Recombinant flagellins with partial deletions of the hypervariable domain lose antigenicity but not mucosal adjuvancy

Flagellin contains conserved N/C domains for TLR5 binding to activate innate immunity and a middle hypervariable domain harboring the major antigenic epitopes. However, conflict results existed in the previous studies as to whether the hypervariable domain was involved in the cytokine production and adjuvancy of flagellin. Here we constructed three flagellin variants (designated as FliCΔ190-278, FliCΔ220-320, and FliCΔ180-400) with deletions in the hypervariable domain. Our data demonstrated that all deletion variants lost substantial antigenicity but not mucosal adjuvancy. Surprisingly, the variant with deletion of amino acids 220-320 (FliCΔ220-320) induced higher production of IL-8, MCP-1, and TNF-α, and showed higher mucosal adjuvancy than full-length FliC flagellin. Our data supported the notion that the hypervariable domain was involved in the cytokine production by flagellin and more importantly demonstrated that the hypervariable domain was important for the mucosal adjuvancy of flagellin.     

TLR2, TLR3, and TLR4 activation specifically alters the oxidative status of intestinal epithelial cells

Intestinal inflammatory diseases are the result of multiple processes, including mucosal oxidative stress and perturbed homeostasis between commensal bacteria and mucosal immunity. Toll-like receptors (TLRs) recognize molecular-associated microorganisms' patterns and trigger innate immunity responses contributing to intestinal homeostasis and inflammatory responses. However, TLRs effects on redox balance in intestinal mucosa remain unknown. Therefore, the present study analyzes the effect of TLR2, TLR3, and TLR4 on both oxidative damage of lipids and proteins, and the activity of antioxidant enzymes in enterocyte-like Caco-2 cells. The results show that the activation of these TLRs increased lipid and protein oxidation levels; however, the effect on the antioxidant enzymes activity is different depending on the TLR activated. These results suggest that the activation of TLR2, TLR3, and TLR4 might affect intestinal inflammation by not only their inherent innate immunity responses, but also their pro-oxidative effects on intestinal epithelial cells.     

Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5

Flagellin is the major structural protein of the flagella of Gram-negative bacteria. Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants. In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5). The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown. In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation. In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity. In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses. These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin.     

Toll‐like receptor 5 is not essential for the promotion of secretory immunoglobulin A antibody responses to flagellated bacteria

Toll‐like receptor 5 recognizes bacterial flagellin, plays a critical role in innate immunity, and contributes to flagellin‐specific humoral immunity. Further, TLR5‐expressing dendritic cells play an important role in IgA synthesis in the intestine; however, the contribution of TLR5 to antigen (Ag)‐specific mucosal immunity remains unclear. Thus, whether TLR5 is essential for the induction of intestinal secretory (S)IgA antibody (Ab) responses against flagellin and bacterial Ags attached to the bacterial surface in response to an oral flagellated bacterium, Salmonella, was explored in this study. Our results indicate that when TLR5 knockout (TLR5^?/?) mice are orally immunized with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella‐Tox C), tetanus toxoid (TT)‐ and flagellin (FliC)‐specific systemic IgG and intestinal SIgA Abs are elicited. The numbers of TT‐specific IgG Ab‐forming cells (AFCs) in the spleen and IgA AFCs in the lamina propria (LP) of TLR5^?/? mice were comparable to those in wild‐type mice. rSalmonella‐Tox C was equally disseminated in TLR5^?/? mice, TLR5^?/? mice lacking Peyer's patches (PPs), and wild‐type mice. In contrast, TLR5^?/? PP‐null mice failed to induce TT‐ and FliC‐specific SIgA Abs in the intestine and showed significantly reduced numbers of TT‐specific IgA AFCs in the LP. These results suggest that TLR5 is dispensable for the induction of flagellin and surface Ag‐specific systemic and mucosal immunity against oral flagellated bacteria. Rather, pathogen recognition, which occurs in PPs, is a prerequisite for the induction of mucosal immunity against flagellated bacteria.

