MicroRNA-326 attenuates hepatic stellate cell activation and liver fibrosis by inhibiting TLR4 signaling

Hepatic fibrosis is a chronic inflammatory and reversible repair reaction of the liver under the continuous action of virus or various injuries. In this study, we aimed at identifying the role of miR-326 in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. In this study, the liver fibrosis mouse model was developed by injecting CCl₄. Liver tissue morphology was observed and the expression level of α-smooth muscle actin, collagen1α1 and miR-326 was measured. Target gene identification was performed by loss-of-function and gain-of-function. The effect of miR-326 on the expression level of the cytokines associated with the TLR4/MyD88/nuclear factor-κB (NF-κB) pathway was assessed in vitro and in vivo. We show that miR-326 was downregulated in CCl₄-induced fibrotic mice and activated HSCs. The target gene of miR-326 is TLR4. Moreover, miR-326 inhibited the activation of HSCs in vitro through TLR4/MyD88/NF-κB signaling. miR-326 attenuated hepatic fibrosis and inflammation of CCl₄-induced mice in vivo. Our results demonstrate for the first time that miR-326 inhibits HSC activation through TLR4/MyD88/NF-κB signaling. Furthermore, miR-326 plays critical roles in attenuating liver fibrosis and inflammation, suggesting the therapeutic potential of miRNAs.     

Phosphatidylinositol 3-kinase inhibitor suppresses inducible nitric oxide synthase expression in bronchiole epithelial cells in asthmatic rats

Inducible nitric oxide synthase (iNOS) is known to produce nitric oxide (NO), which is a main contributor to asthmatic airway inflammation. Recent studies have shown that phosphatidylinositol 3-kinase (PI3K) is ubiquitously expressed in airway epithelial cells and its inhibition could relieve airway inflammation and hyperresponsiveness. This study aimed to explore the interaction of PI3K and NO signaling in allergic asthma. We investigated the effects of PI3K inhibitor wortmannin on iNOS expression in bronchiole epithelial cells and NO, IL-4 and IFN-γ levels in lung tissues of asthmatic rat model, which was prepared by 10% OVA solution sensitization and 1% OVA aerosol challenge. Our results showed that the ratio of eosinophils to total cells in BALF, PI3K activity, NO and IL-4 levels in lung tissues was increased after OVA sensitization and challenge, but then was attenuated by the administration of wortmannin. In contrast, IFN-γ level in lung tissues was decreased after OVA sensitization and challenge and increased after the administration of wortmannin. The expression of iNOS protein in bronchiole epithelial cells, iNOS mRNA level and iNOS activity in lung tissues was markedly upregulated after OVA sensitization and challenge, but the upregulation was significantly antagonized by wortmannin. Taken together, these data provide evidence that PI3K functions upstream to modulate iNOS/NO signaling, which then promotes the development of airway inflammation in asthmatic animal model. PI3K inhibitor wortmannin could lead to reduced iNOS expression and NO production, therefore inhibiting airway inflammatory responses.     

Nitrogen dioxide promotes allergic sensitization to inhaled antigen

Allergen sensitization and allergic airway disease are likely to come about through the inhalation of Ag with immunostimulatory molecules. However, environmental pollutants, including nitrogen dioxide (NO2), may promote adaptive immune responses to innocuous Ags that are not by themselves immunostimulatory. We tested in C57BL/6 mice whether exposure to NO2, followed by inhalation of the innocuous protein Ag, OVA, would result in allergen sensitization and the subsequent development of allergic airway disease. Following challenge with aerosolized OVA alone, mice previously exposed via inhalation to NO2 and OVA developed eosinophilic inflammation and mucus cell metaplasia in the lungs, as well as OVA-specific IgE and IgG1, and Th2-type cytokine responses. One hour of exposure to 10 parts per million NO2 increased bronchoalveolar lavage fluid levels of total protein, lactate dehydrogenase activity, and heat shock protein 70; promoted the activation of NF-kappaB by airway epithelial cells; and stimulated the subsequent allergic response to Ag challenge. Furthermore, features of allergic airway disease were not induced in allergen-challenged TLR2-/- and MyD88-/- mice exposed to NO2 and aerosolized OVA during sensitization. These findings offer a mechanism whereby allergen sensitization and asthma may result under conditions of high ambient or endogenous NO2 levels.     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

The microRNA-1278/SHP-1/STAT3 pathway is involved in airway smooth muscle cell proliferation in a model of severe asthma both intracellularly and extracellularly

