TLR5 and Ipaf: dual sensors of bacterial flagellin in the innate immune system

The innate immune system precisely modulates the intensity of immune activation in response to infection. Flagellin is a microbe-associated molecular pattern that is present on both pathogenic and nonpathogenic bacteria. Macrophages and dendritic cells are able to determine the virulence of flagellated bacteria by sensing whether flagellin remains outside the mammalian cell, or if it gains access to the cytosol. Extracellular flagellin is detected by TLR5, which induces expression of proinflammatory cytokines, while flagellin within the cytosol of macrophages is detected through the Nod-like receptor (NLR) Ipaf, which activates caspase-1. In macrophages infected with Salmonella typhimurium or Legionella pneumophila, Ipaf becomes activated in response to flagellin that appears to be delivered to the cytosol via specific virulence factor transport systems (the SPI1 type III secretion system (T3SS) and the Dot/Icm type IV secretion system (T4SS), respectively). Thus, TLR5 responds more generally to flagellated bacteria, while Ipaf responds to bacteria that express both flagellin and virulence factors.     

TLR5 ligation by flagellin converts tolerogenic dendritic cells into activating antigen-presenting cells that preferentially induce T-helper 1 responses

Dendritic cells (DCs) differentiated in the presence of IL-10 preferentially induce regulatory T-cells and tolerance. Whether the tolerogenic properties displayed by these DCs (Tol-DCs) can be overcome has not been fully explored. Here we show for the first time that Tol-DCs express higher levels of TLR5 mRNA, but not TLR4 or TLR9 mRNA relative to DCs differentiated with GM-CSF and IL-4 (BM-DCs). In response to flagellin, a natural TLR-5 ligand, Tol-DCs produced IL-12 but not IL-10. Unlike Tol-DCs stimulated with LPS, which produce high levels of IL-10 and fail to generate a cognate inflammatory response in CD4⁺ T-cells, flagellin-stimulated Tol-DCs promoted the differentiation of CD4⁺ T cells with a T-helper 1 phenotype. The divergent T-cell outcomes induced by Tol-DCs in response to different TLR-ligands highlights not only their plasticity, but also points to TLR5 ligation as a potential strategy to overcome tolerance in environments that are otherwise conducive to immune unresponsiveness.     

Effects of flagellin on innate and adaptive immunity

Flagella are locomotive organelles present on a wide range of bacteria and are important for the pathogenesis of many species. Cells of the innate immune system lack memory perse, but recognize conserved pathogen-associated molecular patterns (PAMPs) through a family of type I membrane receptors known as Toll-like receptors (TLRs). Flagellin, the major structural component of flagella, is a highly conserved protein recognized in hosts by TLR5. Signaling of flagellin via TLR5/TLR4 heteromeric complexes enhances the diversity of the response, likely by engaging MyD88-independent adaptors to activate the interferon pathway. Flagellin is a potent immune activator, stimulating diverse biologic effects that mediate both innate inflammatory responses as well as the development of adaptive immunity. Binding of flagellin to the extracellular domain of TLR5 rapidly induces a signal cascade that culminates in the production of proinflammatory mediators such as cytokines, chemokines, and costimulatory molecules. This review focuses on the mechanisms of action of flagellin and its effects on both innate and adaptive immunity.     

Replacement of two aminoacids in the bovine Toll-like receptor 5 TIR domain with their human counterparts partially restores functional response to flagellin

Flagellin potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Recently, we were able to show that stimulation of bovine TLR5 resulted in neither NFκB signalling nor CXCL8 production. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Analysis of available TLR5 sequences revealed substitutions in all artiodactyl sequences at amo acid (AA) position 798 and 799. Interestingly, a putative binding site for PI3K was identified at tyrosine 798 in the human TLR5 TIR domain, analogous to the PI3K recruitment domain in the IL-1 receptor. Mutation of the artiodactyl residues at position 798, 799 or both with their corresponding human counterparts partially restored the response of bovine (bo)TLR5 to flagellin as well as phosphorylation of PI3K. Together, our results suggest a potential lack of phosphorylation of F798 and H799 in boTLR5 partially explains the lack in observed response.     

