Through Toll-like Receptors Ligands Research Articles

Different interleukin-17-secreting Toll-like receptor+ T-cell subsets are associated with disease activity in multiple sclerosis.

Signalling through Toll-like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR-2, -4 and -9 was significantly higher on CD4+ and CD8+ T-cells from MS patients compared to healthy individuals. Following in-vitro activation, the proportion of interleukin (IL)-17+ and IL-6+ CD4+ and CD8+ T-cells was higher in the patients. In addition, the proportion of IFN-γ-secreting TLR+ CD8+ T-cells was increased in MS patients. Among different IL-17+ T-cell phenotypes, the proportion of IL-17+ TLR+ CD4+ and CD8+ T-cells producing IFN-γ or IL-6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4+ and CD8+ T-cells with ligands for TLR-2 (Pam3Csk4), TLR-4 [lipopolysaccharide (LPS)] and TLR-9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen-associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL-6, IFN-γ, IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels produced by Pam3Csk4-activated CD4+ cells were directly associated with disease activity. A similar correlation was observed with regard to IL-17 levels released by Pam3Csk4-stimulated CD8+ T-cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR-2, -4 and -9 is associated with MS disease activity, and reveals a preferential ability of TLR-2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.

Alpha(v) Integrins Engage Autophagy Components to Regulate B cell Immune Response

Abstract Signaling through Toll-like receptors (TLR) has been implicated in activation of immune cell populations such as B lymphocytes. TLR ligands associated with self-antigens can drive increased self-reactive B cell responses however TLR ligands present in pathogenic products or vaccines can also enhance protective antibody responses. Our recent work has identified a novel mechanism by which αv integrins, a family of adhesion molecules and the autophagy proteins limit excessive B cell TLR signaling. Specifically, we have found that αvβ3 integrin regulates TLR signaling (NFKB and IRF7 activation) by directing maturation of TLR containing endosomes, through activation of components of the autophagy pathway (LC3 and Atg5). B cells lacking either αv integrins or autophagy components show enhanced TLR signaling and increased proliferative responses to TLR ligands. Mice with disruption in this pathway develop increased autoantibodies with age and develop accelerated autoimmunity in murine lupus models. Therefore, we propose that αv-mediated regulation of TLR signaling exists to limit excessive B cell responses to self-antigens. In addition, we find that mice in which αv is specifically deleted from B cells mount stronger antibody responses when immunized with exogenous antigens containing TLR-ligand adjuvants. Immune responses to these antigens are characterized by increased expansion of germinal center cells, increased somatic hyper-mutation and higher levels of high-affinity class-switched antibodies. Therefore this αv mediated regulation of TLR signaling not only regulates B cell responses to self-antigens but it is also important for regulating germinal center B cell responses and production of high-affinity antibodies.

B cell αv integrins regulate development of autoimmunity in murine lupus models

Abstract B cell activation by self-antigen and signaling through Toll-like receptors (TLR) such as TLR7 and TLR9 has been implicated in the development of autoimmunity. We have recently identified a mechanism by which αvβ3 integrin limit excessive B cell responses to antigens containing TLR ligands, including self-antigens. We have previously reported that mice in which αv integrins are selectively deleted from B cells develop increased levels of autoantibodies to RNA and double stranded DNA. Based on these findings, we hypothesized that deletion of αv integrins would accelerate TLR dependent autoimmunity. To study this further, we crossed mice lacking αv integrins on B cells (αv-CD19 mice) with mice expressing increased levels of TLR7 (TLR7.1tg mice) that develop Lupus-like autoimmunity. We find that the resulting αv-CD19 TLR7tg mice had increased spleen weight in comparison with age-matched TLR7tg littermates. αv-CD19 TLR7tg mice also had increased number of IgG2c+ plasma cells and increased autoantibodies to RNA, all indicative of enhanced B Cell activation and development of autoimmunity. In addition, deletion of αv integrin on B Cells in this model leads to enhanced activation of T Cells. Currently, we are investigating the role of αv integrins on B Cell subpopulations in driving T Cell activation and antigen presentation in this model of autoimmunity. B Cells contribute to autoimmunity by activating T Cells in addition to producing autoantibodies and cytokines. These studies allow us to understand how altered B Cell TLR signaling can lead to enhanced immune activation in autoimmunity and highlight the importance of αv integrins in regulating this process.