This study investigated the regulatory effects of microRNA-1278 (miR-1278) on airway inflammation, airway reconstruction, and the proliferation and apoptosis of airway smooth muscle cells (ASMCs) induced by transforming growth factor β1 (TGF-β1). The results showed that miR-1278 was upregulated in the blood and lung tissues (LTs) of patients with asthma compared with that in healthy volunteers; miR-1278 expression was also upregulated in asthmatic mice, and miR-1278 inhibition improved the LTs of asthmatic mice. Moreover, miR-1278 inhibited inflammation in asthmatic mice and counteracted the effect of TGF-β1 of induced proliferation and reduced apoptosis in ASMCs. DLRA indicated that miR-1278 targeted the 3′-UTR of Src-homology 2-containing phosphatase 1 (SHP-1). Furthermore, miR-1278 promoted ASMC proliferation, in which TGF-β1 played an important role by regulating the SHP-1/STAT3 signaling pathway. In conclusion, this study showed that miR-1278 played a critical role in the processes of airway remodeling and reduction of apoptosis by targeting SHP-1.

     

TLR4在气道上皮细胞诱导的哮喘气道平滑肌细胞迁移中的作用

目的:探讨Toll样受体4(TLR4)的激活在气道上皮细胞诱导的哮喘气道平滑肌细胞(ASMCs)迁移中的作用。方法:细胞消化法培养原代哮喘ASMCs,TNF-α刺激上皮细胞系RTE细胞收集细胞培养上清液,检测上清液中IL-8和RANTES的含量,改良Boyden趋化小室检测哮喘ASMCs的跨膜迁移,以TLR4抗体作为工具药,观察其在上皮细胞诱导的哮喘ASMCs跨膜迁移中的作用。结果:各TNF-α组培养上清液中IL-8和RANTES水平均显著增高,20 ng/ml组较其他组显著增高(P<0.01)。各组哮喘ASMCs跨膜迁移较正常组均增加(P<0.01);哮喘组和TNF-α+TLR4抗体组哮喘ASMCs跨膜迁移数较TNF-α组显著减少(P<0.01)。TLR4抗体组哮喘ASMCs跨膜迁移数较哮喘组增加(P<0.05)。结论:气道上皮细胞可能通过分泌细胞因子激活哮喘ASMCs表面的TLR4,诱导增强ASMCs的跨膜迁移,在哮喘的气道重构中发挥一定的作用。     

CD4+CD25+FOXP3+ T cells,Foxp3 gene and protein expression contribute to antiasthmatic effects of San’ao decoction in mice model of asthma

ObjectiveSan’ao decoction (SAD) is a commonly used traditional combinatorial formula composed of Herba Ephedrae, Radix Glycyrrhizae and Amygdalus Communis Vas. Early studies showed that in the OVA sensitization asthmatic mice model its compatibility could lower airway reactivity and airway inflammatory cell infiltration. Based on the above results, this study mainly discussed San’ao decoction's immunomodulatory effects on Tregs.MethodsUPLC–PDA–TOF-MS was applied to analyze chemicals of SAD, and under the optimized chromatographic and MS condition, the major components in SAD were well separated and detected within 22 min. An asthma model was established in BALB/c mice that were sensitized and challenged with ovalbumin. After 2 weeks’ treatment, peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed for inflammatory cell counts; histological change of lung tissue were detected; flow cytometry detection of splenic CD4⁺CD25⁺Foxp3⁺ Treg cells of the mice were counted; Foxp3 expression in lung tissues were examined as well.Results22 Peaks signal chemical components in SAD were identified by UPLC–QTO-MS method. In terms of the percentage of eosinophile in peripheral blood and bronchoalveolar lavage fluid (BALF), SAD groups were significantly lower (p < 0.01) than model group. Compared with model group, lung histological changes of SAD groups were reduced; the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺ cells of asthmatic mice also decreased; SAD significantly increased the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells and promoted Foxp3 expression in a mouse model of asthma.ConclusionThese results suggest that the antiasthmatic effects of SAD are at least partially associated with CD4⁺CD25⁺Foxp3⁺ Treg cells.     

Endotoxin Exposure during Sensitization to Blomia tropicalis Allergens Shifts TH2 Immunity Towards a TH17-Mediated Airway Neutrophilic Inflammation: Role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.     

