Modulatory Effects of Vasoactive Intestinal Peptide on Intestinal Mucosal Immunity and Microbial Community of Weaned Piglets Challenged by an Enterotoxigenic Escherichia coli (K88)

Toll-like receptors (TLRs) recognize microbial pathogens and trigger immune response, but their regulation by neuropeptide-vasoactive intestinal peptide (VIP) in weaned piglets infected by enterotoxigenic Escherichia coli (ETEC) K88 remains unexplored. Therefore, the study was conducted to investigate its role using a model of early weaned piglets infected by ETEC K88. Male Duroc×Landrace×Yorkshire piglets (n = 24) were randomly divided into control, ETEC K88, VIP, and ETEC K88+VIP groups. On the first three days, ETEC K88 and ETEC K88+VIP groups were orally administrated with ETEC K88, other two groups were given sterile medium. Then each piglet from VIP and ETEC K88+VIP group received 10 nmol VIP intraperitoneally (i.p.) once daily, on day four and six. On the seventh day, the piglets were sacrificed. The results indicated that administration of VIP improved the growth performance, reduced diarrhea incidence of ETEC K88 challenged pigs, and mitigated the histopathological changes of intestine. Serum levels of IL-2, IL-6, IL-12p40, IFN-γ and TNF-α in the ETEC K88+ VIP group were significantly reduced compared with those in the ETEC group. VIP significantly increased IL-4, IL-10, TGF-β and S-IgA production compared with the ETEC K88 group. Besides, VIP could inhibit the expression of TLR2, TLR4, MyD88, NF-κB p65 and the phosphorylation of IκB-α, p-ERK, p-JNK, and p-38 induced by ETEC K88. Moreover, VIP could upregulate the expression of occludin in the ileum mucosa compared with the ETEC K88 group. Colon and caecum content bacterial richness and diversity were lower for pigs in the ETEC group than the unchallenged groups. These results demonstrate that VIP is beneficial for the maturation of the intestinal mucosal immune system and elicited local immunomodulatory activities. The TLR2/4-MyD88 mediated NF-κB and MAPK signaling pathway may be critical to the mechanism underlying the modulatory effect of VIP on intestinal mucosal immune function and bacterial community.     

Tumor Progression Locus 2-dependent Oxidative Burst Drives Phosphorylation of Extracellular Signal-regulated Kinase during TLR3 and 9 Signaling

Signal transduction via NFκB and MAP kinase cascades is a universal response initiated upon pathogen recognition by Toll-like receptors (TLRs). How activation of these divergent signaling pathways is integrated to dictate distinct immune responses to diverse pathogens is still incompletely understood. Herein, contrary to current perception, we demonstrate that a signaling pathway defined by the inhibitor of κB kinase β (IKKβ), MAP3 kinase tumor progression locus 2 (Tpl2/MAP3K8), and MAP kinase ERK is differentially activated by TLRs. TLRs 2, 4, and 7 directly activate this inflammatory axis, inducing immediate ERK phosphorylation and early TNFα secretion. In addition to TLR adaptor proteins, IKKβ-Tpl2-ERK activation by TLR4 is regulated by the TLR4 co-receptor CD14 and the tyrosine kinase Syk. Signals from TLRs 3 and 9 do not initiate early activation of IKKβ-Tpl2-ERK pathway but instead induce delayed, NADPH-oxidase-dependent ERK phosphorylation and TNFα secretion via autocrine reactive oxygen species signaling. Unexpectedly, Tpl2 is an essential regulator of ROS production during TLR signaling. Overall, our study reveals distinct mechanisms activating a common inflammatory signaling cascade and delineates differences in MyD88-dependent signaling between endosomal TLRs 7 and 9. These findings further confirm the importance of Tpl2 in innate host defense mechanisms and also enhance our understanding of how the immune system tailors pathogen-specific gene expression patterns.     

Human Parainfluenza Virus Type 2 V Protein Inhibits TRAF6-Mediated Ubiquitination of IRF7 To Prevent TLR7- and TLR9-Dependent Interferon Induction

Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.     

Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKβ subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKβ, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the “NEMO-binding domain” at the C terminus of IKK2/IKKβ. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKβ through this secondary interaction in vitro and for full activation of IKK2/IKKβ in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKβ within its scaffold-dimerization domain proximal to the kinase domain–Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture–based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKβ for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling.     

Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance

Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.     

