Rac2 is involved in bleomycin-induced lung inflammation leading to pulmonary fibrosis

Background

Pulmonary fibrotic diseases induce significant morbidity and mortality, for which there are limited therapeutic options available. Rac2, a ras-related guanosine triphosphatase expressed mainly in hematopoietic cells, is a crucial molecule regulating a diversity of mast cell, macrophage, and neutrophil functions. All these cell types have been implicated in the development of pulmonary fibrosis in a variety of animal models. For the studies described here we hypothesized that Rac2 deficiency protects mice from bleomycin-induced pulmonary fibrosis.

Methods

To determine the role of Rac2 in pulmonary fibrosis we used a bleomycin-induced mouse model. Anesthetized C57BL/6 wild type and rac2 ^-/- mice were instilled intratracheally with bleomycin sulphate (1.25 U/Kg) or saline as control. Bronchoalveolar lavage (BAL) samples were collected at days 3 and 7 of treatment and analyzed for matrix metalloproteinases (MMPs). On day 21 after bleomycin treatment, we measured airway resistance and elastance in tracheotomized animals. Lung sections were stained for histological analysis, while homogenates were analyzed for hydroxyproline and total collagen content.

Results

BLM-treated rac2 ^-/- mice had reduced MMP-9 levels in the BAL on day 3 and reduced neutrophilia and TNF and CCL3/MIP-1α levels in the BAL on day 7 compared to BLM-treated WT mice. We also showed that rac2 ^-/- mice had significantly lower mortality (30%) than WT mice (70%) at day 21 of bleomycin treatment. Lung function was diminished in bleomycin-treated WT mice, while it was unaffected in bleomycin-treated rac2 ^-/- mice. Histological analysis of inflammation and fibrosis as well as collagen and hydroxyproline content in the lungs did not show significant differences between BLM-treated rac2 ^-/- and WT and mice that survived to day 21.

Conclusion

Rac2 plays an important role in bleomycin-induced lung injury. It is an important signaling molecule leading to BLM-induced mortality and it also mediates the physiological changes seen in the airways after BLM-induced injury.     

Melatonin Inhibits Endoplasmic Reticulum Stress and Epithelial-Mesenchymal Transition during Bleomycin-Induced Pulmonary Fibrosis in Mice

Several reports indicate that melatonin alleviates bleomycin (BLM)-induced pulmonary fibrosis in rodent animals. Nevertheless, the exact mechanism remains obscure. The present study investigated the effects of melatonin on endoplasmic reticulum (ER) stress and epithelial-mesenchymal transition (EMT) during BLM-induced lung fibrosis. For the induction of pulmonary fibrosis, mice were intratracheally injected with a single dose of BLM (5.0 mg/kg). Some mice were intraperitoneally injected with melatonin (5 mg/kg) daily for a period of 3 wk. Twenty-one days after BLM injection, lung fibrosis was evaluated. As expected, melatonin significantly alleviated BLM-induced pulmonary fibrosis, as evidenced by Sirius red staining. Moreover, melatonin significantly attenuated BLM-induced EMT to myofibroblasts, as determined by its repression of α-SMA expression. Further analysis showed that melatonin markedly attenuated BLM-induced GRP78 up-regulation and elevation of the cleaved ATF6 in the lungs. Moreover, melatonin obviously attenuated BLM-induced activation of pulmonary eIF2α, a downstream target of the PERK pathway. Finally, melatonin repressed BLM-induced pulmonary IRE1α phosphorylation. Correspondingly, melatonin inhibited BLM-induced activation of XBP-1 and JNK, two downstream targets of the IRE1 pathway. Taken together, these results suggest that melatonin alleviates ER stress and ER stress-mediated EMT in the process of BLM-induced pulmonary fibrosis.     

