Polydatin ameliorates pulmonary fibrosis by suppressing inflammation and the epithelial mesenchymal transition via inhibiting the TGF-β/Smad signaling pathway

Pulmonary fibrosis is a chronic and progressive lung disease which results in a loss of pulmonary function and eventually respiratory failure. Inflammation and epithelial mesenchymal transition (EMT) play important roles in the pathogenesis of pulmonary fibrosis. This study aimed to investigate the therapeutic effect of polydatin (PD) in bleomycin-induced pulmonary fibrosis. A bleomycin-induced pulmonary fibrosis animal model used SD rats. Morphological changes were analyzed by hematoxylin-eosin staining. RT-qPCR and western blot were used for the detection of the expression of TGF-β1, collagen I, collagen III, E-cadherin, fibronectin and the ratios of p-Smad2/Smad2, p-Smad3/Smad3. The concentrations of PICP, PIIINP, TNF-α, IL-1β, IL-6 and IL-17 were measured by enzyme linked immunosorbent assay (Elisa) assay. Results showed that PD attenuated bleomycin-induced pulmonary fibrosis. The beneficial effect of PD was possibly related to the inhibition of inflammation and EMT through suppressing the TGF-β/Smad signaling pathway. Our findings suggested that PD might be a potential therapeutic candidate in the treatment of pulmonary fibrosis.

Pulmonary fibrosis is a chronic and progressive lung disease which results in a loss of pulmonary function and eventually respiratory failure.     

MyD88-dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals

While it is known that monosodium urate (MSU) crystals cause the disease gout, the mechanism by which these crystals stimulate this inflammatory condition has not been clear. Here we find that the Toll/IL-1R (TIR) signal transduction adaptor myeloid differentiation primary response protein 88 (MyD88) is required for acute gouty inflammation. In contrast, other TIR adaptor molecules, TIRAP/Mal, TRIF, and TRAM, are not required for this process. The MyD88-dependent TLR1, -2, -4, -6, -7, -9, and -11 and IL-18 receptor (IL-18R) are not essential for MSU-induced inflammation. Moreover, MSU does not stimulate HEK cells expressing TLR1-11 to activate NF-kappaB. In contrast, mice deficient in the MyD88-dependent IL-1R showed reduced inflammatory responses, similar to those observed in MyD88-deficient mice. Similarly, mice treated with IL-1 neutralizing antibodies also showed reduced MSU-induced inflammation, demonstrating that IL-1 production and IL-1R activation play essential roles in MSU-triggered inflammation. IL-1R deficiency in bone marrow-derived cells did not affect the inflammatory response; however, it was required in non-bone marrow-derived cells. These results indicate that IL-1 is essential for the MSU-induced inflammatory response and that the requirement of MyD88 in this process is primarily through its function as an adaptor molecule in the IL-1R signaling pathway.     

IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice

Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of proinflammatory cytokines, including IL-1β. IL-1β, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) are all important regulators of the IL-1 signaling complex, which plays a role in inflammation. Furthermore, IL-1β maturation is dependent on caspase-1 (Casp-1). Using IL-1Ra–treated mice as well as 3 mouse models deficient in regulators of IL-1β activation (Casp-1 and ASC) or signaling (IL-1R1), we found that IL-1β signaling is required for the development of alcohol-induced liver steatosis, inflammation, and injury. Increased IL-1β was due to upregulation of Casp-1 activity and inflammasome activation. The pathogenic role of IL-1 signaling in ALD was attributable to the activation of the inflammasome in BM-derived Kupffer cells. Importantly, in vivo intervention with a recombinant IL-1Ra blocked IL-1 signaling and markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. Furthermore, physiological doses of IL-1β induced steatosis, increased the inflammatory and prosteatotic chemokine MCP-1 in hepatocytes, and augmented TLR4-dependent upregulation of inflammatory signaling in macrophages. In conclusion, we demonstrated that Casp-1–dependent upregulation of IL-1β and signaling mediated by IL-1R1 are crucial in ALD pathogenesis. Our findings suggest a potential role of IL-1R1 inhibition in the treatment of ALD.     

Dual function of MyD88 in RAS signaling and inflammation, leading to mouse and human cell transformation

Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for RAS-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, Erk, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical RAS pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated Erk in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in RAS signaling, cell-cycle control, and cell transformation.     

Increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury

Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-β activation that was mediated by proteinase-activated receptor–1 (PAR1) and integrin α_vβ₅. PAR1, α_vβ₅, and α-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.     

IL-36 in chronic inflammation and fibrosis — bridging the gap?

