Enhanced lysosomal activity is involved in Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress response and cell death against ER stress: involvement of vacuolar H+-ATPase (V-ATPase)

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

Bax inhibitor-1 regulates the expression of P450 2E1 through enhanced lysosome activity

In this study, we explored the role of Bax inhibitor-1 (BI-1) on the expression of P450 2E1 and related ROS production. P450 2E1 protein, not mRNA, was expressed at relatively low levels in BI-1 plasmid-transfected cells (BI-1 cells) compared with neomycin-resistant vector-transfected cells (Neo cells). When exposed to ER stress, P450 2E1 expression and activity and ER membrane lipid peroxidation increased in both Neo cells and BI-1 cells, but to a lesser degree in BI-1 cells. This observation correlated with the lower level of ER stress in BI-1 cells than Neo cells. To examine the BI-1-associated P450 2E1 degradation mechanism, cells were treated with the lysosome inhibitor, bafilomycin and the proteasome inhibitor, MG132. Bafilomycin recovered the reduced P450 2E1 expression in BI-1 cells, but did not affect P450 2E1 expression in Neo cells. Next, proteosomal and lysosomal activities in Neo cells were compared to those in BI-1 cells. Although proteosomal activity was similar between Neo and BI-1 cells, LysoTracker and acridine orange labeling, lysosomal V-ATPase activity, and lysosomal cathepsin B expression were higher in BI-1 cells than in Neo cells. In the presence of ER stress, lysosomal activities decreased in Neo cells but did not change in BI-1 cells. P450 2E1 expression and ER membrane lipid peroxidation were greater in the hepatocytes and livers of BI-1 knock-out mice than in BI-1 wild-type cells and mice. Our results suggest that the BI-1-mediated enhancement of lysosomal activity regulates P450 2E1 expression and resultant ROS accumulation.     

Expression of 150-kDa oxygen-regulated protein (ORP150) stimulates bleomycin-induced pulmonary fibrosis and dysfunction in mice

Idiopathic pulmonary fibrosis (IPF) involves pulmonary injury associated with inflammatory responses, fibrosis and dysfunction. Myofibroblasts and transforming growth factor (TGF)-β1 play major roles in the pathogenesis of this disease. Endoplasmic reticulum (ER) stress response is induced in the lungs of IPF patients. One of ER chaperones, the 150-kDa oxygen-regulated protein (ORP150), is essential for the maintenance of cellular viability under stress conditions. In this study, we used heterozygous ORP150-deficient mice (ORP150(+/-) mice) to examine the role of ORP150 in bleomycin-induced pulmonary fibrosis. Treatment of mice with bleomycin induced the expression of ORP150 in the lung. Bleomycin-induced inflammatory responses were slightly exacerbated in ORP150(+/-) mice compared to wild-type mice. On the other hand, bleomycin-induced pulmonary fibrosis, alteration of lung mechanics and respiratory dysfunction was clearly ameliorated in the ORP150(+/-) mice. Bleomycin-induced increases in pulmonary levels of both active TGF-β1 and myofibroblasts were suppressed in ORP150(+/-) mice. These results suggest that although ORP150 is protective against bleomycin-induced lung injury, this protein could stimulate bleomycin-induced pulmonary fibrosis by increasing pulmonary levels of TGF-β1 and myofibroblasts.     

Thalidomide prevents bleomycin-induced pulmonary fibrosis in mice

Pulmonary fibrosis in humans can occur as a result of a large number of conditions. In idiopathic pulmonary fibrosis (IPF), pulmonary function becomes progressively compromised resulting in a high mortality rate. Currently there are no proven effective treatments for IPF. We have recently reported that IL-6 and TGF-beta(1) plays an important role in proliferation and differentiation of lung fibroblasts, and all-trans-retinoic acid (ATRA) prevented bleomycin-induced lung fibrosis through the inhibition of these cytokines. Thalidomide (Thal) has been used in the treatment of multiple myeloma through the inhibitory effect on IL-6-dependent cell growth and angiogenesis. In this study, we examined the preventive effect of Thal on bleomycin-induced pulmonary fibrosis in mice. We performed histological examinations and quantitative measurements of IL-6, TGF-beta(1), collagen type Ialpha1 (COL1A1), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in bleomycin-treated mouse lung tissues with or without the administration of Thal. Thal histologically ameliorated bleomycin-induced fibrosis in mouse lung tissues. Thal decreased the expressions of IL-6, TGF-beta(1), VEGF, Ang-1 Ang-2, and COL1A1 mRNA in mouse lung tissues. In addition, Thal inhibited angiogenesis in the lung. In vitro studies disclosed that Thal reduced 1) production of IL-6, TGF-beta(1), VEGF, Ang-1, and collagen synthesis from human lung fibroblasts, and 2) both IL-6-dependent proliferation and TGF-beta(1)-dependent transdifferentiation of the cells, which could be the mechanism underlying the preventive effect of Thal on pulmonary fibrosis. These data may provide a rationale to explore clinical use of Thal for the prevention of pulmonary fibrosis.     