TLR4 mediates the impairment of ubiquitin-proteasome and autophagy-lysosome pathways induced by ethanol treatment in brain

New evidence indicates the involvement of protein degradation dysfunctions in neurodegeneration, innate immunity response and alcohol hepatotoxicity. We recently demonstrated that ethanol increases brain proinflammatory mediators and causes brain damage by activating Toll-like receptor 4 (TLR4) signaling in glia. However, it is uncertain if the ubiquitin-proteasome and autophagy-lysosome pathways are involved in ethanol-induced brain damage and whether the TLR4 response is implicated in proteolytic processes. Using the cerebral cortex of WT and TLR4-knockout mice with and without chronic ethanol treatment, we demonstrate that ethanol induces poly-ubiquitinated proteins accumulation and promotes immunoproteasome activation by inducing the expression of β2i, β5i and PA28α, although it decreases the 20S constitutive proteasome subunits (α2, β5). Ethanol also upregulates mTOR phosphorylation, leading to a downregulation of the autophagy-lysosome pathway (ATG12, ATG5, cathepsin B, p62, LC3) and alters the volume of autophagic vacuoles. Notably, mice lacking TLR4 receptors are protected against ethanol-induced alterations in protein degradation pathways. In summary, the present results provide the first evidence demonstrating that chronic ethanol treatment causes proteolysis dysfunctions in the mouse cerebral cortex and that these events are TLR4 dependent. These findings could provide insight into the mechanisms underlying ethanol-induced brain damage.     

The Alpha-Melanocyte-Stimulating Hormone Suppresses TLR2-Mediated Functional Responses through IRAK-M in Normal Human Keratinocytes

Alpha-melanocyte stimulating hormone (α-MSH) is a highly conserved 13-aa neuropeptide derived from pro-opiomelanocortin by post-translational processing, which has been reported to exhibit potent anti-inflammatory activity and a wide range of immunosuppressive activities in the skin. However, the regulatory effect of α-MSH is not completely clear in cutaneous innate immunity. In this study, we investigate the functional regulation of α-MSH in TLR2-mediated inflammatory responses in normal human keratinocytes (HKs). α-MSH pretreatment down-regulated the Staphylococcus aureus LTA-induced expression of both TLR2 and IL-8 as well as NF-κB nuclear translocation in HK cells. The inhibitory effect of α-MSH was blocked by agouti signaling protein (ASP), an α-MSH receptor-1 antagonist. To investigate the mechanism of this response in more detail, siRNA of IRAK-M, a negative regulator of TLR signaling, was utilized in these studies. The α-MSH suppressive effect on IL-8 production and NF-κB transactivation was inhibited by IRAK-M siRNA transfection in HK cells. These results indicate that α-MSH is capable of suppressing keratinocyte TLR2-mediated inflammatory responses induced by S. aureus-LTA, thus demonstrating another novel immunomodulatory activity of α-MSH in normal human keratinocytes.     

Bruton's Tyrosine Kinase and Protein Kinase C μ Are Required for TLR7/9-Induced IKKα and IRF-1 Activation and Interferon-β Production in Conventional Dendritic Cells

Stimulation of TLR7/9 by their respective ligands leads to the activation of IκB kinase α (IKKα) and Interferon Regulatory Factor 1 (IRF-1) and results in interferon (IFN)-β production in conventional dendritic cells (cDC). However, which other signaling molecules are involved in IKKα and IRF-1 activation during TLR7/9 signaling pathway are not known. We and others have shown that Bruton''s Tyrosine Kinase (BTK) played a part in TLR9-mediated cytokine production in B cells and macrophages. However, it is unclear if BTK participates in TLR7/9-induced IFN-β production in cDC. In this study, we show that BTK is required for IFN-β synthesis in cDC upon TLR7/9 stimulation and that stimulated BTK-deficient cDC are defective in the induction of IKKα/β phosphorylation and IRF-1 activation. In addition, we demonstrate that Protein Kinase C µ (PKCµ) is also required for TLR7/9-induced IRF-1 activation and IFN-β upregulation in cDC and acts downstream of BTK. Taken together, we have uncovered two new molecules, BTK and PKCµ, that are involved in TLR7/9-triggered IFN-β production in cDC.     

Antinociceptive Effect of Tetrandrine on LPS-Induced Hyperalgesia via the Inhibition of IKKβ Phosphorylation and the COX-2/PGE2 Pathway in Mice

Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid that is isolated from the Stephania Tetrandra. It is known to possess anti-inflammatory and immunomodulatory effects. We have shown that TET can effectively suppress the production of bacterial lipopolysaccharide (LPS)-induced inflammatory mediators, including cyclooxygenases (COXs), in macrophages. However, whether TET has an antinociceptive effect on LPS-induced hyperalgesia is unknown. In the present study, we investigated the potential antinociceptive effects of TET and the mechanisms by which it elicits its effects on LPS-induced hyperalgesia. LPS effectively evoked hyperalgesia and induced the production of PGE₂ in the sera, brain tissues, and cultured astroglia. TET pretreatment attenuated all of these effects. LPS also activated inhibitor of κB (IκB) kinase β (IKKβ) and its downstream components in the IκB/nuclear factor (NF)-κB signaling pathway, including COX-2; the increase in expression levels of these components was significantly abolished by TET. Furthermore, in primary astroglia, knockdown of IKKβ, but not IKKα, reversed the effects of TET on the LPS-induced increase in IκB phosphorylation, P65 phosphorylation, and COX-2. Our results suggest that TET can effectively exert antinociceptive effects on LPS-induced hyperalgesia in mice by inhibiting IKKβ phosphorylation, which leads to the reduction in the production of important pain mediators, such as PGE₂ and COX-2, via the IKKβ/IκB/NF-κB pathway.     

