Expression of Toll-like receptors and their association with cytokine responses in peripheral blood mononuclear cells of children with acute rotavirus diarrhoea

To understand virus and host interactions and host responses to rotavirus infection in children, we analysed by real-time polymerase chain reaction (PCR) the expression of mRNA for five Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR7 and TLR8) and four T helper (Th)1 and Th2 cytokines [interleukin (IL)-2, IL-12, interferon (IFN)-gamma and IL-4) in peripheral blood mononuclear cells (PBMC) of children with acute rotavirus diarrhoea. We observed significantly higher expression of genes encoding TLR2, TLR3, TLR4, TLR7 and TLR8 in PBMC of 41% (31/75) patients within 3 days of illness onset than those in healthy children. After 3 days of illness onset, only TLR3 and TLR8 mRNA expressions were still significantly (P<0.05) increased in 59% (44/75) children with diarrhoea. We also observed significantly (P<0.05) elevated expression of IL-12p40 and IFN-gamma in PBMC of patients during the entire period of illness and the first 3 days of illness, respectively. We further demonstrated a weak but significant association between elevated levels of gene expression of four TLRs (TLR2, TLR3, TLR4 and TLR8) and IFN-gamma. Our results suggest that multiple TLRs may modulate the immune response in the acute phase of rotavirus infection and play a role in the activation of IFN-gamma.     

Toll‐like receptors and CD40 modulate each other's expression affecting Leishmania major infection

Toll‐like receptors (TLRs) recognize pathogen‐associated molecular patterns and results in innate immune system activation that results in elicitation of the adaptive immune response. One crucial modulator of the adaptive immune response is CD40. However, whether these molecules influence each other's expression and functions is not known. Therefore, we examined the effects of TLRs on CD40 expression on macrophages, the host cell for the protozoan parasite L eishmania major. While polyinosinic‐polycytidylic acid [poly (I:C)], a TLR‐3 ligand, lipopolysaccharide (LPS), a TLR‐4 ligand, imiquimod, a TLR‐7/8 ligand and cytosine–phosphate–guanosine (CpG), a TLR‐9 ligand, were shown to enhance CD40 expression, CD40 stimulation enhanced only TLR‐9 expression. Therefore, we tested the synergism between CD40 and CpG in anti‐leishmanial immune response. In L eishmania‐infected macrophages, CpG was found to reduce CD40‐induced extracellular stress‐regulated kinase (ERK)1/2 activation; with the exception of interleukin (IL)‐10, these ligands had differential effects on CD40‐induced IL‐1α, IL‐6 and IL‐12 production. CpG significantly enhanced the anti‐leishmanial function of CD40 with differential effects on IL‐4, IL‐10 and interferon (IFN)‐γ production in susceptible BALB/c mice. Thus, we report the first systematic study on CD40–TLR cross‐talk that regulated the experimental L . major infection.     

Toll-like receptor 2, 3, and 4 expression and function in human airway smooth muscle

BACKGROUND: Host defense against microbial pathogens is elicited through the innate immune system by means of Toll-like receptors (TLRs). Airway smooth muscle cells (ASMCs) display proinflammatory and immunomodulatory functions. ASMCs might participate in airway inflammatory responses associated with innate immune activation. OBJECTIVES: We determined the effects of cytokines, TLR ligands, and corticosteroids on TLR expression and function in human ASMCs. METHODS: Real-time PCR and flow cytometry were used to assess TLR mRNA and protein expression, respectively. ASMCs were stimulated with TLR ligands, and chemokine release was measured by means of ELISA. RESULTS: ASMCs expressed TLR1 to TLR10 mRNA, and TLR2 and TLR3 protein expression was demonstrated. TNF-alpha and double-stranded RNA (dsRNA; TLR3 ligand) were potent inducers of TLR2 and TLR3 mRNA expression, and both stimuli had additive or synergistic effects with IFN-gamma on TLR2 and TLR4, but not TLR3, mRNA expression. Peptidoglycan (TLR2 ligand) and LPS (TLR4 ligand) weakly enhanced TLR2 mRNA expression. Peptidoglycan, dsRNA, and LPS induced IL-8 and eotaxin release, with dsRNA being most potent. dsRNA also modulated cytokine-induced chemokine release in a differential manner. Dexamethasone inhibited cytokine- and ligand-induced TLR2, TLR3, and TLR4 expression and chemokine release. However, dexamethasone potentiated TLR2 expression induced by combined IFN-gamma and TNF-alpha stimulation. CONCLUSION: Expression of TLR2, TLR3, and TLR4 is regulated by cytokines and TLR ligands, and their activation mediates chemokine release in ASMCs. CLINICAL IMPLICATIONS: Proinflammatory responses mediated by activation of pathogen-recognition receptors in ASMCs might contribute to infectious exacerbations of airway inflammatory conditions, such as asthma and chronic obstructive pulmonary disease.     

