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Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-β (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNβ, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways. [ABSTRACT FROM AUTHOR]

The cornea must maintain homeostasis, enabling rapid response to injury and microbial insult, to protect the eye from insult and infection. Toll-like receptors (TLRs) are critical to this innate immune response through the recognition and response to pathogens. Myeloid differentiation primary response (MyD88) is a key signaling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-mediated immune defense and has been shown to be necessary for corneal defense during infection. Here, we examined the intrinsic role of TLR signaling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88-/-). In addition, cytokine, chemokine, and matrix metalloproteinase (MMP) expression was determined in ocular surface cells exposed to a panel of TLR agonists. Compared to wild-type (WT) animals, MyD88-/- mice expressed lower MMP-9 levels in the cornea and conjunctiva. Corneal IL-1α, TNFα, and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were also significantly reduced. Additionally, CXCL1 and RANTES expression was lower in both MyD88-/- tissues compared to WT and IL-1R-/- mice. Interestingly, MyD88-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm2) than both WT (0.59±0.16 gm/mm2) and IL-1R-/- (0.52±0.08 gm/mm2). Following Pseudomonas aeruginosa challenge, MyD88-/- mice had better clinical scores (0.5±0.0) compared to IL-1R-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal bacterial isolates. However, no signs of infection were detected in inoculated uninjured corneas from either MyD88 or IL-1R-deficient mice. This work furthers our understanding of the importance of TLR signaling in corneal defense and immune homeostasis, showing that a lack of MyD88 may compromise the baseline innate response to insult. [ABSTRACT FROM AUTHOR]

Immunomodulating proteins and peptides are formed during the hydrolysis of cow’s milk proteins. These proteins are potential ingredients in functional foods used for the management of a range of immune related problems, both in infants and adults. However, the mechanism behind these effects is unknown. We hypothesize that the interaction of peptides with Toll-like receptors (TLRs) can induce immune effects, since these receptors are known to sample many dietary molecules in the intestine in order to regulate immune effects. To investigate this, we compared the immune effects and TLR activation and inhibition by whey and casein hydrolysates with different hydrolysis levels. We first measured cytokine production in primary peripheral blood mononuclear cells stimulated with either whey, casein, or their hydrolysates. IL-10 and TNFα were induced by whey hydrolysates (decreasing with increasing hydrolysis level), but not by casein hydrolysates. Next, the activation of TLR 2, 3, 5 and 9 receptors were observed by intact and mildly hydrolysed whey proteins only and not by casein hydrolysates in TLR reporter cell lines. Many casein hydrolysates inhibited TLR signaling (mainly TLR 5 and 9). These results demonstrate that the effects of cow’s milk proteins on the immune system are protein type and hydrolysis dependent. TLR signaling is suggested as a possible mechanism for differences in effect. This knowledge contributes to a better understanding of the immune effects of hydrolysates and the design of infant formula, and nutrition in general, with specific immunoregulatory effects. [ABSTRACT FROM AUTHOR]

