Potentiation of TLR9 responses for human naïve B-cell growth through RP105 signaling

Toll-like receptor 9 (TLR9) signals induce important pathways in the early defense against microbial pathogens. Although TLR9 signaling can activate memory B cells directly, efficient naïve B cell responses seem to require additional, but as yet unidentified, signals. We explored the effects of RP105 (CD180) on CpG DNA-activated naïve and memory B cells from normal controls and patients with common variable immunodeficiency (CVID). RP105 dramatically enhanced CpG DNA-induced proliferation/survival by naïve B cells but not by memory B cells. This enhancement was mediated by TLR9 upregulation induced by RP105, leading to Akt activation and sustained NF-κB activation. CpG DNA-activated CVID B cells showed enhancement of proliferation/survival by RP105 and produced specific IgM antibody to Streptococcus pneumoniae polysaccharides in response to interleukin-21 stimulation. Thus, RP105 strongly affects expansion of the naïve B-cell pool, and suggests that the putative RP105 ligand (s) upon cytokine stimulation facilitates antibody-mediated acute pathogen clearance.     

Toll-like receptor 1 inhibits Toll-like receptor 4 signaling in endothelial cells

Toll-like receptor 4 (TLR4) is the signal-transducing component of the LPS recognition complex and is essential for LPS-induced septic shock. Here we demonstrate that TLR1 has the capacity to abrogate TLR4 signaling. Human microvascular endothelial cells express TLR4 but not TLR1 and respond to LPS through TLR4. The ability of these cells to respond to LPS was lost, however, when they were transfected with TLR1. Inhibition was specific for TLR1 because TL5 failed to block TLR4 function. Moreover, TLR1 had no effect upon TNF-alpha signaling, indicating that TLR1 operated at a step upstream of the convergence between the two pathways. Inhibition of TLR4 signaling was mediated by the extracellular, but not cytoplasmic domain of TLR1. In addition, TLR1 physically associated with TLR4 in co-precipitation experiments. These findings suggest that TLR1 might restrain potentially dangerous innate response to LPS by binding to TLR4 and preventing the formation of active signaling complexes.     

The RP105/MD-1 complex is indispensable for TLR4/MD-2-dependent proliferation and IgM-secreting plasma cell differentiation of marginal zone B cells

Marginal zone (MZ) B cells mount rapid T-cell-independent (T-I) immune responses against microbial components such as LPS. While Toll-like receptor 4 (TLR4) is essential for LPS responses, MZ B cells uniquely express high levels of another LPS sensor Radioprotective 105 (RP105). However, little is known about how RP105 is used by MZ B cells. In this study, we investigated TLR4- or RP105-dependent MZ B cell responses by utilizing agonistic monoclonal antibodies (mAbs) to each receptor. Cross-linking TLR4 and RP105 at the same time with the mAbs induced robust IgM-secreting plasma cell generation as lipid A moiety of LPS. In contrast, stimulation with either mAb alone did not elicit such responses. RP105-deficient MZ B cells failed to produce IgM-secreting plasma cells in response to lipid A. TLR4 or lipid A stimulation of MZ B cells up-regulated their B lymphocyte-induced maturation protein 1 (Blimp-1) and X-box-binding protein 1 (Xbp-1) mRNA expression. RP105 stimulation alone did not give these responses and in fact decreased TLR4-mediated their expression. Compared with wild-type (WT) MZ B cells, RP105-deficient MZ B cells exhibited increased levels of Blimp-1 and Xbp-1 mRNA expression in response to lipid A. Lipid A or TLR4 plus RP105 stimulation induced massive proliferation and expression of Bcl-xL and c-Myc in WT but not RP105-deficient MZ B cells. These responses contributed to TLR4-mediated anti-apoptotic responses in MZ B cells. Thus, RP105 contributes in a unique way to the TLR4-dependent survival, proliferation and plasma cell generation of MZ B cells.     