IL-36 is a member of the IL-1 superfamily and consists of three agonists and one receptor antagonist (IL-36Ra). The three endogenous agonists, IL-36α, –β, and –γ, act primarily as proinflammatory cytokines, and their signaling through the IL-36 receptor (IL-36R) promotes immune cell infiltration and secretion of inflammatory and chemotactic molecules. However, IL-36 signaling also fosters secretion of profibrotic soluble mediators, suggesting a role in fibrotic disorders. IL-36 isoforms and IL-36 have been implicated in inflammatory diseases including psoriasis, arthritis, inflammatory bowel diseases, and allergic rhinitis. Moreover, IL-36 has been connected to fibrotic disorders affecting the kidney, lung, and intestines. This review summarizes the expression, cellular source, and function of IL-36 in inflammation and fibrosis in various organs, and proposes that IL-36 modulation may prove valuable in preventing or treating inflammatory and fibrotic diseases and may reveal a mechanistic link between inflammation and fibrosis.     

Interleukin 1 receptor dependence of serum transferred arthritis can be circumvented by toll-like receptor 4 signaling

Inflammatory arthritis is associated with the release of a network of key cytokines. In T cell receptor transgenic K/BxN mice interleukin (IL)-1 plays a key role in joint swelling and destruction, as suggested by the ability of anti-IL-1receptor (IL-1R) antibody treatment to delay the onset and slow the progression of this disease. This mechanism is dependent on the signaling pathway intermediary myeloid differentiation factor 88 (MyD88), such that neither IL-1R nor MyD88-deficient mice developed visually detectable synovitis after transfer of arthritogenic sera. The Toll-like receptors (TLRs) share the same signaling pathway through MyD88 as the IL-1R. The administration of a TLR-4 ligand, lipopolysaccharide, concomitant with arthritogenic serum in IL-1 receptor-deficient mice resulted in acute paw swelling, but not in MyD88-deficient mice. Also, serum transferred arthritis was not sustained in TLR-4 mutant mice compared with controls. These results suggest that innate immune functions via TLR-4 might perpetuate inflammatory mechanisms and bypass the need for IL-1 in chronic joint inflammation.     

Alpinumisoflavone attenuates lipopolysaccharide-induced acute lung injury by regulating the effects of anti-oxidation and anti-inflammation both in vitro and in vivo

Alpinumisoflavone (AIF) is a plant-derived pyranoisoflavone that exhibits a number of pharmacological activities, but the protective effects of AIF against pulmonary inflammation are still unknown. This study aimed to investigate the anti-inflammatory effects and possible molecular mechanisms of AIF in both lipopolysaccharide (LPS)-stimulated macrophages and mice. The results revealed that AIF dramatically suppressed the production of pro-inflammatory mediators [including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, IL-17, intercellular adhesion molecule-1 (ICAM-1), and nitric oxide (NO)] and increased the levels of anti-oxidative enzymes [including catalase (CAT), heme oxygenase-1 (HO-1), glutathione peroxidase (GPx), and superoxide dismutase (SOD)] both in vitro and in vivo. Additionally, pre-treatment with AIF could not only significantly prevent histopathological changes and neutrophil infiltration but also decreased the expression levels of nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), and the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, as well as IL-17 production in LPS-induced lung tissues. The anti-inflammatory effects of AIF were mediated by up-regulating anti-oxidative enzymes and suppressing the NF-κB, MAPK, NLRP3 inflammasome and IL-17 signaling pathways. This is the first study to reveal that AIF has a protective effect against LPS-induced lung injury in mice.

The present study demonstrated that alpinumisoflavone exerts the significant effects of anti-inflammatory and anti-oxidative in both LPS-induced RAW264.7 macrophages and a mouse model of acute lung injury.     

Epithelial cell α3β1 integrin links β-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis

Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-β1. As the ECM critically regulates AEC responses to TGF-β1, we explored the role of the prominent epithelial integrin α3β1 in experimental fibrosis by generating mice with lung epithelial cell–specific loss of α3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through β-catenin has been implicated in EMT; we found that in primary AECs, α3 integrin was required for β-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654–β-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654–β-catenin/pSmad2 complexes and showed accumulation of pY654–β-catenin in myofibroblasts. These findings demonstrate epithelial integrin–dependent profibrotic crosstalk between β-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.     