Epithelium: at the interface of innate and adaptive immune responses

Several diseases of the airways have a strong component of allergic inflammation in their cause, including allergic rhinitis, asthma, polypoid chronic rhinosinusitis, eosinophilic bronchitis, and others. Although the roles played by antigens and pathogens vary, these diseases have in common a pathology that includes marked activation of epithelial cells in the upper airways, the lower airways, or both. Substantial new evidence indicates an important role of epithelial cells as both mediators and regulators of innate immune responses and adaptive immune responses, as well as the transition from innate immunity to adaptive immunity. The purpose of this review is to discuss recent studies that bear on the molecular and cellular mechanisms by which epithelial cells help to shape the responses of dendritic cells, T cells, and B cells and inflammatory cell recruitment in the context of human disease. Evidence will be discussed that suggests that secreted products of epithelial cells and molecules expressed on their cell surfaces can profoundly influence both immunity and inflammation in the airways.     

Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice

Flagellin is a potent activator of a broad range of cell types that are involved in innate and adaptive immunity. Therefore, it is a good adjuvant candidate for vaccines, and it might function as a biological protectant against both major acute radiation syndrome during cancer radiotherapy and a mitigator of radiation emergencies. However, accumulating evidence has implicated flagellin in the occurrence of some inflammatory diseases, such as acute lung inflammation, cardiovascular collapse and inflammatory bowel disease. The aim of this study was to elucidate whether only flagellin-TLR5 signaling activation plays a role in the pathophysiology of liver or whether some other flagellin activity also contributes to liver injury either via bacterial infections or during clinical applications. Recombinant flagellin proteins with or without TLR5-stimulating activity were used to evaluate the role of flagellin-TLR5 signaling in liver injury in wild-type and TLR5 KO mice. Gross lesions and large areas of hepatocellular necrosis were observed in liver tissue 12 h after the intraperitoneal administration of 100 or 200 µg flagellin (FliC) in a dose- and time-dependent manner in wild-type mice, but not in TLR5 KO mice. Deletion of the N-terminal or TLR5 binding domain of flagellin inhibited flagellin-induced inflammatory responses and the subsequent acute liver function abnormality and damage. These data confirmed that flagellin is an essential determinant of liver injury and demonstrated that the over-activation of TLR5 signaling by high-dose flagellin caused acute inflammatory responses, neutrophil accumulation and oxidative stress in the liver, which contributes to the progression and severity of flagellin-induced liver injury.     

Pathogen recognition and Toll-like receptor targeted therapeutics in innate immune cells

The innate immune system deploys a variety of pattern-recognition receptors (PRRs) which include Toll-like receptors (TLRs), RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors to detect the invasion of pathogens and initiate protective responses. The intercellular and intracellular orchestration of signals from different PRRs, their endogenous or microbial ligands and accessory molecules determine the stimulatory or inhibitory responses. Progressing over the last two decades, considerable research on the molecular mechanisms underlying host–pathogen interactions has led to a paradigm shift of our understanding of TLR signaling in the innate immune system. Given that a significant amount of evidence implicates TLRs in the pathogenesis of immune diseases and cancer, and their activation occurs early in the inflammatory cascade, they are attractive targets for novel therapeutic agents. In this review, we discuss the recent advances in TLR signaling cross talks and the mechanism of pathogen recognition with special emphasis on the role of TLRs in tumor immunity and TLR-targeted therapeutics.     

mTOR-driven glycolysis governs induction of innate immune responses by bronchial epithelial cells exposed to the bacterial component flagellin