Entamoeba histolytica Cysteine Proteinase 5 Binds Integrin on Colonic Cells and Stimulates NFκB-mediated Pro-inflammatory Responses

Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 10⁵ deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to α_Vβ₃ integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to α_Vβ₃ integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2^−/− mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for α_Vβ₃ integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.     

Endotoxin Exposure during Sensitization to Blomia tropicalis Allergens Shifts TH2 Immunity Towards a TH17-Mediated Airway Neutrophilic Inflammation: Role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.     

Negative Regulation of TLR Inflammatory Signaling by the SUMO-deconjugating Enzyme SENP6

The signaling of Toll-like receptors (TLRs) induces host defense against microbial invasion. Protein posttranslational modifications dynamically shape the strength and duration of the signaling pathways. It is intriguing to explore whether de-SUMOylation could modulate the TLR signaling. Here we identified SUMO-specific protease 6 (SENP6) as an intrinsic attenuator of the TLR-triggered inflammation. Depletion of SENP6 significantly potentiated the NF-κB-mediated induction of the proinflammatory genes. Consistently, SENP6-knockdown mice were more susceptible to endotoxin-induced sepsis. Mechanistically, the small ubiquitin-like modifier 2/3 (SUMO-2/3) is conjugated onto the Lysine residue 277 of NF-κB essential modifier (NEMO/IKKγ), and this impairs the deubiquitinase CYLD to bind NEMO, thus strengthening the inhibitor of κB kinase (IKK) activation. SENP6 reverses this process by catalyzing the de-SUMOylation of NEMO. Our study highlights the essential function of the SENP family in dampening TLR signaling and inflammation.     

Involvement of DNA-PKcs in the Type I IFN Response to CpG-ODNs in Conventional Dendritic Cells in TLR9-Dependent or -Independent Manners

CpG-ODNs activate dendritic cells (DCs) to produce interferon alpha (IFNα) and beta (IFNβ). Previous studies demonstrated that Toll-like receptor 9 (TLR9) deficient DCs exhibited a residual IFNα response to CpG-A, indicating that yet-unidentified molecules are also involved in induction of IFNα by CpG-A. Here, we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) but not Ku70 deficient BMDCs showed defective IFNα and IFNβ responses to CpG-A or CpG-B. Loss of both DNA-PKcs and TLR9 further reduced the IFNα response to CpG-A. These DNA-PKcs and TLR9 effects were mediated by their downstream Akt/mTORC1 pathway and downstream events IRAK1 and IKKα. Loss of DNA-PKcs, TLR9, MyD88 or IRAK4 impaired phosphorylation of Akt(S473), S6K, S6, IRAK1, or IKKα in BMDCs in response to CpG-ODNs. The residual IFNα and IFNβ in DNA-PKcs-deficient BMDCs were partially responsible for the induction of IL-6 and IL-12 by CpG-ODNs and their stimulatory effect was blocked by IFNAR1 neutralizing antibodies. Further analysis indicated that CpG-ODN associated with DNA-PKcs and Ku70, and induced DNA-PKcs’s interaction with TRAF3. Intriguingly, DNA-PKcs but not Ku70 expression level was reduced in TLR9-deficient BMDCs. Taken together, our data suggest that DNA-PKcs is an important mediator in the type I IFN response to CpG-ODNs in TLR9-dependent or -independent fashions.     

Dose-Dependent Effects of Lactobacillus rhamnosus on Serum Interleukin-17 Production and Intestinal T-Cell Responses in Pigs Challenged with Escherichia coli