TLR7 ligand prevents allergen-induced airway hyperresponsiveness and eosinophilia in allergic asthma by a MYD88-dependent and MK2-independent pathway

Asthma is one of the leading causes of childhood hospitalization, and its incidence is on the rise throughout the world. Currently, the standard treatment for asthma is the use of corticosteroids to try to suppress the inflammatory reaction taking place in the bronchial tree. Using a murine model of atopic allergic asthma employing a methacholine-hyperresponsive (A/J) as well as a hyporesponsive (C57BL/6) strain of mice sensitized and challenged with ovalbumin, we show that treatment with a synthetic Toll-like receptor 7 (TLR7) ligand (S-28463, a member of the imidazoquinoline family) prevents development of the asthmatic phenotype. Treatment with S-28463 resulted in a reduction of airway resistance and elastance following ovalbumin sensitization and challenge. This was accompanied by a dramatic reduction in infiltration of leukocytes, especially eosinophils, into the lungs of both C57BL/6 and A/J mice following OVA challenge. Treatment with S-28463 also abolished both the elevation in serum IgE level as well as the induction of IL-4, IL-5, and IL-13 by OVA challenge. The protective effects of S-28463 were also observed in MK2 knockout, but not MYD88 knockout, mice. We did not observe a switch in cytokine profile from T(H)2 to T(H)1, as both IL-12p70 and IFN-gamma levels were reduced following S-28463 treatment. These results clearly demonstrate the anti-inflammatory effect of imidazoquinolines in an allergic asthma model as well as the clinical potential of TLR7 ligands in the treatment of allergic diseases.     

Ligation of TLR2 and TLR4 on murine bone marrow-derived mesenchymal stem cells triggers differential effects on their immunosuppressive activity

Mesenchymal stem cells (MSCs) have potent regulatory effects on immune and inflammatory responses. Recently the findings of functional TLR expression on MSC implicates these receptors in the function established for MSCs. Here we specially investigated the effects of TLR2, 4 ligation in mice MSC on migration, modulation of allogeneic mixed lymphocytes reaction (allo-MLR) and inducing Treg cells. We demonstrated that ligation of TLR2, but not TLR4, could significantly inhibit migration of MSC, impair MSC-mediated immunosuppression on allo-MLR, and reduce MSC-mediated expansion of CD4+CD25+Foxp3+ regulatory T cells. Compared with TLR4 activated MSCs and non-TLR activated MSC, TLR2 activation induced a relatively lower level of CXCL-10 mRNA and protein expressions which has been elucidated to act in concert with other soluble factor in MSC-mediated immunomodulation. These data indicate that TLR2 and TLR4 ligation had different effects on immunomodulatory capability of murine BMSCs, which should be considered in their use for treating inflammatory diseases.     

当归挥发油对哮喘BALB/c小鼠的平喘作用及对Th17免疫活性的影响

目的：探讨当归挥发油对哮喘BALB/c小鼠的平喘作用及对IL-17A、RORγt等Th17细胞活性因子的影响。方法：取雌性小鼠随机分为7组（n=12）：正常对照组、模型对照组、地塞米松组、当归挥发油高中低剂量组及当归油中+地米组,通过注射卵清白蛋白（OVA）致敏、吸入OVA激发的方法来复制哮喘动物模型,在当归挥发油的防治下,考察当归挥发油对哮喘行为学、肺功能、肺组织病理学、血清IL-17A及肺组织RORγt表达的影响。结果：60、120、240 mg/kg当归挥发油可维持哮喘小鼠体重的正常增长,改善哮喘行为学、肺功能及肺组织病理学变化,抑制IL-17A与RORγt的过度表达（P<0.05, 0.01）。同时,当归挥发油与地塞米松配伍后对维持体重的正常增长、缓解IL-17A与RORγt的过度表达具有一定的协同作用。结论：当归挥发油具有明显的防治哮喘作用,抑制IL-17、RORγt过度表达而抑制Th17细胞免疫活性是其作用机制之一;而当归挥发油与糖皮质激素配伍后有一定的协同作用。     

Periostin regulates goblet cell metaplasia in a model of allergic airway inflammation

Periostin is a 90-kDa member of the fasciclin-containing family and functions as part of the extracellular matrix. Periostin is expressed in a variety of tissues and expression is increased in airway epithelial cells from asthmatic patients. Recent studies have implicated a role for periostin in allergic eosinophilic esophagitis. To further define a role for periostin in Th2-mediated inflammatory diseases such as asthma, we studied the development of allergic pulmonary inflammation in periostin-deficient mice. Sensitization and challenge of periostin-deficient mice with OVA resulted in increased peripheral Th2 responses compared with control mice. In the lungs, periostin deficiency resulted in increased airway resistance and significantly enhanced mucus production by goblet cells concomitant with increased expression of Gob5 and Muc5ac compared with wild type littermates. Periostin also inhibited the expression of Gob5, a putative calcium-activated chloride channel involved in the regulation of mucus production, in primary murine airway epithelial cells. Our studies suggest that periostin may be part of a negative-feedback loop regulating allergic inflammation that could be therapeutic in the treatment of atopic disease.     