Human bronchial epithelial (HBE) cells play an essential role during bacterial infections of the airways by sensing pathogens and orchestrating protective immune responses. We here sought to determine which metabolic pathways are utilized by HBE cells to mount innate immune responses upon exposure to a relevant bacterial agonist. Stimulation of HBE cells by the bacterial component flagellin triggered activation of the mTOR pathway resulting in an increased glycolytic flux that sustained the secretory activity of immune mediators by HBE cells. The mTOR inhibitor rapamycin impeded glycolysis and limited flagellin-induced secretion of immune mediators. The role of the mTOR pathway was recapitulated in vivo in a mouse model of flagellin-triggered lung innate immune responses. These data demonstrate that metabolic reprogramming via the mTOR pathway modulates activation of the respiratory epithelium, identifying mTOR as a potential therapeutic target to modulate mucosal immunity in the context of bacterial infections.     

TLR8 and TLR7 are involved in the host's immune response to human parechovirus 1

Toll-like receptors (TLR) have a key role in regulating immunity against microbial agents. Engagement of TLR by bacterial, viral or fungal components leads to the production and release of inflammatory cytokines. In this study we show that mainly TLR8 and also TLR7 act as the host sensors for human parechovirus 1, a single-stranded RNA (ssRNA) virus. Furthermore, we see that the viral ssRNA genome is detected in endosomal compartments by these TLR, which activate signalling that lead to the synthesis of pro-inflammatory molecules by the host.     

TLR9-/- and TLR9+/+ mice display similar immune responses to a DNA vaccine

Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9-/-) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-gamma (IFN-gamma)-secreting cells in TLR9-/- mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.     

Treponema pallidum flagellin FlaA2 induces IL-6 secretion in THP-1 cells via the Toll-like receptor 2 signaling pathway

Treponema pallidum subsp. pallidum membrane proteins are considered as potent inducers in the initiation and development of inflammation. In the present study, the mechanism that leads to the production of interleukin 6 (IL-6), one of the key proinflammatory cytokines, by human monocytic THP-1 cells when these cells are treated with T. pallidum flagellin FlaA2 was investigated. Stimulation with flagellin FlaA2 can induce IL-6 expression in human monocytes and augment the phosphorylation of ERK, p38, and NF-κB, but has no effect on the phosphorylation of JNK. Likewise, FlaA2-induced IL-6 production was found to be attenuated by inhibitors for ERK, p38, and NF-κB, but not by JNK inhibitor. Immunofluorescence analysis showed that flagellin FlaA2 could stimulate the translocation of IκBα from the cytosol to the nucleus, and this phenomenon could be inhibited by the specific inhibitor BAY11-7082. FlaA2–induced IL-6 expression was also proved to be abrogated by transfection with dominant negative (DN) plasmid of MyD88. We further demonstrated that transfection with DN-TLR2 was sufficient to attenuate IL-6 expression and the phosphorylation of ERK, p38, and IκBα. These results suggest that flagellin FlaA2 induces IL-6 production via signaling pathways involving TLR2, MyD88, ERK, p38, and NF-κB in monocytes, which could contribute to the pathogenesis of T. pallidum.     

Vitamin D modulation of innate immune responses to respiratory viral infections

Vitamin D, in addition to its classical functions in bone homeostasis, has a modulatory and regulatory role in multiple processes, including host defense, inflammation, immunity, and epithelial repair. Patients with respiratory disease are frequently deficient in vitamin D, implying that supplementation might provide significant benefit to these patients. Respiratory viral infections are common and are the main trigger of acute exacerbations and hospitalization in children and adults with asthma and other airways diseases. Respiratory monocytes/macrophages and epithelial cells constitutively express the vitamin D receptor. Vitamin D, acting through this receptor, may be important in protection against respiratory infections. Whether the in vitro findings can be translated into a substantial in vivo benefit still remains uncertain. Here we review the in vitro data on the role of vitamin D in antiviral innate immunity, the data concerning the deficient levels of vitamin D in lung diseases, and the in vivo role of supplementation as protection against respiratory viral infections in healthy individuals and in patients with chronic respiratory diseases. Finally, we suggest ways of improving the effectiveness of vitamin D as an adjuvant in the prevention and treatment of acute respiratory infections.     