Self-assembly of immune signals to induce and control tolerance

Abstract Multiple sclerosis (MS) is an autoimmune disease in which myelin insulating neurons is attacked. New research in humans shows excess inflammation through toll like receptors (TLR) contribute to disease. In animals, co-administration of self-antigens and regulatory cues leads to more specific tolerance. Polymers and other biomaterials can enable this co-delivery, along with attractive features such as tunable cargo loading, targeting, and cargo protection. However, these materials often exhibit intrinsic properties that can cause inflammation; features that might exacerbate disease. Thus, we designed immune polyelectrolyte multilayers (iPEMs) mimicking attractive features of biomaterial, but constructed entirely from immune signals. iPEMs were built by electrostatic interactions between myelin-derived, cationic peptides with an anionic, regulatory TLR9 antagonist (GpG). We hypothesized iPEMs would reduce TLR-driven inflammation during T cell differentiation, biasing expanding myelin-reactive T cells away from effector cells (TH1, TH17) and toward regulatory T cells (TREG) that control disease specifically. In culture, iPEMs co-deliver each signal to dendritic cells, reduce TLR9 levels, deactivate DCs, and promote myelin-specific TREG. These results depend on the presence of both GpG and myelin. In mice, iPEMs reduce TH17 and TH1 cells, expand TREG, and reverse MS-like disease (EAE). In samples from human MS patients, iPEMs also activate T cells, but polarize cytokine profiles toward tolerance and away from inflammation. This approach represents a simple, modular platform for combatting autoimmunity by programming the combinations and relative concentrations of self-antigens and regulatory TLR ligands.

Lipopolysaccharides-Induced Suppression of Innate-Like B Cell Apoptosis Is Enhanced by CpG Oligodeoxynucleotide and Requires Toll-Like Receptors 2 and 4.

Innate-like B lymphocytes play an important role in innate immunity in periodontal disease through Toll-like receptor (TLR) signaling. However, it is unknown how innate-like B cell apoptosis is affected by the periodontal infection-associated innate signals. This study is to determine the effects of two major TLR ligands, lipopolysaccharide (LPS) and CpG-oligodeoxynucleotides (CpG-ODN), on innate-like B cell apoptosis. Spleen B cells were isolated from wild type (WT), TLR2 knockout (KO) and TLR4 KO mice and cultured with E. coli LPS alone, P. gingivalis LPS alone, or combined with CpG-ODN for 2 days. B cell apoptosis and expressions of specific apoptosis-related genes were analyzed by flow cytometry and real-time PCR respectively. P. gingivalis LPS, but not E. coli LPS, reduced the percentage of AnnexinV+/7-AAD- cells within IgMhighCD23lowCD43-CD93- marginal zone (MZ) B cell sub-population and IgMhighCD23lowCD43+CD93+ innate response activator (IRA) B cell sub-population in WT but not TLR2KO or TLR4KO mice. CpG-ODN combined with P. gingivalis LPS further reduced the percentage of AnnexinV+/7-AAD- cells within MZ B cells and IRA B cells in WT but not TLR2 KO or TLR4 KO mice. Pro-apoptotic CASP4, CASP9 and Dapk1 were significantly down-regulated in P. gingivalis LPS- and CpG-ODN-treated B cells from WT but not TLR2 KO or TLR4 KO mice. Anti-apoptotic IL-10 was significantly up-regulated in P. gingivalis LPS- and CpG-ODN-treated B cells from WT and TLR2 KO but not TLR4 KO mice. These results suggested that both TLR2 and TLR4 signaling are required for P. gingivalis LPS-induced, CpG-ODN-enhanced suppression of innate-like B cell apoptosis.