Natural Killer Cell-Dependent Anti-Fibrotic Pathway in Liver Injury via Toll-Like Receptor-9

The toll-like receptor-9 (TLR9) agonist cytosine phosphate guanine (CpG), activates hepatic stellate cells (HSCs) and mediates fibrosis. We investigated the TLR9 effects on lymphocyte/HSCs interactions. Liver fibrosis was induced in wild-type (WT) mice by intra-peritoneal carbon-tetrachloride (CCl₄) induction for 6 weeks. Fibrotic groups were intravenously treated by a vehicle versus CpG along last 2 weeks. Compared to vehicle-treated fibrotic WT, the in-vivo CpG-treatment significantly attenuated hepatic fibrosis and inflammation, associated with decreased CD8 and increased NK liver cells. In-vitro, co-cultures with vehicle-treated fibrotic NK cells increased HSCs proliferation (P<0.001) while their CpG-treated counterparts achieved a significant decrease. To investigate the role of lymphocytes, TLR9^-/- mice induced-hepatic fibrosis were used. Although TLR9^-/- mice manifested lower fibrotic profile as compared to their wild-type (WT) counterparts, senescence (SA-β-Gal activity) in the liver and ALT serum levels were significantly greater. In an adoptive transfer model; irradiated WT and TLR9^-/- recipients were reconstituted with naïve WT or TLR9^-/- lymphocytes. The adoptive transfer of TLR9^-/- versus WT lymphocytes led to increased fibrosis of WT recipients. TLR9^-/- fibrotic recipients reconstituted with TLR9^-/- or WT lymphocytes showed no changes in hepatic fibrosis severity or ALT serum levels. TLR9 activation had inconsistent effects on lymphocytes and HSCs. The net balance of TLR9 activation in WT, displayed significant anti-fibrotic activity, accompanied by CD8 suppression and increased NK-cells, activity and adherence to HSCs. The pro-fibrotic and pro-inflammatory properties of TLR9^-/- lymphocytes fail to activate HSCs with an early senescence in TLR9^-/- mice.      

Prostaglandin E2 Induction during Mouse Adenovirus Type 1 Respiratory Infection Regulates Inflammatory Mediator Generation but Does Not Affect Viral Pathogenesis

Respiratory viruses cause substantial disease and are a significant healthcare burden. Virus-induced inflammation can be detrimental to the host, causing symptoms during acute infection and leading to damage that contributes to long-term residual lung disease. Prostaglandin E₂ (PGE₂) is a lipid mediator that is increased in response to many viral infections, and inhibition of PGE₂ production during respiratory viral infection often leads to a decreased inflammatory response. We tested the hypothesis that PGE₂ promotes inflammatory responses to mouse adenovirus type 1 (MAV-1) respiratory infection. Acute MAV-1 infection increased COX-2 expression and PGE₂ production in wild type mice. Deficiency of the E prostanoid 2 receptor had no apparent effect on MAV-1 pathogenesis. Virus-induced induction of PGE₂, IFN-γ, CXCL1, and CCL5 was reduced in mice deficient in microsomal PGE synthase-1 (mPGES-1^-/- mice). However, there were no differences between mPGES-1^+/+ and mPGES-1^-/- mice in viral replication, recruitment of leukocytes to airways or lung inflammation. Infection of both mPGES‑1^+/+ and mPGES-1^-/- mice led to protection against reinfection. Thus, while PGE₂ promotes the expression of a variety of cytokines in response to acute MAV-1 infection, PGE₂ synthesis does not appear to be essential for generating pulmonary immunity.     

Secretoglobin 3A2 Exhibits Anti-Fibrotic Activity in Bleomycin-Induced Pulmonary Fibrosis Model Mice

Objective

Secretoglobin (SCGB) 3A2 is a novel lung-enriched cytokine, previously shown to exhibit anti-inflammatory, growth factor, and anti-fibrotic activities. The latter activity was demonstrated using exogenously-administered recombinant SCGB3A2 in the bleomycin (BLM)-induced pulmonary fibrosis model. Whether SCGB3A2 exhibits anti-fibrotic activity in vivo is not known.

Methods

Mice null for the Scgb3a2 gene were subjected to the BLM-induced pulmonary fibrosis model, and the severity of pulmonary fibrosis determined using histological and biochemical methods.

