fibrosis.