αv integrins engage autophagy components to regulate B cell immune response

Abstract Signaling through Toll-like receptors (TLR) such as TLR7 and TLR9 has been implicated in B cell activation by self-antigens and development of autoimmunity. Our recent work based on murine models has identified a novel mechanism by which a family of adhesion molecules called αv integrins and the autophagy pathway limit excessive B cell responses to antigens containing TLR ligands and self-derived antigens. Specifically, we have found that αvβ3 integrin regulates B cell TLR signaling by directing trafficking and maturation of TLR containing endosomes and engaging components of the autophagy pathway. When B cells lack either αv integrins or autophagy components LC3 and Atg5 endosomal trafficking of the TLRs is disrupted and leads to enhanced TLR signaling and increased B cell responses. We have used av conditional knockout mice to determine the consequences of loss of this αvβ3-mediated immune regulation. Mice lacking B cell av show increased antibody responses to antigens containing TLR-ligands and develop increased levels of autoantibodies. We therefore propose that αv-mediated TLR regulation serves to maintain the balance between protective immunity to microbes and potential pathological autoimmune responses. We are currently studying the role of αv signaling and autophagy components in development of class switched, high affinity antibodies in response to antigens in the context of infection with a pathogen or in response to self-antigens.

The Effect of Chloroquine on Immune Activation and Interferon Signatures Associated with HIV-1.

Immune activation associated with HIV-1 infection contributes to morbidity and mortality. We studied whether chloroquine, through Toll-like receptor (TLR) antagonist properties, could reduce immune activation thought to be driven by TLR ligands, such as gut-derived bacterial elements and HIV-1 RNAs. AIDS Clinical Trials Group A5258 was a randomized, double-blind, placebo-controlled study in 33 HIV-1-infected participants off antiretroviral therapy (ART) and 37 participants on ART. Study participants in each cohort were randomized 1:1 to receive chloroquine 250 mg orally for the first 12 weeks then cross over to placebo for 12 weeks or placebo first and then chloroquine. Combining the periods of chloroquine use in both arms of the on-ART cohort yielded a modest reduction in the proportions of CD8 T cells co-expressing CD38 and DR (median decrease = 3.0%, p = .003). The effect on immune activation in the off-ART cohort was likely confounded by increased plasma HIV-1 RNA during chloroquine administration (median 0.29 log10 increase, p < .001). Transcriptional analyses in the off-ART cohort showed decreased expression of interferon-stimulated genes in 5 of 10 chloroquine-treated participants and modest decreases in CD38 and CCR5 RNAs in all chloroquine-treated participants. Chloroquine modestly reduced immune activation in ART-treated HIV-infected participants. Clinical Trials Registry Number: NCT00819390.

Contribution of MyD88-dependent PKD1 to the development of arthritis (THER2P.954)

Abstract Rheumatoid arthritis (RA) may be initiated in part by signaling through Toll-like receptors (TLRs). Disruption of TLR signaling may provide an opportunity to halt the RA process in the earliest stages. We investigated whether protein kinase D1 (PKD1), which is essential for proinflammatory responses mediated by MyD88-dependent TLRs, plays a role in the development of experimental arthritis. The presence of TLR ligands in joint cavities induces development of reactive arthritis that is characterized by accumulation of activated inflammatory cells in the joints. Deletion of PKD1 in myeloid lineage cells prevented TLR-mediated accumulation of inflammatory cells in joints, thus protecting mice from developing reactive arthritis. Arthritis spontaneously occurring in IL-1R antagonist-deficient (IL-1rn-/-) mice was dependent on MyD88, and suppression of PKD1 expression resulted in inhibition of TLR-mediated cytokine production in IL-1rn-/- macrophages. Daily treatment with a PKD inhibitor substantially reduced the incidence and severity of arthritis in IL-1rn-/- mice. In addition, a PKD inhibitor inhibited the synergistic effect of T cell receptor and TLR2 on IL-6 production in splenocytes, and significantly reduced the incidence and severity of collagen-induced arthritis that closely mimics the pathophysiologic process of human RA. This implies that PKD1 might be one of the key factors that modulate proinflammatory responses in RA, and can be a therapeutic target for RA.