Toll样受体7作为新靶点在过敏性及自身免疫性疾病中的研究进展

Toll样受体(Toll-like receptors,TLRs)属于模式识别受体(pattern recognition receptors,PRRs)家族,通过迅速识别入侵微生物病原相关分子模式(pathogen-associated molecular patterns,PAMP)激活下游信号传导途径,引发机体的免疫反应,是连接固有免疫与适应性免疫的桥梁.目前在人类发现10种不同TLRs,分别命名为TLR1 ～TLR10.近年的研究显示,TLR7与过敏性疾病、自身免疫性疾病—过敏性哮喘、系统性红斑狼疮(systemic lupus erythematosus,SLE)等有密切的联系.TLR7的激活状态影响上述疾病的发生、发展和预后.这些证据预示TLR7可能作为潜在靶点为哮喘和SLE的治疗提供新的方向.     

The role of macrophage IL‐10/innate IFN interplay during virus‐induced asthma

Activation through different signaling pathways results in two functionally different types of macrophages, the pro‐inflammatory (M1) and the anti‐inflammatory (M2). The polarization of macrophages toward the pro‐inflammatory M1 phenotype is considered to be critical for efficient antiviral immune responses in the lung. Among the various cell types that are present in the asthmatic airways, macrophages have emerged as significant participants in disease pathogenesis, because of their activation during both the inflammatory and resolution phases, with an impact on disease progression. Polarized M1 and M2 macrophages are able to reversibly undergo functional redifferentiation into anti‐inflammatory or pro‐inflammatory macrophages, respectively, and therefore, macrophages mediate both processes. Recent studies have indicated a predominance of M2 macrophages in asthmatic airways. During a virus infection, it is likely that M2 macrophages would secrete higher amounts of the suppressor cytokine IL‐10, and less innate IFNs. However, the interactions between IL‐10 and innate IFNs during virus‐induced exacerbations of asthma have not been well studied. The possible role of IL‐10 as a therapy in allergic asthma has already been suggested, but the divergent roles of this suppressor molecule in the antiviral immune response raise concerns. This review attempts to shed light on macrophage IL‐10–IFNs interactions and discusses the role of IL‐10 in virus‐induced asthma exacerbations. Whereas IL‐10 is important in terminating pro‐inflammatory and antiviral immune responses, the presence of this immune regulatory cytokine at the beginning of virus infection could impair the response to viruses and play a role in virus‐induced asthma exacerbations. © 2014 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd.     

Antiviral responses induced by the TLR3 pathway

Antiviral responses are successively induced in virus‐infected animals, and include primary innate immune responses such as type I interferon (IFN) and cytokine production, secondary natural killer (NK) cell responses, and final cytotoxic T lymphocyte (CTL) responses and antibody production. The endosomal Toll‐like receptors (TLRs) and cytoplasmic RIG‐I‐like receptors (RLRs), which recognize viral nucleic acids, are responsible for virus‐induced type I IFN production. RLRs are expressed in most tissues and cells and are primarily implicated in innate immune responses against various viruses through type I IFN production, whereas nucleic acid‐sensing TLRs, TLRs 3, 7, 8 and 9, are expressed on the endosomal membrane of dendritic cells (DCs) and play distinct roles in antiviral immunity. TLR3 recognizes viral double‐stranded RNA taken up into the endosome and serves to protect the host against viral infection by the induction of a range of responses including type I IFN production and DC‐mediated activation of NK cells and CTLs, although the deteriorative role of TLR3 has also been reported in some virus infections. Here, we review the current knowledge on the role of TLR3 during viral infection, and the current understanding of the TLR3‐signalling cascade that operates via the adaptor protein TICAM‐1 (also called TRIF). Copyright © 2011 John Wiley & Sons, Ltd.     