Results

BLM treatment caused weight loss of both Scgb3a2-null and wild-type mice, however, the loss was far more pronounced in BLM-treated Scgb3a2-null than wild-type mice, and the weight of day 21 of BLM-treated Scgb3a2-null mice was about half of that of BLM-treated wild-type mice. Hematoxylin & Eosin, Masson Trichrome, and Sirius Red staining of lung sections, Ashcroft fibrosis scores, hydroxyproline contents, and the levels of mRNAs encoding various collagens demonstrated that BLM-treated Scgb3a2-null mouse lungs had more severe fibrosis than those of wild-type mouse lungs. Total and differential inflammatory cell numbers in bronchoalveolar lavage fluids, and levels of lung mRNAs including those encoding Th2 cytokines such as IL-4 and profibrotic cytokines such as TGFβ were higher in BLM-treated Scgb3a2-null mouse lungs as compared to those of wild-type mouse lungs. In contrast, mRNAs encoding surfactant proteins A, B, C, and D, and SCGB1A1 did not differ between BLM-treated Scgb3a2-null and wild-type mouse lungs.

Conclusion

The role of SCGB3A2 in fibrosis was revisited using Scgb3a2-null mice and littermate controls in the BLM-induced pulmonary fibrosis model. The pulmonary fibrosis in the Scgb3a2-null mice was more severe than the wild-type controls, thus establishing that SCGB3A2 has anti-fibrotic activity in vivo. Importantly, surfactant proteins and SCGB1A1 appear not to be involved in the susceptibility of Scgb3a2-null mice to BLM-induced pulmonary fibrosis.     

Microsomal prostaglandin E synthase-1 aggravates inflammation and demyelination in a mouse model of multiple sclerosis

Microsomal prostaglandin synthetase-1 (mPGES-1) is an inducible terminal enzyme required for prostaglandin E₂ (PGE₂) biosynthesis. In this study, we examined the role of mPGES-1 in the inflammation and demyelination observed in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We induced EAE with myelin oligodendrocyte glycoprotein_35–55 peptide in mPGES-1-deficient (mPGES-1^−/−) and wild-type (WT) mice. First, we examined the histopathology in the early and late phases of EAE progression. Next, we measured the concentration of PGE₂ in the spinal cord and investigated the expression of mPGES-1 using immunohistochemistry. In addition, we examined the progression of the severity of EAE using an EAE score to investigate a correlation between pathological features and paralysis. In this paper, we demonstrate that WT mice showed extensive inflammation and demyelination, whereas mPGES-1^−/− mice exhibited significantly smaller and more localized changes in the perivascular area. The mPGES-1 protein was induced in vascular endothelial cells and microglia around inflammatory foci, and PGE₂ production was increased in WT mice but not mPGES-1^−/− mice. Furthermore, mPGES-1^−/− mice showed a significant reduction in the maximum EAE score and improved locomotor activity. These results suggest that central PGE₂ derived from non-neuronal mPGES-1 aggravates the disruption of the vessel structure, leading to the spread of inflammation and local demyelination in the spinal cord, which corresponds to the symptoms of EAE. The inhibition of mPGES-1 may be useful for the treatment of human MS.     

Blockade of advanced glycation end product formation attenuates bleomycin-induced pulmonary fibrosis in rats

Background

Advanced glycation end products (AGEs) have been proposed to be involved in pulmonary fibrosis, but its role in this process has not been fully understood. To investigate the role of AGE formation in pulmonary fibrosis, we used a bleomycin (BLM)-stimulated rat model treated with aminoguanidine (AG), a crosslink inhibitor of AGE formation.

Methods

Rats were intratracheally instilled with BLM (5 mg/kg) and orally administered with AG (40, 80, 120 mg/kg) once daily for two weeks. AGEs level in lung tissue was determined by ELISA and pulmonary fibrosis was evaluated by Ashcroft score and hydroxyproline assay. The expression of heat shock protein 47 (HSP47), a collagen specific molecular chaperone, was measured with RT-PCR and Western blot. Moreover, TGFβ1 and its downstream Smad proteins were analyzed by Western blot.

