Expression of toll-like receptors by human muscle cells in vitro and in vivo:TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release and up-regulation of NKG2D-ligands

The particular microenvironment of the skeletal muscle can be the site of complex immune reactions. Toll-like receptors (TLRs) mediate inflammatory stimuli from pathogens and endogenous danger signals and link the innate and adaptive immune system. We investigated innate immune responses in human muscle. Analyzing TLR1-9 mRNA in cultured myoblasts and rhabdomyosarcoma cells, we found constitutive expression of TLR3. The TLR3 ligand Poly (I:C), a synthetic analog of dsRNA, and IFN-gamma increased TLR3 levels. TLR3 was mainly localized intracellularly and regulated at the protein level. Poly (I:C) challenge 1) activated nuclear factor-kappaB (NF-kappaB), 2) increased IL-8 release, and 3) up-regulated NKG2D ligands and NK-cell-mediated lysis of muscle cells. We examined muscle biopsy specimens of 6 HIV patients with inclusion body myositis/polymyositis (IBM/PM), 7 cases of sporadic IBM and 9 nonmyopathic controls for TLR3 expression. TLR3 mRNA levels were elevated in biopsy specimens from patients with IBM and HIV-myopathies. Muscle fibers in inflammatory myopathies expressed TLR3 in close proximity of infiltrating mononuclear cells. Taken together, our study suggests an important role of TLR3 in the immunobiology of muscle, and has substantial implications for the understanding of the pathogenesis of inflammatory myopathies or therapeutic interventions like vaccinations or gene transfer.

Expression of Toll-Like Receptors 2 and 4 is Upregulated During Hospital Admission in Traumatic Patients:Lack of Correlation with Blunted Innate Immune Responses

Background: There are reports with conflicting results on the expression of toll-like receptors (TLRs) in trauma patients. In addition, these studies analyzed TLR expression only at patients hospital admission but not later when complications usually arise. Objectives: To analyze the surface expression of TLR2 and TLR4 on circulating monocytes from trauma patients during the hospitalization period and to correlate this with cytokine production after stimulation with TLR2 and TLR4 agonists. The phagocytic capacity of monocytes was analyzed at the same time points of TLR expression analysis; to correlate these molecular findings with the presence or absence of infections. Methods: Prospective and observational study from June 2005 to June 2007. In all analysis, a control group composed of healthy subjects was included. Results: We studied 70 trauma patients admitted to the intensive care unit (ICU) of a tertiary hospital, and 30 healthy volunteers. Blood samples were collected at hospital admission, on day 7 and 14. Forty-four patients (63%) developed at least one episode of infection. Monocytes from trauma patients expressed higher levels of TLR2 and TLR4 than monocytes from control subjects at all time points. Expression of TLR2 and TLR4 in monocytes from those patients who developed any infection was significantly lower than in those patients without infection but still significantly higher than in control subjects. Cellular responses to TLR4 agonist were impaired. Monocytes from traumatic patients phagocytosized less efficiently than monocytes from control subjects. Conclusions: These results indicate that trauma patients present a dysregulation of the innate immune system that persists during the first 14 days after hospital admission. Copyright © 2010 by Lippincott Williams & Wilkins.

Klebsiella pneumoniae increases the levels of Toll-like receptors 2 and 4 in human airway epithelial cells

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IkappaBalpha-dependent NF-kappaBeta pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells

The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-?B. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF.

Glutamine as a modulator of the immune system of critical care patients:effect on Toll-like receptor expression. A preliminary study

We evaluated the expression of Toll-like receptors 2 and 4 (TLR-2 and TLR-4) in circulating monocytes from peripheral blood of critical care patients treated with and without glutamine. Because no research has been published to date on the effect of glutamine on TLR receptors in critical patients, it was determined in an initial sample of 30 patients.

Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterised by pulmonary and systemic inflammation which flare-up during episodes of acute exacerbation (AECOPD). Given the role of Toll-like receptors (TLRs) in the induction of inflammatory responses we investigated the involvement of TLRs in COPD pathogenesis.

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Type-I interferons induce lung protease responses following respiratory syncytial virus infection via RIG-I-like receptors

The role of proteases in viral infection of the lung is poorly understood. Thus, we examined matrix metalloproteinases (MMPs) and cathepsin proteases in respiratory syncytial virus (RSV)-infected mouse lungs. RSV-induced gene expression for MMPs -2, -3, -7, -8, -9, -10, -12, -13, -14, -16, -17, -19, -20, -25, -27, and -28 and cathepsins B, C, E, G, H, K, L1, S, W, and Z in the airways of Friend leukemia virus B sensitive strain mice. Increased proteases were present in the bronchoalveolar lavage fluid (BALF) and lung tissue during infection. Mitochondrial antiviral-signaling protein (MAVS) and TIR-domain-containing adapter-inducing interferon-β-deficient mice were exposed to RSV. Mavs-deficient mice had significantly lower expression of airway MMP-2, -3, -7, -8, -9, -10, -12, -13, and -28 and cathepsins C, G, K, S, W, and Z. In lung epithelial cells, retinoic acid-inducible gene-1 (RIG-I) was identified as the major RIG-I-like receptor required for RSV-induced protease expression via MAVS. Overexpression of RIG-I or treatment with interferon-β in these cells induced MMP and cathepsin gene and protein expression. The significance of RIG-1 protease induction was demonstrated by the fact that inhibiting proteases with batimastat, E64 or ribavirin prevented airway hyperresponsiveness and enhanced viral clearance in RSV-infected mice.