Heme oxygenase-1-mediated CD4+CD25high regulatory T cells suppress allergic airway inflammation

Heme oxygenase-1 (HO-1) has anti-inflammatory effects in asthma. CD4+CD25(high) regulatory T cells (Treg) are a potent immunoregulator that suppresses the immune response. We studied the effects of HO-1-mediated CD4+CD25(high) Treg on suppression of allergic airway inflammation by comparing mice treated with hemin, OVA, Sn-protoporphyrin (SnPP), and hemin plus SnPP. Airway responsiveness, airway eosinophil infiltration, the level of OVA-specific IgE, and the numbers of cells in general and eosinophils in particular in bronchial alveolar lavage fluid were lower in the hemin group than in the OVA, SnPP, and hemin plus SnPP groups. The expressions of HO-1 mRNA and protein in the lung were increased by repeated administrations of hemin and SnPP. However, the activity of HO-1 was highest in hemin mice. The percentage and suppressive function of CD4+CD25(high) Treg and the expression of Foxp3 mRNA were obviously enhanced after treatment with hemin. This increase was diminished by the administration of SnPP. The concentration of serum IL-10 was higher in the hemin group than in the other groups, whereas the level of serum TGF-beta did not significantly differ across groups. Furthermore, the ratio of IFN-gamma/IL-4 mRNA in the lung was higher in hemin-treated mice than in OVA and SnPP mice. The suppressive capacity of CD4+CD25(high) Treg was not enhanced in the IL-10-deficient mice treated with hemin. In conclusion, our experiments in the animal model demonstrated that HO-1 has anti-inflammatory effects, probably via enhancement of the secretion of IL-10 and promotion of the percentage of CD4+CD25(high) Treg.     

咪喹莫特对慢性哮喘模型小鼠肺组织中基质金属蛋白酶-9-表达的影响

目的：观察Toll样受体7配体咪喹莫特对慢性哮喘小鼠模型气道重塑及肺组织中基质金属蛋白酶MMP-9表达的影响。方法：36只BALB／c小鼠按随机原则分成正常对照组、哮喘模型组、咪喹莫特组,每组12只。通过卵蛋白致敏,气道激发8周,末次激发24h后,检测各组小鼠气道反应性,HE染色观察气道炎症变化;Masson三色染色观察气道纤维化的改变;real-timePCR和western—blot分别检测肺组织中MMP-9的mRNA和蛋白表达。结果：慢性哮喘组小鼠气道炎症、气道高反应性和气道重塑较正常对照小鼠明显加重,而咪喹莫特组小鼠模型的气道炎症和气道反应性及气道重塑均较哮喘模型组小鼠减少或降低。慢性哮喘组小鼠肺组织MMP-9的mRNA和蛋白水平均较正常对照小鼠明显增加（P〈0．05）,而咪喹莫特治疗可显著降低哮喘小鼠肺组织MMP-9的mRNA和蛋白水平（P〈0．05）。结论：咪喹莫特能够显著抑制慢性哮喘小鼠模型的气道炎症、降低气道高反应性并减轻气道重塑,这可能与其抑制MMP-9的表达有关。     

ICOS Mediates the Generation and Function of CD4+CD25+Foxp3+ Regulatory T Cells Conveying Respiratory Tolerance

Costimulatory molecules like ICOS are crucial in mediating T cell differentiation and function after allergen contact and thereby strongly affect the immunologic decision between tolerance or allergy development. In this study, we show in two independent approaches that interruption of the ICOS signaling pathway by application of a blocking anti-ICOSL mAb in wild-type (WT) mice and in ICOS(-/-) mice inhibited respiratory tolerance development leading to eosinophilic airway inflammation, mucus hypersecretion, and Th2 cytokine production in response to OVA sensitization. Respiratory Ag application almost doubled the number of CD4(+)Foxp3(+) regulatory T cells (Tregs) in the lung of WT mice with 77% of lung-derived Tregs expressing ICOS. In contrast, in ICOS(-/-) mice the number of CD4(+)CD25(+)Foxp3(+) Tregs did not increase after respiratory Ag application, and ICOS(-/-) Tregs produced significantly lower amounts of IL-10 than those of WT Tregs. Most importantly, in contrast to WT Tregs, ICOS(-/-) Tregs did not convey protection when transferred to "asthmatic" recipients demonstrating a strongly impaired Treg function in the absence of ICOS signaling. Our findings demonstrate a crucial role of ICOS for the generation and suppressive function of Tregs conveying respiratory tolerance and support the importance of ICOS as a target for primary prevention strategies.