Mast cell-mediated immune responses through IgE antibody and Toll-like receptor 4 by malarial peroxiredoxin

In this study, 2-Cys Plasmodium berghei ANKA (PbA) peroxiredoxin (Prx) was identified as an antigenic protein recognized by an anti-PbA IgE antibody using two-dimensional polyacrylamide gel electrophoresis and proteomic analysis. Innate immune responses to PbAPrx were examined using cells from mice deficient in Toll-like receptors (TLR) or related molecules, and it was demonstrated that responses were severely impaired in TLR4(-/-), MyD88(-/-) and MD-2(-/-) mice, but not in Toll/IL-1 receptor domain-containing adaptor inducing IFN-gamma (TRIF)(-/-), TLR2(-/-) or radioprotective 105 (RP105)(-/-) mice. An association between PbAPrx and TLR4 was observed following immunoprecipitation and immunoblotting, suggesting that PbAPrx was associated with TLR4/MD-2. Interactions between Prx and TLR4/MD-2 were also examined by flow cytometry using TLR4/MD-2- or TLR2-expressing cells. NFkappaB/GFP activity was observed in TLR4/MD-2- but not in TLR2-expressing cells following stimulation with Prx. However, this effect was not observed after treatment with proteinase K, suggesting that PbAPrx is a protein ligand for TLR4 and that the PbAPrx activity observed in this study is not due to contamination with LPS. These findings indicate that malarial Prx induces IgE-mediated protection through FcepsilonRI on mast cells and innate immunity through TLR4 with MyD88 and MD-2, suggesting a novel function for malarial Prx in innate and acquired immune responses in malaria.     

Toll-like receptor]

Toll-like receptors (TLRs) have been revealed to recognize specific patterns of microbial components. Recognition of microbial components by TLRs initiates signal transduction pathways, triggering expression of genes, which products control innate immune responses and further instruct development of antigen-specific acquired immunity. TIR domain-containing adaptors, such as MyD88, TIRAP, TRIF, and TRAM, play pivotal roles in TLR signaling pathways. Differential utilization of these TIR domain-containing adaptors provides specificity of individual TLR-mediated signaling pathways. TLR-mediated activation of innate immunity, when in excess, leads to immune disorders such as inflammatory bowel diseases. Therefore, several mechanisms that negatively control TLR signaling pathways and thereby prevent overactivation of innate immunity have been elucidated. Nuclear IkappaB proteins, such as Bcl-3 and IkappaBNS, have been revealed to be responsible for this process, by differentially inhibiting TLR-dependent cytokine production.     

TLR accessory molecules

Accessory molecules are required for microbial recognition by Toll-like receptor (TLR), subsequent signaling, and regulation of ensuing immune responses. Accessory molecules regulate TLRs on the cell surface (MD-2 and RP105), or in the endoplasmic reticulum (ER) (Unc93B, PRAT4A, and gp96). Other types of accessory molecules modulate TLR responses by acting directly on TLR ligands (CD14, CD36, HMGB1, and the antimicrobial peptide LL37). These molecules cooperate with TLR, inducing appropriate defense mechanisms. It is important to understand how TLR signaling is controlled by these accessory molecules. These accessory molecules could be promising targets for therapeutic intervention in infectious disease and immune disorders.     

Endotoxin recognition molecules, Toll-like receptor 4-MD-2

Toll-like receptors (TLRs) are innate pathogen recognition molecules for microbial products. Lipopolysaccharide (LPS), a membrane constituent of Gram-negative bacteria, is one of the most potent microbial products. LPS is recognized by TLR4 and MD-2. TLR4 is a transmembrane protein, the extracellular domain of which is composed of a protein motif called leucine-rich repeats (LRR). MD-2 is an extracellular molecule that is associated with the extracellular LRR of TLR4. MD-2 has a role in cell surface expression of TLR4 and interaction with LPS. TLR4-MD-2 contributes to containment of infections by Gram-negative bacteria by activating immune responses.     

Evasion of Toll-like receptor 2 activation by staphylococcal superantigen-like protein 3

Toll-like receptors (TLRs) are crucial for our host defense against microbial infections. TLR2 is especially important to fight bacterial infections, as it specifically recognizes bacterial lipoproteins of both Gram-positive and Gram-negative origin. Present on a variety of immune cells, TLR2 is critical for host protection against several bacterial infections, including those caused by Staphylococcus aureus. This major human pathogen causes increasing health care problems due to its increased resistance to antibiotics. S. aureus secretes a wide variety of proteins that inhibit innate immune responses. Recently, several staphylococcal superantigen-like proteins (SSLs) have been described to mediate immune evasive properties. Here, we describe that SSL3 specifically binds and inhibits TLR2 activation on human and murine neutrophils and monocytes. Through binding of the extracellular TLR2 domain, SSL3 inhibits IL-8 production by HEK cells expressing TLR1/2 and TLR2/6 dimers, stimulated with their specific ligands. The SSL3-TLR2 interaction is partially glycan dependent as binding of SSL3 to TLR2 is affected upon removal of sialic acid residues. Moreover, the SSL3(R308A) mutant lacking glycan-binding properties shows lower TLR2 inhibition. An SSL3 mutant, lacking the N-terminal 126 amino acids, still retains full TLR2 inhibiting activity. Of other SSLs tested, only SSL4, which shares the highest homology with SSL3, blocks TLR2 activation. SSL3 is the first-described bacterial protein that blocks TLR2 activation through direct extracellular interaction with the receptor. This unique function of SSL3 adds to the arsenal of immune evasive molecules that S. aureus can employ to subvert both innate and adaptive immunity.     