     

Adenovirus vector-induced innate inflammatory mediators, MAPK signaling, as well as adaptive immune responses are dependent upon both TLR2 and TLR9 in vivo

Adenovirus (Ad) vectors are promising candidates for both gene transfer and vaccine applications. In this study, we investigated the role of TLR2 in innate and adaptive immune responses to Ad and/or the transgene it expresses following systemic injection. We found that Ad directly activates ERK1/2 in vivo, but that initiation of ERK1/2 activation is primarily a MyD88/TLR2-independent, but Kupffer cell-dependent, event. The complexity of Ad-induced innate immune responses was confirmed when we also found that both TLR2 and MyD88 functions are required for the sustained activation of ERK1/2. Although we found that the initial activation of NF-kappaB by Ads is dependent upon MyD88, but independent of TLR2 in (non-Kupffer cells) the liver, TLR2 significantly influenced the Ad-induced late phase NF-kappaB activation. These very rapid responses were positively correlated with subsequent innate immune responses to the Ad vector, as our results confirmed that the induction of several cytokines and chemokines, and the expression of innate immune response genes following Ad injection were TLR2 dependent in vivo. The requirement of TLR2 in Ad-induced innate responses also correlated with significantly altered adaptive immune responses. For example, our results demonstrate that the generation of Ad-neutralizing Abs, and anti-transgene-specific Abs elicited subsequent to Ad vector treatments, are both dependent upon TLR2 functionality. Finally, we found that several Ad-induced innate immune responses are dependent on both TLR2 and TLR9. Therefore, this study confirms that several (but not all) Ad-induced innate and adaptive immune responses are TLR dependent.     

Humoral immune response to flagellin requires T cells and activation of innate immunity

Bacterial flagellin, the primary structural component of flagella, is a dominant target of humoral immunity upon infection by enteric pathogens and in Crohn's disease. To better understand how such responses may be regulated, we sought to define, in mice, basic mechanisms that regulate generation of flagellin-specific Igs. We observed that, in response to i.p. injection with flagellin, generation of flagellin-specific Ig required activation of innate immunity in that these responses were ablated in MyD88-deficient mice and that flagellin from Helicobacter pylori, which is known not to activate TLR5, also did not elicit Abs. Mice lacking alphabeta T cells (TCRbeta(null)) were completely deficient in their ability to make flagellin Abs in various contexts indicating that, in contrast to common belief, generation of flagellin-specific Ig is absolutely T cell dependent. In contrast to Ab responses to whole flagella (H serotyping), responses to flagellin monomers displayed only moderate serospecificity. Whereas neither oral nor rectal administration of flagellin elicited a strong serum Ab response, induction of colitis with dextran sodium sulfate resulted in a MyD88-dependent serum Ab response to endogenous flagellin, suggesting that, in an inflammatory milieu, TLR signaling promotes acquisition of Abs to intestinal flagellin. Thus, acquisition of a humoral immune response to flagellin requires activation of innate immunity, is T cell dependent, and can originate from flagellin in the intestinal tract in inflammatory conditions in the intestine.     

Toll-like receptor 9, CpG DNA and innate immunity

Innate immunity provides the first line of defense against invading pathogens and is essential for survival in the absence of adaptive immune responses. Innate immune recognition relies on a limited number of germ-line encoded receptors, such as Toll-like receptors (TLRs), that evolved to recognize conserved molecular patterns of microbial origin. To date, ten transmembrane proteins in the TLR family have been described. It is becoming increasingly clear that bacterial CpG DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG are potent inducers of the innate immune system including dendritic cells (DCs), macrophages, and natural killer (NK) and NKT cells. Recent studies indicate that mucosal or systemic delivery of CpG DNA can act as a potent adjuvant in a vaccine combination or act alone as an anti-microbial agent. Recently, it was shown that TLR9 is essential for the recognition of unmethylated CpG DNA since cells from TLR9-deficient mice are unresponsive to CpG stimulation. Although the effects of CpG DNA on bone marrow-derived cells are beginning to unfold, there has been little or no information regarding the mechanisms of CpG DNA function on non-immune cells or tissues. This review focuses on the recent advances in CpG-DNA/TLR9 signaling effects on the activation of innate immunity.     

Endless Hide-and-Seek： Dynamic Co-evolution in Plant-Bacterium Warfare

Plants possess innate Immune systems to prevent most potential Infections. The ancient and conserved innate immune responses are triggered by microbe-associated molecular patterns （MAMPs） and play important roles in broad-spectrum defenses. However, successful bacterial pathogens evolved type Ⅲ virulence effectors to suppress MAMP-medlated immunity. To survive, plants further developed highly specific resistance （R） genes to trigger gene-for-gene-mediated immunity and turn the virulent pathogens into avirulent ones. We summarize here the very recent advances in this dynamic coevolution of plantbacterium interaction.     