Esophageal adenocarcinoma (EAC) develops in an inflammatory microenvironment with reduced microbial diversity, but mechanisms for these influences remain poorly characterized. We hypothesized that mutations targeting the Toll-like receptor (TLR) pathway could disrupt innate immune signaling and promote a microenvironment that favors tumorigenesis. Through interrogating whole genome sequencing data from 171 EAC patients, we showed that non-synonymous mutations collectively affect the TLR pathway in 25/171 (14.6%, PathScan p = 8.7x10-5) tumors. TLR mutant cases were associated with more proximal tumors and metastatic disease, indicating possible clinical significance of these mutations. Only rare mutations were identified in adjacent Barrett’s esophagus samples. We validated our findings in an external EAC dataset with non-synonymous TLR pathway mutations in 33/149 (22.1%, PathScan p = 0.05) tumors, and in other solid tumor types exposed to microbiomes in the COSMIC database (10,318 samples), including uterine endometrioid carcinoma (188/320, 58.8%), cutaneous melanoma (377/988, 38.2%), colorectal adenocarcinoma (402/1519, 26.5%), and stomach adenocarcinoma (151/579, 26.1%). TLR4 was the most frequently mutated gene with eleven mutations in 10/171 (5.8%) of EAC tumors. The TLR4 mutants E439G, S570I, F703C and R787H were confirmed to have impaired reactivity to bacterial lipopolysaccharide with marked reductions in signaling by luciferase reporter assays. Overall, our findings show that TLR pathway genes are recurrently mutated in EAC, and TLR4 mutations have decreased responsiveness to bacterial lipopolysaccharide and may play a role in disease pathogenesis in a subset of patients. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS)-binding protein (LBP) as an acute-phase protein plays a crucial role in innate host response to bacterial challenge. Our previous study shows that LBP expression in human gingiva is associated with periodontal status. Porphyromonas gingivalis is a keystone periodontopathogen, and its LPS with lipid A structural heterogeneity critically accounts for periodontal pathogenesis. This study investigated the effects of LBP and its interactions with two featured isoforms of P. gingivalis LPS (tetra-acylated LPS1435/1449 and penta-acylated LPS1690) on the expression of pro-inflammatory cytokines in human oral keratinocytes (HOKs), and the involvement of Toll-like receptor (TLR) signaling. HOKs were pre-incubated with recombinant human LBP (rhLBP) at 10ng/ml, 100ng/ml and 1μg/ml for 1 h, followed by the treatment of P. gingivalis LPS1690 or LPS1435/1449 for 3h or 24h respectively. The expression of IL-6 and IL-8, and involvements of TLR2 and TLR4 were analyzed. The genes associated with TLR signaling were assessed by PCR array. Interestingly, rhLBP per se significantly up-regulated the expression of IL-6 and IL-8 in HOKs (p<0.05), which was blocked by TLR2 antibody (p<0.001). LPS1435/1449 down-regulated more significantly rhLBP-induced IL-6 and IL-8 mRNAs with reference to P. gingivalis LPS1690 (approximately 80% vs. 40%, p<0.05; and 90% vs. 36%, p<0.001, respectively). Moreover, rhLBP markedly down-regulated the gene expression of TLRs and their adaptors such as CD180 (-2.44 folds) and MD-1 (-9.62 folds), while the interaction of P. gingivalis LPS1435/1449 with rhLBP greatly up-regulated both transcripts (7.11 and 4.05 folds, respectively). Notably, P. gingivalis LPS1690-rhLBP interaction dramatically up-regulated CD180 transcript (20.86 folds) and significantly down-regulated MD-1 transcript (-6.93 folds). This pioneering study shows that rhLBP enables to enhance the expression of pro-inflammatory cytokines in HOKs through TLR2 signaling pathway. P. gingivalis LPS with different lipid A structures down-regulates to different extents rhLBP-induced cytokine expression, possibly through fine-tuning of the CD180-MD1 complex and relevant TLRs. [ABSTRACT FROM AUTHOR]

The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS) in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2’-5’ oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence. [ABSTRACT FROM AUTHOR]

Background: Toll-like receptor (TLR) signalling begins early in subarachnoid haemorrhage (SAH), and plays a key role in inflammation following cerebral aneurysm rupture. Available studies suggest significance of endogenous first-line blockers of a TLR pathway—soluble TLR2 and 4. Methods: Eighteen patients with SAH and acute hydrocephalus underwent endovascular coiling and ventriculostomy; sTLR2 and 4 levels were assayed in cerebrospinal fluid (CSF) collected on post-SAH days 0–3, 5, and 10–12. Release kinetics were defined. CSF levels of sTLR2 and 4 were compared with a control group and correlated with the clinical status on admission, the findings on imaging, the degree of systemic inflammation and the outcome following treatment. Results: None of study group showed detectable levels of sTLR2 and 4 on post-SAH day 0–3. 13 patients showed increased levels in subsequent samples. In five SAH patients sTLR2 and 4 levels remained undetectable; no distinctive features of this group were found. On post-SAH day 5 the strongest correlation was found between sTLR2 level and haemoglobin level on admission (cc = -0.498, P = 0.037). On post-SAH day 10–12 the strongest correlation was revealed between sTLR2 and treatment outcome (cc = -0.501, P = 0.076). Remaining correlations with treatment outcome, status at admission, imaging findings and inflammatory markers on post-SAH day 5 and 10–12 were negligible or low (-0.5 ≤ cc ≤ 0.5). Conclusions: In the majority of cases, rupture of a cerebral aneurysm leads to delayed release of soluble TLR forms into CSF. sTLR2 and 4 seem to have minor role in human post-SAH inflammation due to delayed release kinetics and low levels of these protein. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease, which can lead to fatal neurological complications in young children and infants. Few gastrointestinal symptoms are observed clinically, suggesting the presence of a unique immunity to EV71 in the gut. We reported a robust induction of interferons (IFNs) in human intestinal epithelial cells (HT-29), which was suppressed in other types such as RD and HeLa cells. The underlying mechanism for the apparent difference remains obscure. In this study we report that in EV71-infected HT-29 cells, TLR/TRIF signaling was essential to IFN induction; viral replication increased and the induction of IFN-α, -β, -ω, -κ, and -ε decreased markedly in TRIF-silenced HT-29 cells. Importantly, TRIF was degraded by viral 3Cpro in RD cells, but resisted cleavage, and IRF3 was activated and translocated into the nucleus in HT-29 cells. Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection. [ABSTRACT FROM AUTHOR]