The contribution of Toll-like receptor 2 to the innate recognition of a Leishmania infantum silent information regulator 2 protein

We have characterized a Leishmania protein belonging to the silent information regulator 2 (SIR2) family [SIR2 related protein 1 (SIR2RP1)] that might play an immunoregulatory role during infection through its capacity to trigger B-cell effector functions. We report here that SIR2RP1 leads to the proliferation of activated B cells, causing increased expression of major histocompatibility complex (MHC) II and the costimulatory molecules CD40 and CD86, which are critical ligands for T-cell cross-talk during the development of adaptive immune responses. In contrast, B cells isolated from Toll-like receptor 2 (TLR2) knockout mice were unable to respond to the SIR2RP1 stimulus. Similarly, SIR2RP1 induced the maturation of dendritic cells (DCs) in a TLR2-dependent manner with the secretion of pro-inflammatory cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)-α] and enhanced the costimulatory properties of DCs. Nevertheless, immunization assays demonstrated that TLR2-deficient mice were able to mount a specific humoral response to SIR2RP1. Interestingly, further investigations showed that macrophages were activated by SIR2RP1 even in the absence of TLR2. Therefore, a different type of interplay between SIR2RP1 and the major antigen-presenting cells in vivo could explain the immune response observed in TLR2-deficient mice. Together, these results demonstrate that TLR2 signalling contributes to SIR2RP1 recognition by innate immune host cells.     

Human airway epithelial cell responses to Neisseria lactamica and purified porin via Toll-like receptor 2-dependent signaling

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.     

Toll-like receptor 4-mediated activation of murine mast cells.

Toll-like receptors (TLRs) are a family of pattern recognition receptors that are critical for cellular responses to a variety of bacterial, viral, and fungal products. Mast cells are important to host survival in a number of models of bacterial infection and might act as sentinel cells in host defense. We therefore examined the expression of TLRs and associated molecules by murine bone marrow-derived mast cells (BMMCs). BMMCs and the murine mast cell line MC/9 expressed mRNA for TLR2, TLR4, and TLR6 but not TLR5 and for both adapter molecule MD-2 and signaling molecule MyD88 but lacked surface CD14. After activation with the TLR2- and TLR4-dependent stimuli Staphylococcus aureus-derived peptidoglycan and Escherichia coli-derived lipopolysaccharide (LPS), respectively, mast cells produced significant levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). To determine whether mast cells require TLR4 for cellular responses to LPS, mast cells were derived from the bone marrow cells of C3H/HeJ and C57Bl/10ScNCr mice containing a point mutation and a null mutation, respectively, in TLR4. Using these models, we demonstrated that the BMMC IL-6 and TNF-alpha responses to LPS were completely dependent on functional TLR4 with no significant LPS response observed in its absence. These findings have important implications for the mechanism of mast cell responses to pathogens and their products and suggest that different TLR4-expressing cells might have different thresholds for activation with LPS.     

Toll-like receptor 9 signaling can sensitize fibroblasts for apoptosis

Toll-like receptors (TLR) are activated by microbial components and transmit signals that induce cell activation and differentiation. A number of recent reports further indicate that TLR also have the potential to induce apoptosis upon ligand binding. Here we investigate the apoptosis-inducing capacity of TLR9, the receptor for microbial CpG-DNA. Unlike ligands for TLR2 and TLR4, CpG-DNA failed to induce apoptosis in RAW264.7 mouse macrophages. In human embryonic kidney fibroblasts transfected stably to express TLR9, CpG-DNA weakly induced apoptosis in one clone but not others without an obvious allocation to differences in TLR-signaling events. Analysis of the apoptotic signaling showed that the mitochondrial pathway of apoptosis was triggered by TLR9, as mitochondrial Bax was activated upstream of caspase-cleavage. CpG-DNA-induced apoptosis was reduced by cycloheximide suggesting that de novo protein synthesis was required. Strikingly, stimulation with CpG-DNA resulted in a strongly increased sensitivity of TLR9-expressing fibroblasts to apoptosis induced by staurosporine and UV-irradiation. These results identify a mitochondrial pathway to apoptosis that can be triggered by TLR9 and that may serve to sensitize cells from the innate immune system to apoptosis in the course of an immune response.     