Modulation of expression and function of Toll-like receptor 3 in A549 and H292 cells by histamine

It was reported recently that histamine induced Toll-like receptor (TLR)2 and TLR4 expression in endothelial cells and enhanced their sensitivity to Gram-positive and Gram-negative bacteria; and that TLRs were expressed in airway epithelial cells and that several inflammatory mediators modulated their expression. However, little is known of potential influence of histamine on TLRs in pulmonary epithelial cells. In the present study, effects of histamine on expression of TLRs in both human A549 and NCI-H292 cell lines were examined by using real-time quantitative RT-PCR analysis, flow cytometry and immunofluorescent staining. The results revealed that both cell types constitutively expressed mRNAs for TLR1-TLR10. Histamine up-regulated the expression of TLR3 mRNA by 12.3- and 11.6-fold, respectively in both cell types. The time course showed that histamine induced TLR3 mRNA expression was initiated at 30 min, nearly reached peak levels after 2 h and was sustained at least until 12 h. Histamine also induced TLR3 protein expression in A549 and NCI-H292 cells. Histamine and poly (I:C), a specific TLR3 ligand stimulated interleukin (IL)-8 secretion from both cell types. Moreover, histamine enhanced poly (I:C)-induced IL-8 secretion and phosphorylation of NF-kappaB in the two cell types, and histamine H1 receptor antagonists inhibited the action of histamine. In conclusion, histamine selectively up-regulated expression of TLR3, and stimulated IL-8 secretion from the cells. Histamine also enhanced poly (I:C) induced IL-8 secretion and phosphorylation of NF-kappaB. These observations suggest that histamine might play an important role in enhancing the innate immune responses of airway to viral infection.     

Polymorphisms of toll like receptors in the genetics of severe RSV associated diseases

Toll like receptors (TLRs) are an essential part of the innate immune response. So far, ten different TLRs were identified in humans. They recognize a wide range of microbial and viral pathogens. Infection by respiratory syncytial virus (RSV) is still a major health problem, about 2% of all children are hospitalised due to RSV bronchiolitis during their first 2 years of live. TLR4 has already been described in association with RSV associated diseases by us and others. Thus we were interested whether other TLRs are also involved in the genetics of severe RSV infection. We genotyped 19 polymorphisms in the autosomal TLRs, these are TLR1, 2, 3, 5, 6, 9 and 10. Association analyses by the Armitage's Trend test revealed weak association of one TLR9 promoter polymorphism with RSV infection (p = 0.013). In addition, association was found with TLR10 haplotypes (p = 0.024). We conclude from our data--that--although we can not rule out a minor involvement of TLR9 polymorphism and TLR10 haplotypes--TLRs other than TLR4 do not play a major role in the genetics of severe RSV associated diseases. Future studies should focus on additional genes of the innate immune response.     

Induction of adaptive immunity by flagellin does not require robust activation of innate immunity

The ability of TLR agonists to promote adaptive immune responses is attributed to their ability to robustly activate innate immunity. However, it has been observed that, for adjuvants in actual use in research and vaccination, TLR signaling is dispensable for generating humoral immunity. Here, we examined the role of TLR5 and MyD88 in promoting innate and humoral immunity to flagellin using a prime/boost immunization regimen. We observed that eliminating TLR5 greatly reduced flagellin‐induced cytokine production, except for IL‐18, and ablated DC maturation but did not significantly impact flagellin's ability to promote humoral immunity. Elimination of MyD88, which will ablate signaling through TLR and IL‐1β/IL‐18 generated by Nod‐like receptors, reduced, but did not eliminate flagellin's promotion of humoral immunity. In contrast, loss of the innate immune receptor for profilin‐like protein (PLP), TLR11, greatly reduced the ability of PLP to elicit humoral immunity. Together, these results indicate that, firstly, the degree of innate immune activation induced by TLR agonists may be in great excess of that needed to promote humoral immunity and, secondly, there is considerable redundancy in mechanisms that promote the humoral immune response upon innate immune recognition of flagellin. Thus, it should be possible to design innate immune activators that are highly effective vaccine adjuvants yet avoid the adverse events associated with systemic TLR activation.     

Toll‐like receptors as novel therapeutic targets for herpes simplex virus infection

Seropositivity for HSV reaches more than 70% within the world population, and yet no approved vaccine exists. While HSV1 is responsible for keratitis, encephalitis, and labialis, HSV2 carriers have a high susceptibility to other STD infections, such as HIV. Induction of antiviral innate immune responses upon infection depends on a family of pattern recognition receptors called Toll‐like receptors (TLR). TLRs bridge innate and adaptive immunity by sensing virus infection and activating antiviral immune responses. HSV adopts smart tricks to evade innate immunity and can also manipulate TLR signaling to evade the immune system or even confer destructive effects in favor of virus replication. Here, we review mechanisms by which HSV can trick TLR signaling to impair innate immunity. Then, we analyze the role of HSV‐mediated molecular cues, in particular, NF‐κB signaling, in promoting protective versus destructive effects of TLRs. Finally, TLR‐based therapeutic opportunities with the goal of preventing or treating HSV infection will be discussed.     