Duvelisib attenuates bleomycin-induced pulmonary fibrosis via inhibiting the PI3K/Akt/mTOR signalling pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that seriously threatens the health of patients. The pathogenesis of IPF is still unclear, and there is a lack of effective therapeutic drugs. Myofibroblasts are the main effector cells of IPF, leading to excessive deposition of extracellular matrix (ECM) and promoting the progression of fibrosis. Inhibiting the excessive activation and relieving autophagy blockage of myofibroblasts is the key to treat IPF. PI3K/Akt/mTOR pathway plays a key regulatory role in promoting fibroblast activation and autophagy inhibition in lung fibrosis. Duvelisib is a PI3K inhibitor that can simultaneously inhibit the activities of PI3K-δ and PI3K-γ, and is mainly used for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma tumour (SLL). In this study, we aimed to examine the effects of Duvelisib on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of Duvelisib on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of Duvelisib in lung fibroblasts in vitro. The in vivo experiments showed that Duvelisib significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro and in vivo pharmacological experiments showed that Duvelisib dose-dependently suppressed lung fibroblast activation and improved autophagy inhibition by inhibiting the phosphorylation of PI3K, Akt and mTOR. Our results indicate that Duvelisib can alleviate the severity of pulmonary fibrosis and provide potential drugs for the treatment of pulmonary fibrosis.     

Citrus alkaline extracts prevent endoplasmic reticulum stress in type II alveolar epithelial cells to ameliorate pulmonary fibrosis via the ATF3/PINK1 pathway

BackgroundIdiopathic pulmonary fibrosis is a chronic, progressive, fibrotic disease. Although the pathogenesis remains unclear, the effect of endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AEC IIs) is increasingly thought to be a critical mechanism.PurposeWe investigated the effects of citrus alkaline extracts (CAE) on AEC IIs and elucidated the underlying mechanism for their possible use in ameliorating pulmonary fibrosis (PF).MethodsA bleomycin-induced mouse model of PF, and an in vitro tunicamycin (TM) -induced ER stress model in A549 cells were successfully established. Accumulation of collagen in lung tissues in vivo was assessed using histological analysis and western blotting. The expression levels of the ER-stress marker BiP and other related proteins were assessed by western blotting and immunofluorescence staining. Mitochondrial membrane potential was assessed to evaluate mitochondrial homeostasis.ResultsCAE mitigated collagen deposition to ameliorate PF in vivo. CAE suppressed the bleomycin or TM-induced increases in ER-stress biomarker, BiP, and PERK pathway proteins, resulting in a decrease in ER stress in mouse lung tissues and A549 cells, respectively. Additionally, CAE treatment suppressed the bleomycin or TM-induced increase in the ER-stress downstream proteins, activating ATF3 and increased the levels of PINK1 in AEC IIs, both in vivo and in vitro. The reduced mitochondrial homeostasis induced by TM was restored by CAE-treatment in A549 cells. Furthermore, conditioned media from TM-treated A549 cells increased collagen deposition in MRC5 cells mainly via TGF-β1. The increased collagen deposition was not seen using conditioned media from CAE-treated A549 cells.ConclusionThese results provide novel insights into the potential mechanism of CAE in inhibiting ER stress in AEC IIs, and suggests that it has great potential to ameliorate PF via the ATF3/PINK1 pathway.     

Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of insidious onset, and is responsible for up to 30,000 deaths per year in the U.S. Excessive production of extracellular matrix by myofibroblasts has been shown to be an important pathological feature in IPF. TGF-β1 is expressed in fibrotic lung and promotes fibroblast to myofibroblast differentiation (FMD) as well as matrix deposition.

Methods

To identify the mechanism of Arsenic trioxide’s (ATO)’s anti-fibrotic effect in vitro, normal human lung fibroblasts (NHLFs) were treated with ATO for 24 hours and were then exposed to TGF-β1 (1 ng/ml) before harvesting at multiple time points. To investigate whether ATO is able to alleviate lung fibrosis in vivo, C57BL/6 mice were administered bleomycin by oropharyngeal aspiration and ATO was injected intraperitoneally daily for 14 days. Quantitative real-time PCR, western blotting, and immunofluorescent staining were used to assess the expression of fibrotic markers such as α-smooth muscle actin (α-SMA) and α-1 type I collagen.