Results

AGEs level in rat lungs, as well as lung hydroxyproline content and Ashcroft score, was significantly enhanced by BLM stimulation, which was abrogated by AG treatment. BLM significantly increased the expression of HSP47 mRNA and protein in lung tissues, and AG treatment markedly decreased BLM-induced HSP47 expression in a dose-dependent manner (p < 0.05). In addition, AG dose-dependently downregulated BLM-stimulated overexpressions of TGFβ1, phosphorylated (p)-Smad2 and p-Smad3 protein in lung tissues.

Conclusion

These findings suggest AGE formation may participate in the process of BLM-induced pulmonary fibrosis, and blockade of AGE formation by AG treatment attenuates BLM-induced pulmonary fibrosis in rats, which is implicated in inhibition of HSP47 expression and TGFβ/Smads signaling.     

Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing

Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3 ^-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3 ^-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3 ^-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3 ^-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3 ^-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3 ^-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3 ^-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3 ^-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3 ^-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression of wound closure.     

Lack of Matrilin-2 Favors Liver Tumor Development via Erk1/2 and GSK-3β Pathways In Vivo

Matrilin-2 (Matn2) is a multidomain adaptor protein which plays a role in the assembly of extracellular matrix (ECM). It is produced by oval cells during stem cell-driven liver regeneration. In our study, the impact of Matn2 on hepatocarcinogenesis was investigated in Matn2^-/- mice comparing them with wild-type (WT) mice in a diethylnitrosamine (DEN) model. The liver tissue was analyzed macroscopically, histologically and immunohistochemically, at protein level by Proteome Profiler Arrays and Western blot analysis. Matn2^-/- mice exhibited higher susceptibility to hepatocarcinogenesis compared to wild-type mice. In the liver of Matn2^-/- mice, spontaneous microscopic tumor foci were detected without DEN treatment. After 15 μg/g body weight DEN treatment, the liver of Matn2^-/- mice contained macroscopic tumors of both larger number and size than the WT liver. In contrast with the WT liver, spontaneous phosphorylation of EGFR, Erk1/2 GSK-3α/β and retinoblastoma protein (p-Rb), decrease in p21/CIP1 level, and increase in β-Catenin protein expression were detected in Matn2^-/- livers. Focal Ki-67 positivity of these samples provided additional support to our presumption that the lack of Matn2 drives the liver into a pro-proliferatory state, making it prone to tumor development. This enhanced proliferative capacity was further increased in the tumor nodules of DEN-treated Matn2^-/- livers. Our study suggests that Matn2 functions as a tumor suppressor in hepatocarcinogenesis, and in this process activation of EGFR together with that of Erk1/2, as well as inactivation of GSK-3β, play strategic roles.     

TLR2 Regulates Neutrophil Recruitment and Cytokine Production with Minor Contributions from TLR9 during Hypersensitivity Pneumonitis

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to environmental antigens. The disease results in alveolitis, granuloma formation and may progress to a fibrotic chronic form, which is associated with significant morbidity and mortality. The severity of the disease correlates with a neutrophil rich influx and an IL-17 response. We used the Saccharopolyspora rectivirgula (SR) model of HP to determine whether Toll-like receptors (TLR) 2 and 9 cooperate in neutrophil recruitment and IL-17-associated cytokine production during the development of HP. Stimulation of bone marrow derived macrophages (BMDMs) from C57BL/6, MyD88^-/- and TLR2/9^-/- mice with SR demonstrate that SR is a strong inducer of neutrophil chemokines and growth factors. The cytokines induced by SR were MyD88-dependent and, of those, most were partially or completely dependent on TLRs 2 and 9. Following in vivo exposure to SR, CXCL2 production and neutrophil recruitment were reduced in TLR2^-/- and TLR2/9^-/- mice suggesting that the response was largely dependent on TLR2; however the reduction was greatest in the TLR2/9^-/- double knockout mice indicating TLR9 may also contribute to the response. There was a reduction in the levels of pro-inflammatory cytokines TNFα and IL-6 as well as CCL3 and CCL4 in the BALF from TLR2/9^-/- mice compared to WT and single knockout (SKO) mice exposed one time to SR. The decrease in neutrophil recruitment and TNFα production in the TLR2/9^-/- mice was maintained throughout 3 weeks of SR exposures in comparison to WT and SKO mice. Both TLRs 2 and 9 contributed to the Th17 response; there was a decrease in Th17 cells and IL-17 mRNA in the TLR2/9^-/- mice in comparison to the WT and SKO mice. Despite the effects on neutrophil recruitment and the IL-17 response, TLR2/9^-/- mice developed granuloma formation similarly to WT and SKO mice suggesting that there are additional mediators and pattern recognition receptors involved in the disease.     