Toll样受体在机体抗病毒免疫反应中的作用

哺乳动物的Toll样受体(TLR)家族具有模式识别受体的功能,其可以识别微生物的保守分子成分,启动机体的固有免疫系统,从而帮助机体清除病原体.利用TLR敲除的动物或细胞模型进行的研究使人们认识到TLR在机体抗病毒免疫反应中发挥着重要作用.病毒与宿主细胞的TLR结合后,通过NF-κB或IRF-3的信号路径激活细胞因子的表达,从而激发免疫应答.研究TLR如何与病原体结合及如何激活下游基因对深入认识病原体所致相关疾病的发病机制、免疫应答及病理生理具有重要的意义,并为病毒性疾病的临床治疗或免疫预防提供新的思路.     

Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells

The objective of this study was to examine the expression of Toll-like receptor 3 (TLR3) by human corneal epithelial cells (HCECs) and to determine whether exposure to the TLR3 agonist polyinosinic-polycytidylic acid [poly(I:C)] induces an antiviral response in these cells. Fluorescence-activated cell sorter (FACS) analysis revealed TLR3 to be constitutively expressed and distributed intracellularly in HCECs. Stimulation of HCECs with the TLR3 agonist poly(I:C) induced the activation of nuclear factor (NF)-kappaB and production of the proinflammatory cytokine interleukin (IL)-6 and the chemokine IL-8. Upon exposure to poly(I:C), HCECs initiated a potent antiviral response resulting in an increase of interferon (IFN)-beta mRNA expression (7-fold). Poly(I:C) stimulation also up-regulated mRNA expression of the antiviral chemokine IFN-gamma inducible protein 10 (IP10), myxovirus resistance gene A and 2',5'-oligoadenylate synthetase (5-, 10- and 9-fold, respectively), and secretion of IP10. These responses were also induced by exogenously added type 1 IFNs, but could not be blocked by pretreatment of the cells with anti-TLR3 monoclonal antibody, suggesting that the receptor was not expressed on the cell surface. Furthermore, incubation of HCECs with an endosomal acidification inhibitor, chloroquine, markedly inhibited poly(I:C)-mediated IFN-beta expression in HCECs. These results suggest that corneal epithelial cells are important sentinels of the corneal innate immune system against viral infection, and that stimulation of TLR3 can induce the expression of key proinflammatory cytokines and chemokines and antiviral genes that help in the defence of the cornea against viral infection.     

Human platelets express Toll-like receptor 3 and respond to poly I:C

Platelets functions in hemostasis have been widely studied. Currently, growing evidence shows that platelets have also a role in the immune innate response. Recently, protein expression of Toll-like receptors (TLR’s) 2, 4, 7, 8, and 9, and the presence of TLRs 1 and 6 mRNA in human platelets was described. Up to now the functionality of TLR-2, 4 and 9 in human platelets has been demonstrated. Due to the relevance of TLRs functions to PAMPS (pathogen-associated molecular patterns) recognizing, we evaluated the presence of TLR3 in human platelets founding low percentages of platelets expressing surface or intracellular TLR3 protein. The activation with thrombin induced an increase in the percentage of platelets expressing surface TLR3 and higher levels of TLR3 expression in the whole population. Human platelets responded to poly I:C by increasing [Ca²⁺]_i, the percentages of cells expressing TLR4 and CD62P, and by releasing CXCL4 and IL-1β in comparison to unstimulated platelets. These results demonstrate that human platelets express TLR3 and are capable of responding to poly I:C, suggesting that these cells might influence the immune innate response when detecting viral dsRNA.     