IL-1beta expression in Int407 is induced by flagellin of Vibrio cholerae through TLR5 mediated pathway

Vibrio cholerae, a noninvasive enteric bacterium, causing inflammatory diarrheal disease cholera, is associated with the secretion of proinflamammatory cytokines including IL-1β in cultured epithelial cells. Incubation of Int407 with live V. cholerae resulted in increased IL-1β mRNA expression as early as 2 h of infection, reached a peak at 3.5 h and decreased thereafter. The identity of the effector molecule(s) is largely unknown. The bacterial culture supernatant showed IL-1β stimulating activity. An engineered aflagellate V. cholerae flaA mutant (O395FLAN) resulted in highly reduced level of IL-1β expression in Int407. The crude flagellar protein of V. cholerae as well as recombinant FlaA induced IL-1β expression in Int407. Infection of Toll-like receptor 5 (TLR5) transfected HeLa cells with O395FLAN showed reduced expression of IL-1β compared to wild-type. Unlike wild-type V. cholerae, O395FLAN did not activate the NF-κB while the recombinant flagellin could activate NF-κB. Finally, the mitogen activated protein kinases (ERK1 and 2, p38) were phosphorylated in wild-type and recombinant flagellin treated Int407 cells and inhibition of the p38 and ERK pathways significantly decreased the IL-1β response induced by wild-type V. cholerae as well as recombinant flagellin. Our data clearly indicate that flagellin of V. cholerae could induce IL-1β expression by recognizing TLR5 that activate NF-κB and MAP kinase in Int407.     

Genetic analysis of the innate immune responses in wild-derived inbred strains of mice

The vertebrate immune system has evolved to recognize nucleic acids of bacterial and viral origin. Microbial DNA, as well as synthetic oligonucleotides based on these motifs, activates innate immune pathways mediated by the family of Toll-like receptors (TLR) initiating a cascade of signals in immune cells necessary for responses to pathogens. However, not all of the proteins that participate in TLR-mediated responses have been identified. In studies described herein, we observed significant variation in innate immune responses among selected wild-derived strains of mice. Specifically, we show that mice of MOLF/Ei, Czech/Ei, and MSM/Ms strains are hypo-responsive to polyinosinic-polycytidylic acid (poly(I:C)) because of a mutation in Tlr3. In addition, we discovered a hypo-response to cytosine guanine dinucleotide in MOLF/Ei mice and established that it is not linked to Tlr9, but to another locus. Further inquiry revealed that this hypo-response is transmitted as a monogenic dominant trait that can be mapped and cloned through positional cloning methods. These results suggest the existence of a novel molecule that can alter pro-inflammatory signals or activate additional signal transduction pathways. In addition, they support the wild-derived mouse strain as a forward genetic tool for the identification of novel immunological phenotypes.     

The affirmative response of the innate immune system to apoptotic cells

Growing evidence exists for a new role for apoptotic cell recognition and clearance in immune homeostasis. Apoptotic cells at all stages, irrespective of membrane integrity, elicit a signature set of signaling events in responding phagocytes, both professional and non-professional. These signaling events are initiated by receptor-mediated recognition of apoptotic determinants, independently of species, cell type, or apoptotic stimulus. We propose that the ability of phagocytes to respond to apoptotic targets with a characteristic set of signaling events comprises a second distinct dimension of innate immunity, as opposed to the traditional innate discrimination of self vs. non-self. We further propose that a loss or abnormality of the signaling events elicited by apoptotic cells, as distinct from the actual clearance of those cells, may predispose to autoimmunity.     