Sm16, a major component of Schistosoma mansoni cercarial excretory/secretory products, prevents macrophage classical activation and delays antigen processing

BackgroundSchistosoma mansoni cercariae penetrate the skin by releasing excretory/secretory (E/S) products known as 0-3hRP, which are associated with immune modulation through Toll like receptor (TLR) signalling. Furthermore, these secretions contain Sm16, which when given to cells as a recombinant protein inhibits human monocyte derived cytokine responses to TLR4 and TLR3 ligands. Nonetheless, the extent and mechanism(s) of these inhibitory effects remain largely uncharacterized.MethodsMurine bone marrow derived macrophages were exposed to different fractions of 0-3hRP, obtained via ultracentrifugation, or recombinant Sm16. These cells were exposed to the parasite molecules in combination with different TLR ligands, or Interferon gamma, and tested for the production of the cytokines IL-10 and IL-12p40, and their ability to process antigen.ResultsThe immunomodulatory function of 0-3hRP is enriched predominantly in the pellet fraction, which contains a greater proportion of Sm16, also corroborating the ability of recombinant Sm16 to inhibit macrophage activation in response to TLR ligands. We further demonstrate that Sm16 blocks classical activation of macrophages to LPS or IFN-γ stimulation in vitro, and that inhibition of macrophage classical activation is independent of TLR2 recognition. Finally we show that Sm16 shares the altered intracellular processing observed for 0-3hRP, and is able to delay antigen processing by macrophages.ConclusionsCollectively, our findings show that Sm16 is a major component of S. mansoni cercarial E/S products, and is partly responsible for its immune-regulatory properties. Moreover, we propose that the mechanism employed by Sm16 to exert its inhibitory function is likely to be linked with alteration of endosomal trafficking and is not dependent on particular TLR receptors. Finally, we suggest that accumulation of Sm16 in the skin after percutaneous infection with S. mansoni cercariae could contribute to limiting dermal inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0608-1) contains supplementary material, which is available to authorized users.

The innate immune response of equine bronchial epithelial cells is altered by training.

Respiratory diseases, including inflammatory airway disease (IAD), viral and bacterial infections, are common problems in exercising horses. The airway epithelium constitutes a major physical barrier against airborne infections and plays an essential role in the lung innate immune response mainly through toll-like receptor (TLR) activation. The aim of this study was to develop a model for the culture of equine bronchial epithelial cells (EBEC) in vitro and to explore EBEC innate immune responses in trained horses. Bronchial epithelial biopsies were taken from 6 adult horses during lower airway endoscopy. EBEC were grown in vitro by an explant method. The innate immune response of EBEC was evaluated in vitro by treatment with TLR ligands. TLR3 is the most strongly expressed TLR at the mRNA level in EBEC and stimulation of EBEC with Poly(I:C), an analog of viral dsRNA, triggers a strong secretion of IFN-β, TNF-α, IL-6 and CXCL8. We further evaluated the EBEC innate immune response in horses that underwent a 4-month-training program. While training had no effect on TLR mRNA expression in EBEC as well as in bronchial biopsies, it increased the production of IFN-β after stimulation with a TLR3 ligand and decreased the secretion of TNF-α and IL-6 after stimulation with a TLR2 and TLR3 ligand. These findings may be implicated in the increased risk for viral and bacterial infections observed in sport horses. Altogether, we report a successful model for the culture of EBEC that can be applied to the investigation of pathophysiologic conditions in longitudinal studies.

Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1.

Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection.

Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.

Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons—a form of cell death called “phagoptosis.” UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1–42, 5 μM peroxynitrite, or 50 μM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons.

Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia

Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.

Expression of Toll-like receptors 2 and 4 is increased in peripheral blood and synovial fluid monocytes of patients with enthesitis-related arthritis subtype of juvenile idiopathic arthritis