Toll-like receptor 2-mediated human B cell differentiation

Human B cells likely have a major role in the adjuvant activity of Toll-like receptor (TLR) 9 agonists by enhancing innate and adaptive immune responses. As several TLR2 ligands are promising vaccine adjuvant candidates, our aim was to characterize the effects of TLR2 stimulation on human B cell activation and differentiation using cells derived from healthy peripheral blood (PB), spleen, and diseased tonsils. We found a subset of partially differentiated TLR2+ PB and splenic B cells which responds to TLR2 agonists by mediating events involved in germinal center formation, such as upregulating CD77 and secreting chemokines. Furthermore, we show that TLR2-activated monocytes induce B cells to secrete significant quantities of IgM. Finally, activated TLR2+ B cells from tonsils are induced to secrete IgM directly by TLR2 ligands. Thus, TLR2 is likely involved in specific B cell-mediated functions and may be a viable vaccine adjuvant target in humans.     

TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll-interleukin 1 receptor (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. Here we have identified a fourth TIR domain-containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-beta production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.     

The cytoplasmic 'linker region' in Toll-like receptor 3 controls receptor localization and signaling

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA and transmits signals to activate NF-kappaB and the interferon (IFN)-beta promoter via the newly identified adaptor, TICAM-1. The extracellular LRR domain of TLR3 is engaged in the ligand recognition, while the intracellular TIR domain is crucial for the adaptor binding and signal transduction upon ligand stimulation. Here, we analyzed TLR3 localization in human monocyte-derived immature dendritic cells (iDCs) and stable transfectants expressing human TLR3 by immunofluorescence staining and confocal microscopy. TLR3 was predominantly localized in specific but as yet unidentified intracellular vesicles where TLR3 signaling was initiated. Expression analysis of TLR3-tail-truncated mutants revealed that the cytoplasmic 'linker' region (residues 730-755) determines the intracellular localization of TLR3. Site-directed mutagenesis of the linker region allowed us to identify the relevant determinants as Arg(740) and Val(741) residues for intracellular expression of TLR3. Furthermore, alanine scanning of the linker region demonstrated that the Phe(732), Leu(742) and Gly(743) in the TLR3 cytoplasmic linker region are essential for ligand-induced NF-kappaB and IFN-beta promoter activation. Thus, the cytoplasmic linker region of TLR3 regulates receptor retention inside the organelle and signaling, which may be closely linked to TLR3 function in DCs.     

Regulatory T cells and Toll-like receptors in tumor immunity

Regulatory T (Treg) cells induce immune tolerance by suppressing host immune responses against self- or non-self-antigens, thus playing critical roles in preventing autoimmune diseases. However, tumor cells may take advantage of Treg cells to protect themselves from immune attack elicited by vaccines. Recent studies demonstrate the presence of Treg cells in various types of cancers and their suppressive function. Therefore, Treg cells at tumor sites have detrimental effects on immunotherapy directed to cancer and infectious diseases. This review will discuss antigen specificity of Treg cells, the factors that contribute to Treg cell generation and suppressive function, and their regulation through Toll-like receptor signaling. It was generally though that TLR-mediated recognition of specific structures of invading pathogens initiate innate as well as adaptive immune responses through dendritic cells. New evidence suggests that TLR signaling may directly regulate the suppressive function of Treg cells. Linking TLR signaling to the functional control of Treg cells opens intriguing opportunities to shift the balance between CD4(+) T-helper and Treg cells, in ways that may improve the outcome of cancer immunotherapy.     

A novel negative regulator for IL-1 receptor and Toll-like receptor 4

The Toll-IL-1 receptor (TIR) superfamily, defined by the presence of an intracellular TIR domain, initiates innate immunity via NF-kappaB activation, leading to production of proinflammatory cytokines. ST2 is a member of the TIR family that does not activate NF-kappaB and has been suggested as an important effector molecule of type 2 T helper cell responses. We have recently demonstrated that the membrane bound form of ST2 (ST2L) negatively regulated IL-1RI and TLR4 but not TLR3 signaling by sequestrating the adaptors MyD88 and Mal. In contrast to wild-type mice, ST2 deficient mice failed to develop endotoxin tolerance. Thus, ST2 suppresses IL-1R and TLR4 signaling via MyD88- and Mal-dependent pathways and modulates innate immunity. The results provide a molecular explanation for the role of ST2 in T(H)2 responses since inhibition of TLRs will promote a T(H)2 response and also identify ST2 as a key regulator of endotoxin tolerance.