Divergence of IL-1, IL-18, and cell death in NLRP3 inflammasomopathies

The inflammasome is a cytoplasmic multiprotein complex that promotes proinflammatory cytokine maturation in response to host- and pathogen-derived signals. Missense mutations in cryopyrin (NLRP3) result in a hyperactive inflammasome that drives overproduction of the proinflammatory cytokines IL-1β and IL-18, leading to the cryopyrin-associated periodic syndromes (CAPS) disease spectrum. Mouse lines harboring CAPS-associated mutations in Nlrp3 have elevated levels of IL-1β and IL-18 and closely mimic human disease. To examine the role of inflammasome-driven IL-18 in murine CAPS, we bred Nlrp3 mutations onto an Il18r-null background. Deletion of Il18r resulted in partial phenotypic rescue that abolished skin and visceral disease in young mice and normalized serum cytokines to a greater extent than breeding to Il1r-null mice. Significant systemic inflammation developed in aging Nlrp3 mutant Il18r-null mice, indicating that IL-1 and IL-18 drive pathology at different stages of the disease process. Ongoing inflammation in double-cytokine knockout CAPS mice implicated a role for caspase-1–mediated pyroptosis and confirmed that CAPS is inflammasome dependent. Our results have important implications for patients with CAPS and residual disease, emphasizing the need to explore other NLRP3-mediated pathways and the potential for inflammasome-targeted therapy.     

Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury

Adenosine has been implicated in the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease. In vitro studies suggest that activation of the A2B adenosine receptor (A2BAR) results in proinflammatory and profibrotic effects relevant to the progression of lung diseases; however, in vivo data supporting these observations are lacking. Adenosine deaminase-deficient (ADA-deficient) mice develop pulmonary inflammation and injury that are dependent on increased lung adenosine levels. To investigate the role of the A2BAR in vivo, ADA-deficient mice were treated with the selective A2BAR antagonist CVT-6883, and pulmonary inflammation, fibrosis, and airspace integrity were assessed. Untreated and vehicle-treated ADA-deficient mice developed pulmonary inflammation, fibrosis, and enlargement of alveolar airspaces; conversely, CVT-6883-treated ADA-deficient mice showed less pulmonary inflammation, fibrosis, and alveolar airspace enlargement. A2BAR antagonism significantly reduced elevations in proinflammatory cytokines and chemokines as well as mediators of fibrosis and airway destruction. In addition, treatment with CVT-6883 attenuated pulmonary inflammation and fibrosis in wild-type mice subjected to bleomycin-induced lung injury. These findings suggest that A2BAR signaling influences pathways critical for pulmonary inflammation and injury in vivo. Thus in chronic lung diseases associated with increased adenosine, antagonism of A2BAR-mediated responses may prove to be a beneficial therapy.     

MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88-/-) mice and TRIF-deficient (TRIF-/-) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1alpha stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF-/- mice, but not MyD88-/- mice. These factors stimulated receptor activator of nuclear factor-kappaB ligand mRNA expression in TRIF-/- osteoblasts, but not MyD88-/- osteoblasts. LPS stimulated IL-6 production in TRIF-/- osteoblasts, but not TRIF-/- macrophages. LPS and IL-1alpha enhanced the survival of TRIF-/- osteoclasts, but not MyD88-/- osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88-/- mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.     

TIR domain--containing adaptors regulate TLR-mediated signaling pathways

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses via signaling pathways mediated by several Toll/IL-1R (TIR) domain-containing adaptors such as MyD88, TIRAP, and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4 that is responsible for type I interferon production in response to double-stranded RNA and LPS, respectively. TIRAP specifically participates in the MyD88-dependent pathways shared by TLR2 and TLR4, and TRAM is essential for the TLR4-mediated MyD88-independent pathway. Thus, TIR domain-containing adaptors play an important role in the TLR mediated signaling pathways.     

Blocking follistatin-like 1 attenuates bleomycin-induced pulmonary fibrosis in mice