Bacterial trigger possibly causes disease exacerbation in enthesitis-related arthritis (ERA) patients. Microbes initiate immune responses through Toll-like receptors (TLRs). We studied TLR expression on blood and SF monocytes and the effect of TLR ligands on peripheral blood (PB) mononuclear cells (PBMCs) in ERA patients. PB from 26 ERA patients and 19 healthy subjects and paired SF from 13 patients were collected. Dual-colour flow cytometry was done for TLR and CD14 expression. Results are expressed as median fluorescence intensity (MFI). Real-time PCR was done for TLRs. PBMCs were stimulated with lipopolysaccharide (LPS) or peptidoglycan and levels of IL-6 and MMP-3 measured in the culture supernatants. PBMCs from ERA patients had higher expression of TLR-2 [MFI 295.5 (48.1-598) vs 179 (68.7-442); P < 0.05] and TLR-4 [MFI 448 (178-2581) vs 402 (229-569); P < 0.05] as compared with controls. TLR-9 expression showed no significant difference between the two groups. In paired samples, SF mononuclear cells (SFMCs) had higher expression of both TLR-2 [MFI 485 (141-1683) vs 353 (118-598); P < 0.05] and TLR-4 [MFI 1016 (42.4-3159) vs 513 (193-2581); P < 0.05] as compared with PBMCs. Difference in TLR-9 expression was not significant. TLR RNA expression data were similar. Patients' PBMCs produced more IL-6 (13.51 vs 6.54 ng/ml) and MMP-3 (61 vs 32.9 ng/ml) as compared with those of the controls, on stimulation by LPS. With peptidoglycan also, IL-6 (30.58 vs 10.84) and MMP-3 (102.54 vs 49.45) were higher than in controls. Increased TLR-2 and TLR-4 expression on PBMCs and SFMCs may recognize microbial/endogenous ligands and up-regulate IL-6 and MMP-3 leading to disease exacerbation.

Activation requirements and responses to TLR ligands in human CD4+ T cells: Comparison of two T cell isolation techniques

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4+ T cells isolated either by IMACS (IMACS-CD4+) or by IMACS followed by FACS (IMACS/FACS-CD4+). As expected, IMACS-CD4+ were less pure than IMACS/FACS-CD4+ (92.5%±1.4% versus 99.7%±0.2%, respectively). Consequently, IMACS-CD4+ proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4+. In addition IMACS-CD4+ but not IMACS/FACS-CD4+ responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4+ and highly purified IMACS-/FACS-CD4+. Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.

Aberrant innate immune responses in TLR-ligand activated HLA-B27 transgenic rat cells

Commensal enteric microbiota initiate and perpetuate immune-mediated colitis in HLA-B27 transgenic (TG) rats but not wildtype (non-TG) littermates. However, the role of the innate immune response to bacterial components has not been established. We examined responses induced by bacterial adjuvants through Toll-like receptor (TLR) and NOD2 signaling in T-cell-depleted splenocytes from HLA-B27 TG rats versus non-TG controls. We found that various bacterial adjuvants induced TNF production by cells obtained from specific pathogen-free (SPF) and germ-free (GF, sterile) TG and non-TG rats. Peptidoglycan-polysaccharide (PG-PS), lipopolysaccharide (LPS), and CpG DNA motifs stimulated higher levels of TNF production by SPF TG rat spleen cells compared to non-TG cells. CD11b/c cell depletion eliminated PG-PS and LPS-induced TNF and dramatically reduced CpG-stimulated TNF production. Both SPF and GF TG rat spleens contain more cells that express high levels of CD11b/c and show enhanced mRNA expression of TLR-2 and TLR-4 compared to non-TG rat spleens. In contrast, constitutive and bacterial-induced IL-10 production was markedly lower in TG cells compared to non-TG cells of rats from the same SPF or GF housing conditions. Notably, the ratio of TNF to IL-10 produced after TLR ligand activation was significantly higher in TG than non-TG cells. HLA-B27 TG rats have an aberrant cell composition, altered functional TLR expression, and an intrinsic defect in IL-10 production in response to TLR ligands, which may result in exaggerated proinflammatory responses to commensal enteric bacteria and uncontrolled inflammation in this colitis model.

Expression and function of Toll-like receptors on dendritic cells and other antigen presenting cells from non-human primates