Regulation of Toll-like receptor signaling in the innate immunity

Toll-like receptors sense invading pathogens by recognizing a wide variety of conserved pathogen-associated molecular patterns (PAMPs). The members of TLR family selectively utilize adaptor proteins MyD88, TRIF, TIRAP and TRAM to activate overlapping but distinct signal transduction pathways which trigger production of different panels of mediators such as proinflammatory cytokines and type I interferon. These mediators not only control innate immunity but also direct subsequently developed adaptive immunity. TLR activation is strictly and finely regulated at multiple levels of the signal transduction pathways.     

Roundtrip ticket for secretory IgA: role in mucosal homeostasis?

An important activity of mucosal surfaces is the production of Ab referred to as secretory IgA (SIgA). SIgA serves as the first line of defense against microorganisms through a mechanism called immune exclusion. In addition, SIgA adheres selectively to M cells in intestinal Peyer's patches, thus mediating the transepithelial transport of the Ab molecule from the intestinal lumen to underlying gut-associated organized lymphoid tissue. In Peyer's patches, SIgA binds and is internalized by dendritic cells in the subepithelial dome region. When used as carrier for Ags in oral immunization, SIgA induces mucosal and systemic responses associated with production of anti-inflammatory cytokines and limits activation of dendritic cells. In terms of humoral immunity at mucosal surfaces, SIgA appears thus to combine properties of a neutralizing agent (immune exclusion) and of a mucosal immunopotentiator inducing effector immune responses in a noninflammatory context favorable to preserve local homeostasis of the gastrointestinal tract.     

Innate stimuli accentuate end-organ damage by nephrotoxic antibodies via Fc receptor and TLR stimulation and IL-1/TNF-alpha production

Innate stimuli are well recognized as adjuvants of the systemic immune response. However, their role in driving end-organ disease is less well understood. Whereas the passive transfer of glomerular-targeting Abs alone elicited minimal renal disease, the concomitant delivery of innate stimuli triggered severe nephritis, characterized by proliferative glomerulonephritis with crescent formation, and tubulointerstitial disease. Specifically, stimulating TLR2, TLR3, TLR4, and TLR5 by using peptidoglycan, poly(I:C), LPS, and flagellin, respectively, all could facilitate anti-glomerular Ab-elicited nephritis. In this model, innate and immune triggers synergistically activated several cytokines and chemokines, including IL-1, IL-6, TNF-alpha, and MCP-1, some of which were demonstrated to be absolutely essential for the development of renal disease. Genetic studies revealed that, whereas the innate trigger is dependent on TLR/IL-1R-associated kinase-mediated signaling, the immune component was contingent on FcR-mediated signals. Importantly, infiltrating leukocytes as well as intrinsic glomerular cells may both serve to integrate these diverse signals. Extrapolating to spontaneous immune-mediated nephritis, although the adaptive immune system may be important in generating end-organ targeting Abs, the extent of damage inflicted by these Abs may be heavily dependent on cues from the innate immune system.     

Metalloproteinase-Dependent TLR2 Ectodomain Shedding is Involved in Soluble Toll-Like Receptor 2 (sTLR2) Production

Toll-like receptor (TLR) 2, a type I membrane receptor that plays a key role in innate immunity, recognizes conserved molecules in pathogens, and triggering an inflammatory response. It has been associated with inflammatory and autoimmune diseases. Soluble TLR2 (sTLR2) variants have been identified in human body fluids, and the TLR2 ectodomain can negatively regulate TLR2 activation by behaving as a decoy receptor. sTLR2 generation does not involve alternative splicing mechanisms, indicating that this process might involve a post-translational modification of the full-length receptor; however, the specific mechanism has not been studied. Using CD14⁺ peripheral human monocytes and the THP-1 monocytic leukemia-derived cell line, we confirm that sTLR2 generation increases upon treatment with pro-inflammatory agents and requires a post-translational mechanism. We also find that the constitutive and ligand-induced release of sTLR2 is sensitive to pharmacological metalloproteinase activator and inhibitors leading us to conclude that metalloproteinase TLR2 shedding contributes to soluble receptor production. By expressing human TLR2 in ADAM10- or ADAM17-deficient MEF cells, we find both enzymes to be implicated in TLR2 ectodomain shedding. Moreover, using a deletion mutant of the TLR2 juxtamembrane region, we demonstrate that this domain is required for sTLR2 generation. Functional analysis suggests that sTLR2 generated by metalloproteinase activation inhibitsTLR2-induced cytokine production by this monocytic leukemia-derived cell line. The identification of the mechanisms involved in regulating the availability of soluble TLR2 ectodomain and cell surface receptors may contribute further research on TLR2-mediated processes in innate immunity and inflammatory disorders.     