Results

Treatment of NHLFs with ATO at very low concentrations (10-20nM) inhibits TGF-β1-induced α-smooth muscle actin (α-SMA) and α-1 type I collagen mRNA and protein expression. ATO also diminishes the TGF-β1-mediated contractile response in NHLFs. ATO’s down-regulation of profibrotic molecules is associated with inhibition of Akt, as well as Smad2/Smad3 phosphorylation. TGF-β1-induced H₂O₂ and NOX-4 mRNA expression are also blocked by ATO. ATO-mediated reduction in Smad3 phosphorylation correlated with a reduction of promyelocytic leukemia (PML) nuclear bodies and PML protein expression. PML-/- mouse embryonic fibroblasts (MEFs) showed decreased fibronectin and PAI-1 expression in response to TGF-β1. Daily intraperitoneal injection of ATO (1 mg/kg) in C57BL/6 mice inhibits bleomycin induced lung α-1 type I collagen mRNA and protein expression.

Conclusions

In summary, these data indicate that low concentrations of ATO inhibit TGF-β1-induced fibroblast to myofibroblast differentiation and decreases bleomycin induced pulmonary fibrosis.     

Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis

Earlier work from this laboratory showed that local generation of angiotensin (ANG) II is required for the pathogenesis of experimental pulmonary fibrosis and that ANG peptides are expressed robustly in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Angiotensin converting enzyme-2 (ACE-2) degrades the octapeptide ANG II to form the heptapeptide ANG1-7 and thereby limits ANG II accumulation. On this basis, we hypothesized that ACE-2 would be protective against experimental lung fibrogenesis and might be downregulated in human and experimental lung fibrosis. In lung biopsy specimens from patients with IPF, ACE-2 mRNA and enzyme activity were decreased by 92% (P<0.01) and 74% (P<0.05), respectively. ACE-2 mRNA and activity were also decreased similarly in the lungs of bleomycin-treated rats and C57-BL6 mice. In mice exposed to low doses of bleomycin, lung collagen accumulation was enhanced by intratracheal administration of either ACE-2-specific small interfering RNAs (siRNAs) or the peptide DX(600), a competitive inhibitor of ACE-2 (P<0.05). Administration of either ACE-2 siRNA or DX(600) significantly increased the ANG II content of mouse lung tissue above the level induced by bleomycin alone. Coadministration of the ANG II receptor antagonist saralasin blocked the DX(600)-induced increase in lung collagen. Moreover, purified recombinant human ACE-2, delivered to mice systemically by osmotic minipump, attenuated bleomycin-induced lung collagen accumulation. Together, these data show that ACE-2 mRNA and activity are severely downregulated in both human and experimental lung fibrosis and suggest that ACE-2 protects against lung fibrogenesis by limiting the local accumulation of the profibrotic peptide ANG II.     

Crosstalk between pleural mesothelial cell and lung fibroblast contributes to pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive and fibrosing interstitial pneumonia of unknown cause. The main feature of IPF is a heterogeneous appearance with areas of sub-pleural fibrosis. However, the mechanism of sub-pleural fibrosis was poorly understood. In this study, our in vivo study revealed that pleural mesothelial cells (PMCs) migrated into lung parenchyma and localized alongside lung fibroblasts in sub-pleural area in mouse pulmonary fibrosis. Our in vitro study displayed that cultured-PMCs-medium induced lung fibroblasts transforming into myofibroblast, cultured-fibroblasts-medium promoted mesothelial-mesenchymal transition of PMCs. Furthermore, these changes in lung fibroblasts and PMCs were prevented by blocking TGF-β1/Smad2/3 signaling with SB431542. TGF-β1 neutralized antibody attenuated bleomycin-induced pulmonary fibrosis. Similar to TGF-β1/Smad2/3 signaling, wnt/β-catenin signaling was also activated in the process of PMCs crosstalk with lung fibroblasts. Moreover, inhibition of CD147 attenuated cultured-PMCs-medium induced collagen-I synthesis in lung fibroblasts. Blocking CD147 signaling also prevented bleomycin-induced pulmonary fibrosis. Our data indicated that crosstalk between PMC and lung fibroblast contributed to sub-pleural pulmonary fibrosis. TGF-β1, Wnt/β-catenin and CD147 signaling was involved in the underling mechanism.     