Progressive tissue fibrosis is a cause of major morbidity and mortality. Pulmonary fibrosis is an epithelial-mesenchymal disorder in which TGF-β1 plays a central role in pathogenesis. Here we show that follistatin-like 1 (FSTL1) differentially regulates TGF-β and bone morphogenetic protein signaling, leading to epithelial injury and fibroblast activation. Haplodeletion of Fstl1 in mice or blockage of FSTL1 with a neutralizing antibody in mice reduced bleomycin-induced fibrosis in vivo. Fstl1 is induced in response to lung injury and promotes the accumulation of myofibroblasts and subsequent fibrosis. These data suggest that Fstl1 may serve as a novel therapeutic target for treatment of progressive lung fibrosis.Progressive tissue fibrosis is an increasing cause of morbidity and mortality worldwide with limited therapeutic options. Idiopathic pulmonary fibrosis (IPF), a particularly severe form of lung fibrosis, is a chronic, progressive, and often fatal interstitial lung disease of unknown etiology with a mean survival of 2–3 yr from diagnosis (ATS/ERS, 2000; Olson et al., 2007). The hallmark of IPF is the unremitting extracellular matrix (ECM) deposition with minimal associated inflammation (Noble and Homer, 2004; Wilson and Wynn, 2009). Although evidence suggests that lung fibrosis is an epithelial-mesenchymal disorder (Selman and Pardo, 2002; Chapman, 2011), the mechanisms by which injured epithelium activates fibroblasts/myofibroblasts are unclear. Epithelial apoptosis pathways are activated in the lungs of patients with acute lung injury, in part by activation of signaling pathways such as Fas ligand–Fas and TGF-β (Hagimoto et al., 2002). In addition, the injured alveolar epithelial cells (AECs) may also be abnormally activated with phenotypic changes (King et al., 2011; Kage and Borok, 2012; Yang et al., 2013). The signals required for this activation are unknown. A recent study suggests that injured kidney epithelial cells produce an increased number of TGF-β–containing exosomes to activate fibroblasts (Borges et al., 2013). We hypothesized that injured pulmonary epithelial cells may activate mesenchymal cells by releasing soluble factors to promote a fibrogenic microenvironment.Both TGF-β (Sime et al., 1997; Gauldie et al., 2007) and bone morphogenetic protein (BMP) signaling pathways (Costello et al., 2010) play a role in the initiation and progression of fibrosis. They regulate both epithelial cell injury and fibroblast proliferation and transdifferentiation into myofibroblasts at the injury site (Leask and Abraham, 2004; Selman et al., 2008; Goodwin and Jenkins, 2009). BMP4 antagonists have been implicated in fibrotic disorders of multiple organs including lung (Dolan et al., 2003; Patella et al., 2006; Costello et al., 2010). The precise mechanisms of TGF-β superfamily members in regulating lung fibrogenesis in specific cell types are largely unclear.Follistatin-like 1 (FSTL1), initially identified as a TGF-β–inducible gene (Shibanuma et al., 1993), encodes a small secreted glycoprotein belonging to a group of matricellular proteins. We recently reported that Fstl1 acts as a BMP4 antagonist to play a key role in lung development (Geng et al., 2011). The role of FSTL1 in lung fibrosis has not been investigated. In this study, we have interrogated the role of FSTL1-regulated TGF-β/BMP signaling in different cell types during lung injury and fibrosis. We report that FSTL1 mediates epithelial-mesenchymal communication at the cellular level. We found that FSTL1 modulated TGF-β but not BMP signaling, leading to fibroblast activation. We provide evidence that targeting FSTL1 may offer a novel therapeutic approach for patients with progressive tissue fibrosis.     

MyD88 L265P 突变与 B 细胞肿瘤简

髓样分化因子88（myeloid differentiation factor,MyD88）是一种介导多数Toll样受体（Toll-like receptors,TLR）、白介素-1受体（interleukin-1 receptor,IL-1R）、白介素-18受体（interleukin-1 8receptor,IL-18R）细胞信号传导的重要衔接蛋白,在固有免疫中起着重要的作用.近年来有研究发现,在约90％的淋巴浆细胞淋巴瘤/华氏巨球蛋白血症和约29％的激活型弥漫大B细胞淋巴瘤等B细胞肿瘤中有功能活化的MYD88 L265P突变,从而提示MyD88信号转导在B细胞肿瘤发生中有重要意义,因而MyD88抑制剂可能成为治疗B细胞肿瘤的新药.本文就MyD88L265P突变在B细胞肿瘤发生中的最新研究进展进行综述.     

IL-17 produced by neutrophils regulates IFN-γ–mediated neutrophil migration in mouse kidney ischemia-reperfusion injury

The IL-23/IL-17 and IL-12/IFN-γ cytokine pathways have a role in chronic autoimmunity, which is considered mainly a dysfunction of adaptive immunity. The extent to which they contribute to innate immunity is, however, unknown. We used a mouse model of acute kidney ischemia-reperfusion injury (IRI) to test the hypothesis that early production of IL-23 and IL-12 following IRI activates downstream IL-17 and IFN-γ signaling pathways and promotes kidney inflammation. Deficiency in IL-23, IL-17A, or IL-17 receptor (IL-17R) and mAb neutralization of CXCR2, the p19 subunit of IL-23, or IL-17A attenuated neutrophil infiltration in acute kidney IRI in mice. We further demonstrate that IL-17A produced by GR-1⁺ neutrophils was critical for kidney IRI in mice. Activation of the IL-12/IFN-γ pathway and NKT cells by administering α-galactosylceramide–primed bone marrow–derived DCs increased IFN-γ production following moderate IRI in WT mice but did not exacerbate injury or enhance IFN-γ production in either Il17a^–/– or Il17r^–/– mice, which suggested that IL-17 signaling was proximal to IFN-γ signaling. This was confirmed by the finding that IFN-γ administration reversed the protection seen in Il17a^–/– mice subjected to IRI, whereas IL-17A failed to reverse protection in Ifng^–/– mice. These results demonstrate that the innate immune component of kidney IRI requires dual activation of the IL-12/IFN-γ and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17A is upstream of IL-12/IFN-γ. These mechanisms might contribute to reperfusion injury in other organs.