Differential recognition of TLR-dependent microbial ligands in human bronchial epithelial cells

Bronchial epithelial cells represent the first line of defense against invading airborne pathogens. They are important contributors to innate mucosal immunity and provide a variety of antimicrobial effectors. However, mucosal surfaces are prone to contact with pathogenic, as well as nonpathogenic microbes, and therefore, immune recognition principles have to be tightly controlled to avoid uncontrolled permanent activation. TLRs have been shown to recognize conserved microbial patterns and to mediate inducible activation of innate immunity. Our experiments demonstrate that bronchial epithelial cells express functional TLR1-6 and TLR9 and thus make use of a common principle of professional innate immune cells. Although it was observed that TLR2 ligands dependent on heterodimeric signaling either with TLR1 or TLR6 were functional, other ligands like lipoteichoic acid were not. Additionally, it was found that bronchial epithelial cells could be stimulated only marginally by Gram-positive bacteria bearing known TLR2 ligands while Gram-negative bacteria were easily recognized. This correlated with low expression of TLR2 and the missing expression of the coreceptor CD36. Transgenic expression of both receptors restored responsiveness to the complete set of TLR2 ligands and Staphylococcus aureus. Additional gene-array experiments confirmed hyporesponsiveness to this bacterium while Pseudomonas aeruginosa and respiratory syncytial virus induced common, as well as pathogen-specific, sets of genes. The findings indicate that bronchial epithelium regulates its sensitivity to recognize microbes by managing receptor expression levels. This could serve the special needs of controlled microbial recognition in mucosal compartments.     

Recombinant MPT83 derived from Mycobacterium tuberculosis induces cytokine production and upregulates the function of mouse macrophages through TLR2

TLR2 recognizes components of Mycobacterium tuberculosis and initiates APC activities that influence both innate and adaptive immunity. M. tuberculosis lipoproteins are an important class of TLR2 ligands. In this study, we focused on recombinant MPT83 (rMPT83) to determine its effects on mouse macrophages. We demonstrated that rMPT83 induced the production of TNF-α, IL-6, and IL-12 p40 and that cytokine induction depended on activated MAPKs, because we observed the rapid phosphorylation of ERK1/2, p38, and JNK in macrophages. Additionally, neutralizing Abs against TLR2 significantly inhibited cytokine secretion and reduced or attenuated the rMPT83-induced activation of p38 and JNK in RAW264.7 cells, a mouse macrophage cell line. Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2(-/-) mice than in macrophages from wild-type mice. We further found that prolonged exposure (>24 h) of RAW264.7 cells or macrophages from wild-type and TLR2(-/-) mice to rMPT83 resulted in a significant enhancement of IFN-γ-induced MHC class II expression and an enhanced ability of macrophages to present the rMPT83 peptide to CD4(+) T cells. These results indicated that rMPT83 is a TLR2 agonist that induces the production of cytokines by macrophages and upregulates macrophage function.     

Administration of a synthetic TLR4 agonist protects mice from pneumonic tularemia

Francisella tularensis is a Gram-negative intracellular pathogen that causes the zoonosis tularemia. Because F. tularensis LPS causes weak TLR4 activation, we hypothesized that administration of a synthetic TLR4 agonist, aminoalkyl glucosaminide phosphate (AGP), would boost the innate immune system and compensate for reduced TLR4 stimulation. Intranasal administration of AGPs induced intrapulmonary production of proinflammatory cytokines and chemokines. Mice treated with AGPs before and after inhalation of Francisella novicida exhibited augmented cytokine and inflammatory responses to infection; reduced bacterial replication in lung, liver, and spleen; and increased survival, whereas all PBS-treated control mice died within 4 days of infection, all AGP-treated mice showed prolonged time-to-death, and 30-60% of AGP-treated mice survived. The protective effect of AGP was lost in mice lacking IFN-gamma. Long-term survivors developed specific Th1 splenocyte responses and specific Abs dominated by IgG2 isotypes. Survivors were fully protected from rechallenge with aerosolized F. novicida. Thus, preventive administration of AGP successfully modulated innate immune responses to aerosolized F. novicida, leading to protective immunity to pneumonic tularemia. This is the first report of the protective effect of a TLR ligand on resistance to F. novicida-induced pneumonic tularemia.     

Essential role of MAPK phosphatase-1 in the negative control of innate immune responses

TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1-/- mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-alpha, IL-6, IL-12, MCP-1, IFN-gamma, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1-/- macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.