Antigen presenting cells (APCs), especially dendritic cells (DCs), play a crucial role in immune responses against infections by sensing microbial invasion through Toll-like receptors (TLRs). In this regard, TLR ligands are attractive candidates for use in humans and animal models as vaccine adjuvants. So far, no studies have been performed on TLR expression in non-human primates such as rhesus macaques. Therefore, we studied the TLR expression patterns in different subsets of APC in rhesus macaques and compared them to similar APC subsets in human. Also, expression was compared with corresponding DC subsets from different organs from mice. Here we show by semi-quantitative RT-PCR, that blood DC subsets of rhesus macaque expressed the same sets of TLRs as those of human but substantially differed from mouse DC subsets. Macaque myeloid DCs (MDCs) expressed TLR3, 4, 7 and 8 whereas macaque plasmacytoid DCs (PDCs) expressed only TLR7 and 9. Additionally, TLR expression patterns in macaque monocyte-derived dendritic cells (mo-DCs) (i.e., TLR3, 4, 8 and 9), monocytes (i.e., TLR4, 7, and 8) and B cells (i.e., TLR4, 7, 8, and 9) were also similar to their human counterparts. However, the responsiveness of macaque APCs to certain TLR ligands partially differed from that of human in terms of phenotype differentiation and cytokine production. Strikingly, in contrast to human mo-DCs, no IL-12p70 production was observed when macaque mo-DCs were stimulated with TLR ligands. In addition, CD40 and CD86 phenotypic responses to TLR8 ligand (poly U) in mo-DCs of macaque were higher than that of human. Despite these functional differences, our results provide important information for a rational design of animal models in evaluating TLR ligands as adjuvant in vivo.

Peptidoglycan and lipopolysaccharide activate PLCγ2, leading to enhanced cytokine production in macrophages and dendritic cells

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

TLR ligands that stimulate the metabolism of vitamin D3 in activated murine dendritic cells can function as effective mucosal adjuvants to subcutaneously administered vaccines

Cathelicidin production by human myeloid cells stimulated through toll-like receptor (TLR) 2/1, the migration of human CD8+ T cells to inflamed skin sites, and the ability of murine dendritic cells (DCs) to migrate from skin sites of vaccination to mucosal lymphoid organs all occur via calcitriol-dependent mechanisms. Herein, we report that murine DCs exposed to TLR3/TLR4 ligands upregulate their expression of 1 alpha-hydroxylase, the enzyme that converts circulating 25(OH)D3 to calcitriol, the active form of vitamin D3. TLR3/TLR4 ligands injected subcutaneously affect DC migration in vivo, allowing their trafficking to both draining and non-draining systemic and mucosal lymphoid organs. Subcutaneously delivered vaccines containing TLR3/TLR4 ligands and antigen stimulate the induction of both systemic and mucosal immune responses. Vaccines containing TLR9 ligands fail to stimulate 1 alpha-hydroxylase protein expression, are incapable of redirecting DC migration into Peyer's patches and do not induce mucosal immune responses. These findings support a hypothesis that active metabolites of vitamin D3 produced locally are able to affect various aspects of innate and acquired immune responses.

Differential inhibition of human cytomegalovirus (HCMV) by toll-like receptor ligands mediated by interferon-beta in human foreskin fibroblasts and cervical tissue

Human cytomegalovirus (HCMV) can be acquired sexually and is shed from the genital tract. Cross-sectional studies in women show that changes in genital tract microbial flora affect HCMV infection and/or shedding. Since genital microbial flora may affect HCMV infection or replication by stimulating cells through Toll-like receptors (TLR), we assessed the effects of defined TLR-ligands on HCMV replication in foreskin fibroblasts and ectocervical tissue. Poly I:C (a TLR3-ligand) and lipopolysaccharide (LPS, a TLR4-ligand) inhibited HCMV and induced secretion of IL-8 and Interferon-beta (IFNβ) in both foreskin fibroblasts and ectocervical tissue. The anti-HCMV effect was reversed by antibody to IFNβ. CpG (TLR9 ligand) and lipoteichoic acid (LTA, TLR2 ligand) also inhibited HCMV infection in ectocervical tissue and this anti-HCMV effect was also reversed by anti-IFNβ antibody. In contrast, LTA and CpG did not inhibit HCMV infection in foreskin fibroblasts. This study shows that TLR ligands induce an HCMV-antiviral effect that is mediated by IFNβ suggesting that changes in genital tract flora may affect HCMV infection or shedding by stimulating TLR. This study also contrasts the utility of two models that can be used for assessing the interaction of microbial flora with HCMV in the genital tract. Clear differences in the response to different TLR ligands suggests the explant model more closely reflects in vivo responses to genital infections.