Toll样受体5基因多态性与中国人群脓毒症的相关性研究

目的 探讨Toll样受体5(Toll-like receptor 5, TLR5)基因多态性位点与脓毒症发生风险及疾病严重程度的相关性.方法 采用病例-对照研究设计,募集了255例脓毒症患者和260例对照个体.应用贝克曼公司的商用SNPstream 分型技术和PCR-RFLP 方法对TLR5基因的3个编码区多态性位点进行分型.采用Logistic 回归分析,校正性别、年龄、吸烟和饮酒、慢性病状态、APACHEⅡ评分和脓毒症病因等混杂因素的影响,评价多态性位点与脓毒症的发生风险,以及脓毒症性休克、死亡和器官功能障碍等表型的遗传相关性.结果 TLR5基因的3个多态性位点在病例和对照组中的基因型分布均呈哈-温平衡状态.这3个编码区的多态性位点与脓毒症的发生风险和疾病严重程度均无遗传学关联.结论 TLR5基因的多态性位点可能在脓毒症的发生、发展和病程转归中不发挥重要作用.     

Rapamycin和cyclosporin A对同种排斥过程中TLR5及Foxp3表达的影响

目的:研究雷帕霉素(Rapamycin,RAPA)和环孢素A(cyclosperin A,CsA)体内处理对同种移植小鼠急性排斥过程中TLR5及Foxp3表达的影响.方法:建立同种皮肤移植模型,术后给予RAPA或CsA,1次/d,连续14 d,同时设生理盐水对照组.每日观察移植物存活状况.术后选取1、7、10、14、21 d共5个时间点,提取受体小鼠脾细胞,RT-PCR检测TLR5及Foxp3 mRNA表达,探讨不同处理组基因表达与移植物生存的相关性.体外实验利用鞭毛蛋白(flagellin)联合RAPA和CsA处理正常小鼠T细胞6 h,RT-PCR检测TLR5表达.结果:RAPA和CsA可明显延长小鼠同种移植皮片存活期.RAPA可促进脾细胞TLR5 mRNA表达升高,停药后的第21天仍明显高于CsA组和对照组(P＜0.05);CsA组在第7、10天有显著升高,第21天降低(P＜0.05);3组中最高表达的时间点为移植后第10天,此时正值排斥高峰期.RAPA可引起Foxp3 mRNA的表达升高(P＜0.05),停药后仍维持较高水平;CsA组仅在移植后第7、10天短暂升高,第14天低于对照组(P＜0.05).移植后1、7、10、21 d 3组的TLR5与Foxp3 mRNA变化趋势正相关.体外实验中,RAPA或CsA与flagellin联合使用,可促进TLR5的表达,以前者的作用更为明显.结论:RAPA和CsA在同种移植急性排斥高峰期可引起TLR5和Foxp3表达的协同升高,且RAPA的作用更加明显和持久,鞭毛蛋白体外可以增强这种作用效果.     

Stroma: the forgotten cells of innate immune memory

All organisms are exposed constantly to a variety of infectious and injurious stimuli. These induce inflammatory responses tailored to the threat posed. While the innate immune system is the front line of response to each stimulant, it has been considered traditionally to lack memory, acting in a generic fashion until the adaptive immune arm can take over. This outmoded simplification of the roles of innate and acquired arms of the immune system has been challenged by evidence of myeloid cells altering their response to subsequent encounters based on earlier exposure. This concept of ‘innate immune memory’ has been known for nearly a century, and is accepted among myeloid biologists. In recent years other innate immune cells, such as natural killer cells, have been shown to display memory, suggesting that innate immune memory is a trait common to several cell types. During the last 30 years, evidence has slowly accumulated in favour of not only haematopoietic cells, but also stromal cells, being imbued with memory following inflammatory episodes. A recent publication showing this also to be true in epithelial cells suggests innate immune memory to be widespread, if under‐appreciated, in non‐haematopoietic cells. In this review, we will examine the evidence supporting the existence of innate immune memory in stromal cells. We will also discuss the ramifications of memory in long‐lived tissue‐resident cells. Finally, we will pose questions we feel to be important in the understanding of these forgotten cells in the field of innate memory.