Emerging evidence for endoplasmic reticulum stress in the pathogenesis of idiopathic pulmonary fibrosis

While the factors that regulate the onset and progression of idiopathic pulmonary fibrosis (IPF) are incompletely understood, recent investigations have revealed that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are prominent in alveolar epithelial cells in this disease. Initial observations linking ER stress and IPF were made in cases of familial interstitial pneumonia (FIP), the familial form of IPF, in a family with a mutation in surfactant protein C (SFTPC). Subsequent studies involving lung biopsy specimens revealed that ER stress markers are highly expressed in the alveolar epithelium in IPF and FIP. Recent mouse modeling has revealed that induction of ER stress in the alveolar epithelium predisposed to enhanced lung fibrosis after treatment with bleomycin, which is mediated at least in part by increased alveolar epithelial cell (AEC) apoptosis. Emerging data also indicate that ER stress in AECs could impact fibrotic remodeling by altering inflammatory responses and inducing epithelial-mesenchymal transition. Although the cause of ER stress in IPF remains unknown, common environmental exposures such as herpesviruses, inhaled particulates, and cigarette smoke induce ER stress and are candidates for contributing to AEC dysfunction by this mechanism. Together, investigations to date suggest that ER stress predisposes to AEC dysfunction and subsequent lung fibrosis. However, many questions remain regarding the role of ER stress in initiation and progression of lung fibrosis, including whether ER stress or the UPR could be targeted for therapeutic benefit.     

Metastasis-associated protein 1 promotes epithelial-mesenchymal transition in idiopathic pulmonary fibrosis by up-regulating Snail expression

Idiopathic pulmonary fibrosis (IPF) is a progressive and usually fatal lung disease that lacking effective interventions. It is well known that aberrant activation of transforming growth factor-beta1 (TGF-β1) frequently promotes epithelial-mesenchymal transition (EMT) in IPF. Metastasis-associated gene 1 (MTA1) has identified as an oncogene in several human tumours, and aberrant MTA1 expression has been related to the EMT regulation. However, its expression and function in IPF remain largely unexplored. Using a combination of in vitro and in vivo studies, we found that MTA1 was significantly up-regulated in bleomycin-induced fibrosis rats and TGF-β1-treated alveolar type Ⅱ epithelial (RLE-6TN) cells. Overexpression of MTA1 induced EMT of RLE-6TN cells, as well as facilitates cell proliferation and migration. In contrast, knockdown of MTA1 reversed TGF-β1-induced EMT of RLE-6TN cells. The pro-fibrotic action of MTA1 was mediated by increasing Snail expression through up-regulating Snail promoter activity. Moreover, inhibition of MTA1 effectively attenuated bleomycin-induced fibrosis in rats. Additionally, we preliminarily found astragaloside IV (ASV), which was previously validated having inhibitory effects on TGF-β1-induced EMT, could inhibit MTA1 expression in TGF-β1-treated RLE-6TN cells. These findings highlight the role of MTA1 in TGF-β1-mediated EMT that offer novel strategies for the prevention and treatment of IPF.     

The effect of carbamazepine on bone structure and strength in control and osteogenesis imperfecta (Col1a2 +/p.G610C ) mice

The inherited brittle bone disease osteogenesis imperfecta (OI) is commonly caused by COL1A1 and COL1A2 mutations that disrupt the collagen I triple helix. This causes intracellular endoplasmic reticulum (ER) retention of the misfolded collagen and can result in a pathological ER stress response. A therapeutic approach to reduce this toxic mutant load could be to stimulate mutant collagen degradation by manipulating autophagy and/or ER‐associated degradation. Since carbamazepine (CBZ) both stimulates autophagy of misfolded collagen X and improves skeletal pathology in a metaphyseal chondrodysplasia model, we tested the effect of CBZ on bone structure and strength in 3‐week‐old male OI Col1a2 ^+/p.G610C and control mice. Treatment for 3 or 6 weeks with CBZ, at the dose effective in metaphyseal chondrodysplasia, provided no therapeutic benefit to Col1a2 ^+/p.G610C mouse bone structure, strength or composition, measured by micro‐computed tomography, three point bending tests and Fourier‐transform infrared microspectroscopy. In control mice, however, CBZ treatment for 6 weeks impaired femur growth and led to lower femoral cortical and trabecular bone mass. These data, showing the negative impact of CBZ treatment on the developing mouse bones, raise important issues which must be considered in any human clinical applications of CBZ in growing individuals.     