Inhibition of α-SMA by the Ectodomain of FGFR2c Attenuates Lung Fibrosis

The soluble ectodomain of fibroblast growth factor receptor-IIIc (sFGFR2c) is able to bind to fibroblast growth factor (FGF) ligands and block the activation of the FGF-signaling pathway. In this study, sFGFR2c inhibited lung fibrosis dramatically in vitro and in vivo. The upregulation of α-smooth muscle actin (α-SMA) in fibroblasts by transforming growth factor-β1 (TGF-β1) is an important step in the process of lung fibrosis, in which FGF-2, released by TGF-β1, is involved. sFGFR2c inhibited α-SMA induction by TGF-β1 via both the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad3 pathways in primary mouse lung fibroblasts and the proliferation of mouse lung fibroblasts. In a mouse model of bleomycin (BLM)-induced lung fibrosis, mice were treated with sFGFR2c from d 3 or d 10 to 31 after BLM administration. Then we used hematoxylin and eosin staining, Masson staining and immunohistochemical staining to evaluate the inhibitory effects of sFGFR2c on lung fibrosis. The treatment with sFGFR2c resulted in significant attenuation of the lung fibrosis score and collagen deposition. The expression levels of α-SMA, p-FGFRs, p-ERK1/2 and p-Smad3 in the lungs of sFGFR2c-treated mice were markedly lower. sFGFR2c may have potential for the treatment of lung fibrosis as an FGF-2 antagonist.     

Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1β is deleterious

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1β, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1β by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3^-/- mice was due to decreased production of IL-18 and IL-1β. In contrast, Nlrc4^-/- mice produced IL-1β and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1β or IL-1 receptor agonist revealed that IL-1β has deleterious effects during melioidosis. The detrimental role of IL-1β appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4^-/- mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1β.     

Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via β₂-adrenoceptor (β₂-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α_2A-and α_2C-AR (α_2A/2C-AR^-/-). These findings suggest that β₂-AR is not the single adrenoceptor involved in bone turnover regulation and show that α₂-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α_2A/2C-AR^-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α₂-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α₂-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α_2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α_2CAR^-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (μCT) analysis showed that α_2C-AR^-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α_2C-AR^-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α_2C-AR^-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α_2C-AR^-/- mice. Altogether, these findings suggest that α_2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α_2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure.     

Asiaticoside might attenuate bleomycin‐induced pulmonary fibrosis by activating cAMP and Rap1 signalling pathway assisted by A2AR

Asiaticoside (AS) has been reported to have protective effect on pulmonary fibrosis (PF). In this study, we aimed to explore the potential mechanism of the therapeutic role of AS and its relationship with A2AR in PF. Adenosine 2A receptor gene knockout (A2AR^?/?) mice and wild‐type (WT) mice were used to establish bleomycin (BLM)‐induced PF models and were then treated with AS (50 mg/kg/d). Pulmonary inflammation and fibrosis were observed in the PF model with much higher severity in A2AR^?/?mice than that in WT mice and AS significantly alleviated lung inflammation and fibrosis; however, it was less effective in A2AR^?/? mice than in WT mice via histopathological analysis. Using RNA sequencing analysis, we found up‐regulated differentially expressed genes (DEGs) in BLM group were enriched in immune and inflammation‐associated pathways compared with control group. There were 242 common DEGs between down‐regulated in BLM vs control group and up‐regulated in BLM + AS vs BLM group, which were enriched in cAMP and Rap1 signalling pathways. Furthermore, the expression of five key factors of these two pathways including adenylate cyclase (ADCY1, ADCY5, ADCY8, cAMP and Rap1) were confirmed up‐regulated by AS with the presence of A2AR. Therefore, AS might attenuate BLM‐induced PF by activating cAMP and Rap1 signalling pathways which is assisted by A2AR, making it a promising therapeutic optional for PF.     