Foxp3+ T cells induce Perforin-Dependent Dendritic Cell Death in Tumor-Draining Lymph Nodes.

Regulatory T (Treg) cells limit the onset of effective antitumor immunity, through yet-ill-defined mechanisms. We showed the rejection of established ovalbumin (OVA)-expressing MCA101 tumors required both the adoptive transfer of OVA-specific CD8(+) T cell receptor transgenic T cells (OTI) and the neutralization of Foxp3(+) T cells. In tumor-draining lymph nodes, Foxp3(+) T cell neutralization induced a marked arrest in the migration of OTI T cells, increased numbers of dendritic cells (DCs), and enhanced OTI T cell priming. Using an in vitro cytotoxic assay and two-photon live microscopy after adoptive transfer of DCs, we demonstrated that Foxp3(+) T cells induced the death of DCs in tumor-draining lymph nodes, but not in the absence of tumor. DC death correlated with Foxp3(+) T cell-DC contacts, and it was tumor-antigen and perforin dependent. We conclude that Foxp3(+) T cell-dependent DC death in tumor-draining lymph nodes limits the onset of CD8(+) T cell responses.

Altered Toll-like Receptor 2-mediated Endotoxin Tolerance Is Related to Diminished Interferon β Production

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-alpha and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-alpha after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-alpha but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-kappa B activation, whereas TNF-alpha expression is blocked at the gene transcription level. Interferon beta plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon beta. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon beta and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Killer immunoglobulin-like Receptors (KIR) haplogroups A and B track with Natural Killer Cells and Cytokine Profile in Aged Subjects: Observations from Octo/Nonagenarians in the Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST)

Background: Natural Killer Cells (NK) play an important role in detection and elimination of virus-infected, damaged or cancer cells. NK cell function is guided by expression of Killer Immunoglobulin-like Receptors (KIRs) and contributed to by the cytokine milieu. KIR molecules are grouped on NK cells into stimulatory and inhibitory KIR haplotypes A and B, through which NKs sense and tolerate HLA self-antigens or up-regulate the NK-cytotoxic response to cells with altered HLA self-antigens, damaged by viruses or tumours. We have previously described increased numbers of NK and NK-related subsets in association with sIL-2R cytokine serum levels in BELFAST octo/nonagenarians. We hypothesised that changes in KIR A and B haplotype gene frequencies could explain the increased cytokine profiles and NK compartments previously described in Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST) octo/nonagenarians, who show evidence of ageing well.

Results: In the BELFAST study, 24% of octo/nonagenarians carried the KIR A haplotype and 76% KIR B haplotype with no differences for KIR A haplogroup frequency between male or female subjects (23% v 24%; p=0.88) or for KIR B haplogroup (77% v 76%; p=0.99). Octo/nonagenarian KIR A haplotype carriers showed increased NK numbers and percentage compared to Group B KIR subjects (p=0.003; p=0.016 respectively). There were no KIR A/ B haplogroup-associated changes for related CD57+CD8 (high or low) subsets. Using logistic regression, KIR B carriers were predicted to have higher IL-12 cytokine levels compared to KIR A carriers by about 3% (OR 1.03, confidence limits CI 0.99–1.09; p=0.027) and 14% higher levels for TGF-ß (active), a cytokine with an anti-inflammatory role, (OR 1.14, confidence limits CI 0.99–1.09; p=0.002).

Conclusion: In this observational study, BELFAST octo/nonagenarians carrying KIR A haplotype showed higher NK cell numbers and percentage compared to KIR B carriers. Conversely, KIR B haplotype carriers, with genes encoding for activating KIRs, showed a tendency for higher serum pro-inflammatory cytokines compared to KIR A carriers. While the findings in this study should be considered exploratory they may serve to stimulate debate about the immune signatures of those who appear to age slowly and who represent a model for good quality survivor-hood.© 2013 Rea et al.; licensee BioMed Central Ltd.

RIP kinases: key decision makers in cell death and innate immunity

Innate immunity represents the first line of defence against invading pathogens. It consists of an initial inflammatory response that recruits white blood cells to the site of infection in an effort to destroy and eliminate the pathogen. Some pathogens replicate within host cells, and cell death by apoptosis is an important effector mechanism to remove the replication niche for such microbes. However, some microbes have evolved evasive strategies to block apoptosis, and in these cases host cells may employ further countermeasures, including an inflammatory form of cell death know as necroptosis. This review aims to highlight the importance of the RIP kinase family in controlling these various defence strategies. RIP1 is initially discussed as a key component of death receptor signalling and in the context of dictating whether a cell triggers a pathway of pro-inflammatory gene expression or cell death by apoptosis. The molecular and functional interplay of RIP1 and RIP3 is described, especially with respect to mediating necroptosis and as key mediators of inflammation. The function of RIP2, with particular emphasis on its role in NOD signalling, is also explored. Special attention is given to emphasizing the physiological and pathophysiological contexts for these various functions of RIP kinases.