Differential signaling of cmvIL‐10 through common variants of the IL‐10 receptor 1

Human IL‐10 (hIL‐10) signaling is mediated by receptors consisting of two subunits, IL‐10 receptor 1 (IL‐10R1) and IL‐10 receptor 2. Two common variants of the IL‐10R1 (Ser 138 Gly (single‐nucleotide polymorphism 3, SNP3) and Gly 330 Arg (SNP4)) are associated with diverse disease phenotypes. Viral homologs to hIL‐10, such as cmvIL‐10, utilize the same IL‐10 receptor complex as part of viral immune evasion strategies. For the present study we hypothesized that IL‐10R1 variants alter the ability of viral IL‐10 to utilize the IL‐10R1 signaling pathway. HeLa cell clones expressing different IL‐10R1 haplotypes (WT or any variant) were incubated with hIL‐10 or cmvIL‐10. In cells expressing IL‐10R1‐WT, cmvIL‐10 (both non‐glycosylated‐ and HeLa‐expressed) resulted in equal or slightly stronger STAT3 phosphorylation compared with hIL‐10. In clones expressing IL‐10R1‐SNP3, IL‐10R1‐SNP4 or IL‐10R1‐SNP3+4, the cmvIL‐10 showed significantly less STAT3 phosphorylation, especially when HeLa‐expressed cytokines were used. Time course experiments demonstrated a slower kinetic of cmvIL‐10 STAT3 activation through the variant IL‐10R1. Similarly, IL‐10R1 variants decreased the cmvIL‐10‐induced SOCS3 and signaling lymphocytic activation molecule mRNA expression. These data suggest that the IL‐10R1 variants differentially reduce the signaling activity of cmvIL‐10 and thereby may affect CMV's ability to escape from the host's immune surveillance.     

TLR7 signaling exacerbates CNS autoimmunity through downregulation of Foxp3+ Treg cells

The innate Toll‐like receptor 7 (TLR7) detects infections by recognizing viral and bacterial single‐stranded RNA. In addition to pathogen‐derived RNA, immune cells expressing high levels of TLR7, such as B cells and dendritic cells (DCs), can be activated by self‐RNA. During myelin‐induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, TLR7 expression is increased within the central nervous system (CNS). To define the contribution of TLR7 to the development of EAE, we evaluated the course of the disease in C57BL/6‐Tlr7‐deficient mice compared with that in WT mice and found that TLR7‐deficient mice had decreased disease severity. This protection was associated with decreased myelin oligodendrocyte glycoprotein‐specific T‐cell activation by primed DCs, decreased circulating autoantibodies, attenuated inflammation within the CNS, and increased Foxp3⁺ regulatory T cells in the periphery and in the CNS. In conclusion, we show that TLR7 is involved in the maintenance of autoimmunity in the pathogenesis of EAE.     

DC‐SIGN reacts with TLR‐4 and regulates inflammatory cytokine expression via NF‐κB activation in renal tubular epithelial cells during acute renal injury

In the pathological process of acute kidney injury (AKI), innate immune receptors are essential in inflammatory response modulation; however, the precise molecular mechanisms are still unclear. Our study sought to demonstrate the inflammatory response mechanisms in renal tubular epithelial cells via Toll‐like receptor‐4 (TLR‐4) and dendritic cell‐specific intercellular adhesion molecule 3‐grabbing non‐integrin 1 (DC‐SIGN) signalling. We found that DC‐SIGN exhibited strong expression in renal tubular epithelial cells of human acute renal injury tissues. DC‐SIGN protein expression was increased significantly when renal tubular epithelial cells were exposed to lipopolysaccharide (LPS) for a short period. Furthermore, DC‐SIGN was involved in the activation of p65 by TLR‐4, which excluded p38 and c‐Jun N‐terminal kinases (JNK). Interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α expression was decreased after DC‐SIGN knock‐down, and LPS induced endogenous interactions and plasma membrane co‐expression between TLR‐4 and DC‐SIGN. These results show that DC‐SIGN and TLR‐4 interactions regulate inflammatory responses in renal tubular epithelial cells and participate in AKI pathogenesis.     

PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN‐α/β) are major actors of the innate anti‐viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN‐inducing TLR3 agonist. PolyI:C plus IL‐2/IL‐12 induced IFN‐β (but not IFN‐α) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti‐IFNAR1 or anti‐IFN‐β Ab prevented the production of IFN‐γ induced by polyI:C plus IL‐2/IL‐12. Similarly, IFN‐γ production induced by polyI:C plus IL‐12 was reduced in NK cells isolated from IFNAR1^?/? compared with WT mice. The ability of polyI:C plus IL‐12 to induce IFN‐γ production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL‐2/IL‐12, produce IFN‐β that induce, in an autocrine manner, the production of IFN‐γ and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.