The mechanism underlying the dose effect of probiotics on ameliorating diarrhea has not been fully elucidated. Here, low (1 × 10⁹ CFU/ml) or high (1 × 10¹¹ CFU/ml) doses of Lactobacillus rhamnosus ATCC 7469 were administered orally to piglets for 1 week before F4 (K88)-positive enterotoxigenic Escherichia coli (F4⁺ ETEC) challenge. Administration of a low, but not a high, dose of L. rhamnosus decreased the percentage of CD3⁺ CD4⁺ CD8⁻ T cells in the peripheral blood. Notably, transiently increased serum concentrations of interleukin-17A (IL-17A) were observed after F4⁺ ETEC challenge in pigs pretreated with a high dose of L. rhamnosus. Administration of L. rhamnosus increased the percentage of the small intestinal lamina propria CD3⁺ CD4⁺ CD8⁻ cells and Peyer''s patch CD3⁺ CD4⁻ CD8⁻ and CD3⁻ CD4⁻ CD8⁺ cells. The percentage of ileal intraepithelial CD3⁺ CD4⁻ CD8⁺ cells increased only in the high-dose piglets. Administration of L. rhamnosus downregulated expression of ileal IL-17A after F4⁺ ETEC challenge but had no effect on expression of gamma interferon (IFN-γ), IL-12, IL-4, and FOXP3 mRNA in the small intestine. Expression of jejunal IL-2, ileal transforming growth factor β1 (TGF-β1), and ileal IL-10 was upregulated in the low-dose piglets after F4⁺ ETEC challenge. Our findings suggest that amelioration of infectious diarrhea in piglets by L. rhamnosus is associated with the generation of lamina propria CD3⁺ CD4⁺ CD8⁻ T cells, the expansion of Peyer''s patch CD3⁺ CD4⁻ CD8⁻ and CD3⁻ CD4⁻ CD8⁺ cells, and the attenuation of F4⁺ ETEC-induced increase in CD3⁺ CD4⁺ CD8⁺ T cells in the small intestine. However, consumption of high doses of L. rhamnosus may increase levels of serum IL-17A after F4⁺ ETEC challenge, thus eliciting a strong proinflammatory response.     

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4^lps-d), mice deficient in TLR9 (TLR9^−/−) and TLR4/9 double mutant mice (TLR4^lps-d/TLR9^−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4^lps-d/TLR9^−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4^lps-d/TLR9^−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.     

Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection

Dengue virus (DV) infection is the most prevalent mosquito-borne viral disease and its manifestation has been shown to be contributed in part by the host immune responses. In this study, pathogen recognition receptors, Toll-like receptor (TLR) 2 and TLR6 were found to be up-regulated in DV-infected human PBMC using immunofluorescence staining, flow cytometry and Western blot analyses. Using ELISA, IL-6 and TNF-α, cytokines downstream of TLR2 and TLR6 signaling pathways were also found to be up-regulated in DV-infected PBMC. IL-6 and TNF-α production by PBMC were reduced when TLR2 and TLR6 were blocked using TLR2 and TLR6 neutralizing antibodies during DV infection. These results suggested that signaling pathways of TLR2 and TLR6 were activated during DV infection and its activation contributed to IL-6 and TNF-α production. DV NS1 protein was found to significantly increase the production of IL-6 and TNF-α when added to PBMC. The amount of IL-6 and TNF-α stimulated by DV NS1 protein was reduced when TLR2 and TLR6 were blocked, suggesting that DV NS1 protein is the viral protein responsible for the activation of TLR2 and TLR6 during DV infection. Secreted alkaline phosphatase (SEAP) reporter assay was used to further confirm activation of TLR2 and TLR6 by DV NS1 protein. In addition, DV-infected and DV NS1 protein-treated TLR6^-/- mice have higher survivability compared to DV-infected and DV NS1 protein-treated wild-type mice. Hence, activation of TLR6 via DV NS1 protein could potentially play an important role in the immunopathogenesis of DV infection.     

TLR4 and NKT Cell Synergy in Immunotherapy against Visceral Leishmaniasis

NKT cells play an important role in autoimmune diseases, tumor surveillance, and infectious diseases, providing in most cases protection against infection. NKT cells are reactive to CD1d presented glycolipid antigens. They can modulate immune responses by promoting the secretion of type 1, type 2, or immune regulatory cytokines. Pathogen-derived signals to dendritic cells mediated via Toll like Receptors (TLR) can be modulated by activated invariant Natural Killer T (iNKT) cells. The terminal β-(1–4)-galactose residues of glycans can modulate host responsiveness in a T helper type-1 direction via IFN-γ and TLRs. We have attempted to develop a defined immunotherapeutic, based on the cooperative action of a TLR ligand and iNKT cell using a mouse model of visceral leishmaniasis. We evaluated the anti-Leishmania immune responses and the protective efficacy of the β-(1–4)-galactose terminal NKT cell ligand glycosphingophospholipid (GSPL) antigen of L. donovani parasites. Our results suggest that TLR4 can function as an upstream sensor for GSPL and provoke intracellular inflammatory signaling necessary for parasite killing. Treatment with GSPL was able to induce a strong effective T cell response that contributed to effective control of acute parasite burden and led to undetectable parasite persistence in the infected animals. These studies for the first time demonstrate the interactions between a TLR ligand and iNKT cell activation in visceral leishmaniasis immunotherapeutic.