B Cell Deficient Mice Are Protected from Biliary Obstruction in the Rotavirus-Induced Mouse Model of Biliary Atresia

A leading theory regarding the pathogenesis of biliary atresia (BA) is that bile duct injury is initiated by a virus infection, followed by an autoimmune response targeting bile ducts. In experimental models of autoimmune diseases, B cells have been shown to play an important role. The aim of this study was to determine the role of B cells in the development of biliary obstruction in the Rhesus rotavirus (RRV)-induced mouse model of BA. Wild-type (WT) and B cell-deficient (Ig-α^-/-) mice received RRV shortly after birth. Ig-α^-/- RRV-infected mice had significantly increased disease-free survival rate compared to WT RRV-infected BA mice (76.8% vs. 17.5%). In stark contrast to the RRV-infected BA mice, the RRV-infected Ig-α^-/- mice did not have hyperbilirubinemia or bile duct obstruction. The RRV-infected Ig-α^-/- mice had significantly less liver inflammation and Th1 cytokine production compared to RRV-infected WT mice. In addition, Ig-α^-/- mice had significantly increased numbers of regulatory T cells (Tregs) at baseline and after RRV infection compared to WT mice. However, depletion of Tregs in Ig-α^-/- mice did not induce biliary obstruction, indicating that the expanded Tregs in the Ig-α^-/- mice were not the sole reason for protection from disease. Conclusion: B cell deficient Ig-α^-/- mice are protected from biliary obstruction in the RRV-induced mouse model of BA, indicating a primary role of B cells in mediating disease pathology. The mechanism of protection may involve lack of B cell antigen presentation, which impairs T-cell activation and Th1 inflammation. Immune modulators that inhibit B cell function may be a new strategy for treatment of BA.     

Epithelial vs myofibroblast differentiation in immortal rat lung cell lines—modulating effects of bleomycin

Two alveolar epithelial cell lines R3/1 and L2 were screened by immunocytochemical and RT-PCR analysis of epithelial and mesenchymal/contractile marker proteins. R3/1 and L2 cells were tested for their sensitivity to bleomycin (BLM), an anticancer drug, which is proposed to induce changes in lung cell differentiation. Both epithelial cell lines exhibited a mixed phenotype consisting of epithelial (E-cadherin, aquaporin-5 and cytokeratin 8) and myofibroblast-like (vimentin, α-SMA and caveolin-3) properties suggesting that the cell lines are arrested in vitro at a certain developmental stage during epithelial–mesenchymal transition (EMT). BLM treatment of R3/1 cells resulted in a partial reversal of this process modifying the cells in an epithelial direction, e.g., upregulation of E-cadherin, aquaporin-5 and other lung epithelial antigens at the mRNA and protein level. L2 cells showed similar alterations following BLM exposure. Immunohistochemical investigation of lung tissue from two different animal models of BLM-induced fibrosis (mouse and rat), revealed no signs of EMT, e.g., myofibroblastic differentiation of alveolar epithelial cells in situ. Immunohistological analysis of tissue samples of the rat model showed a heterogeneous population of myofibroblasts (α-SMA⁺/caveolin-3⁺, α-SMA^-/caveolin-3⁺, and α-SMA⁺/caveolin-3⁻). These results suggest that BLM, on one hand, induces fibrosis and on the other hand possibly suppresses EMT during fibrogenesis.