TGF-beta driven lung fibrosis is macrophage dependent and blocked by Serum amyloid P

The pleiotropic growth factor TGFβ(1) promotes many of the pathogenic mechanisms observed in lung fibrosis and airway remodeling, such as aberrant extracellular matrix deposition due to both fibroblast activation and fibroblast to myofibroblast differentiation. Serum amyloid P (SAP), a member of the pentraxin family of proteins inhibits bleomycin-induced lung fibrosis through an inhibition of pulmonary fibrocyte and pro-fibrotic alternative (M2) macrophage accumulation. It is unknown if SAP has effects downstream of TGFβ(1), a major mediator of pulmonary fibrosis. Using the lung specific TGFβ(1) transgenic mouse model, we determined that SAP inhibits all of the pathologies driven by TGFβ(1) including apoptosis, airway inflammation, pulmonary fibrocyte accumulation and collagen deposition, without affecting levels of TGFβ(1). To explore the role of monocyte derived cells in this model we used liposomal clodronate to deplete pulmonary macrophages. This led to pronounced anti-fibrotic effects that were independent of fibrocyte accumulation. Administration of SAP mirrored these effects and reduced both pulmonary M2 macrophages and increased chemokine IP10/CXCL10 expression in a SMAD 3-independent manner. Interestingly, SAP concentrations were reduced in the circulation of IPF patients and correlated with disease severity. Last, SAP directly inhibited M2 macrophage differentiation of monocytes obtained from these patients. These data suggest that the beneficial anti-fibrotic effects of SAP in TGFβ(1)-induced lung disease are via modulating monocyte responses.     

TSSK4 upregulation in alveolar epithelial type-II cells facilitates pulmonary fibrosis through HSP90-AKT signaling restriction and AT-II apoptosis

Alveolar epithelial injury is one of the important pathological changes in idiopathic pulmonary interstitial fibrosis (IPF), but the regulatory mechanism remains unclear. Here, we reported that alveolar epithelial type-II cells (AT II) play important roles in pathological process of pulmonary fibrosis. Through iTRAQ (isobaric tagging for relative and absolute quantification) quantitative proteomics, TSSK4 was identified to be upregulated in bleomycin-induced fibrotic mice model, which was further confirmed in clinical IPF patients’ tissue specimens. TSSK4 is a germ-related protein, but its expression in other tissues and the association with other diseases are not reported. Immunofluorescence staining showed that TSSK4 selectively expressed in AT-II cells, which are essential for inflammation-induced AT-II loss during fibrosis. Luciferase assay and other molecular biological experiments proved that TSSK4 expression is regulated by TNF-α-mediated NF-κB signaling. The TSSK4 kinase activity is found to be closely related to the function of HSP90-AKT pathway that TSSK4 can phosphorylate its substrate HSP90β on serine 255, to inhibit the ATPase activity of HSP90β and reduce its molecular chaperone function on AKT. Under this condition, kinase activity of AKT is diminished to interfere its survival function, subsequently facilitating AT-II cellular apoptosis through the mitochondrial death machinery. Our findings highlight the importance of TSSK4 in regulating pulmonary fibrosis by facilitating AT-II loss through HSP90-AKT signaling, all of which suggest TSSK4 and the regulating mechanism as attractive targets for the clinical intervention of pulmonary injury and fibrosis.Subject terms: Apoptosis, Stress signalling     

M2 macrophages promote myofibroblast differentiation of LR-MSCs and are associated with pulmonary fibrogenesis

Background

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the histopathological pattern of usual interstitial pneumonia and is associated with a high mortality rate. Recently, lung resident mesenchymal stem cells (LR-MSCs) have been identified as an important contributor to myofibroblast activation in pulmonary fibrosis. Macrophages are also believed to play a critical role in pulmonary fibrosis. However, the underlying connections between LR-MSCs and macrophages in the pathogenesis of pulmonary fibrosis are still elusive.

Methods

In this study, we investigated the interaction between LR-MSCs and macrophages using a bleomycin-induced mouse pulmonary fibrosis model and a coculture system.

Results

Here, we show that blocking pulmonary macrophage infiltration attenuated bleomycin-induced pulmonary fibrosis. In addition, as determined by flow cytometry, we discovered that the recruited macrophages in fibrotic lungs of bleomycin-treated mice were mainly M2 macrophages. In particular, we found that M2, rather than M1 macrophages, promoted myofibroblast differentiation of LR-MSCs. Moreover, we demonstrated that suppression of the Wnt/β-catenin signaling pathway could attenuate myofibroblast differentiation of LR-MSCs induced by M2 macrophages and bleomycin-induced pulmonary fibrosis. Tissue samples from IPF patients confirmed the infiltration of M2 macrophages and activation of Wnt/β-catenin signaling pathway.

Conclusion

In summary, this study furthered our understanding of the pulmonary fibrosis pathogenesis and highlighted M2 macrophages as a critical target for treating pulmonary fibrosis.