Role Of TLR4 Research Articles

Association between Toll-Like Receptor 4 (Thr399Ile and Asp299Gly), Toll-Like Receptor 2 196-174 Deletion Polymorphisms with cryptosporidiosis

Toll-like receptors (TLRs) 2 and 4 specifically recognize lipopolysaccharide motifs on surfaces of microorganisms. Polymorphims in the TLR genes result in structural variations in the proteins, abnormal host-pathogen interactions and hence, altered immune response. Opportunistic parasite Cryptosporidium spp. infects immuno-compromised patients who present with heterogeneous clinical manifestations ranging from mild infection resolving in a few weeks, to severe form characterized by voluminous diarrhoea, prolonged symptoms and recurrences. The present study investigates the role of TLR2 196_174del, TLR4 Asp299Gly and Thr399Ile polymorphisms in pathogenesis of cryptosporidiosis. Stool samples were collected from 210 immuno-compromised (renal transplant recipients and patients with Human immunodeficiency virus infection) both with and without cryptosporidiosis, and 200 healthy subjects for detection of Cryptosporidium spp. Blood samples were also collected from the patients and healthy subjects for genotyping of Δ22 (TLR2 196_174del) polymorphism, A896G (TLR4 Asp299Gly) and C1196T (Thr399Ile) single nucleotide polymorphisms (SNPs) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Cryptosporidium spp. was detected in 70 immunocompromised patients, while it was absent in all the healthy controls. No significant difference was observed in Δ22, A896G and C1196T alleles and genotypes (P > 0.05), between cases and controls. Thus, TLR2 196_174del, TLR4 Asp299Gly and Thr399Ile polymorphisms are not significantly associated with the pathogenesis or progression of cryptosporidiosis, in the Indian population included in the study.

TLR3 or TLR4 Activation Enhances MSC-Mediated Treg Generation Via Notch Signaling

Mesenchymal stromal cells (MSCs) are used as cell therapy for a variety of disorders, largely because of their immunosuppressive and regenerative functions by exerting immune effects via direct and indirect interactions with many types of immune cells. MSCs recruit and promote the generation of regulatory T cells (Tregs) both in vitro and in vivo. Toll-like receptors (TLRs), known for roles in innate and adaptive immunity, are involved in numerous pathological conditions, including graft-versus-host disease (GVHD). Several TLRs, especially TLR3 and TLR4, are highly expressed on MSCs and affect immunomodulatory functions and possibly, therapeutic potency. Indeed, two distinct anti- and pro-inflammatory MSC phenotypes have been reported after activation of TLR3 and TLR4, respectively. The role of TLRs on MSC-mediated Treg generation, however, is not known.In this study, we investigated the role of TLR3 and TLR4 in the MSC-mediated generation of Tregs in an allogeneic co-culture model. Data for each experiment were collected from 1 PBMC donor and 3 MSC donors. We found that pre-activation of TLR3 and TLR4 by their ligands (poly I:C for TLR3, LPS for TLR4) enhanced the generation of Tregs by MSCs: 1.2 ± 0.2% in CD4+ cells cultured alone, 3.9 ± 0.3% in co-culture with control MSCs, 6.04 ± 0.1% in co-culture with TLR3-activated MSCs and 6.6 ± 0.4% in co-culture with TLR4-activated MSCs. siRNA-mediated silencing of TLR3 and TLR4 reduced Tregs by 51.7% and 61.8% in co-culture with poly I:C- and LPS-primed MSCs, respectively. Treg levels for the poly I:C-activated group were 6.3 ± 0.2% for co-cultures with control MSCs, 5.2 ± 0.3% for MSCs treated with scrambled RNA and 3 ± 0.3% for MSCs treated with TLR3-siRNA. For the LPS-activated group, Treg levels were 6.7 ± 0.3% with control MSCs, 5.7 ± 0.5% with MSCs treated with scrambled RNA and 2.5 ± 0.3% for MSCs treated with TLR4-siRNA. MSC-mediated Treg induction required cell-cell contact as conditioned media (CM) from TLR-activated or control MSCs failed to induce Tregs among CD4+ enriched cells: 4.75 ± 0.1% in direct co-culture vs 2.72 ± 0.3%, P= 0.004 in CM from control MSCs, 6.35 ± 0.2% in direct co-culture vs 2.97 ± 0.2%, P=0.0008 in CM from TLR3-activated MSCs, 6.7 ± 0.3% in direct co-culture vs 3.2 ± 0.3, P=0.001 in CM from the TLR4-activated group. We showed that the notch pathway is activated in CD4+ cells co-cultured with TLR-activated, but not control MSCs, and inhibition of notch signaling reduced MSC-mediated Tregs in co-cultures with TLR3- and TLR4-activated, but not control MSCs: 4.75 ± 0.1% vs 3.76 ± 0.4%, P=0.09 in control MSCs, 6.35 ± 0.2% vs 4.43 ± 0.3%, P=0.012 in TLR3-activated MSCs, 6.7 ± 0.3% vs 3.97 ± 0.1%, P=0.001 in TLR4-activated MSCs. Our data show a new role for TLR3 and TLR4 in the immunoregulatory function of human MSCs, and indicate the involvement of notch signaling as a mechanism for the further induction of Tregs in TLR3- and TLR4-activated MSCs. These studies have implications for the use of TLR-activated MSCs in the enhanced generation of Tregs such as for the treatment of acute GVHD. DisclosuresNo relevant conflicts of interest to declare.

Abstract 12135: Toll-like Receptor Signalling Mediates C-kit+ Cell Migration in vivo Following Ischemia-reperfusion Injury via the Myd88 Pathway

Introduction: Toll-like-receptor (TLR) mediated signalling has been shown to regulate tissue damage following cardiac ischemia-reperfusion (I-R) injury. Whether progenitor cell based cardiac regeneration might also depend on TLR-signaling has not been described conclusively. As sufficient progenitor cell migration represents a crucial step of cell-based therapy protocols, we aimed at analyzing the migratory capacity of c-kit+ bone marrow stem cells in vivo following I-R injury as well as the impact of endothelial TLR's and related signalling pathways (MyD88,TRIF)on c-kit+ cell migration. Methods: Murine c-kit+ stem cells were isolated from the bone marrow. In vivo migration of fluorescently labeled c-kit+ following different extents of local I-R injury was visualized by intravital fluorescence microscopy, utilizing the cremaster muscle model in male mice.C-kit+ cell migration was quantified by analyzing early (endothelial rolling) and late (firm endothelial adhesion) c-kit+ cell interaction with the local endothelium. The role of TLR-2 and TLR-4, as well as MyD88 and TRIF on c-kit+ migration following I-R-injury was analyzed by applying specific knock-out models. Results: A sequence of 15 min ischemia followed by 15 min reperfusion induced relevant interactions of c-kit+ cells with the microvascular endothelium(Firm endothelial adhesion:5.6±1.3 cells/mm2 in control vs. 30.2±10.1 cells/mm2 in IR, p<0.05). Prolonged I-R periods did not result in an additional gain of c-kit+ cell-endothelial cell interaction. Knock-out of TLR-2 and TLR-4 diminished I-R induced c-kit+ - endothelial cell interaction.Subsequent knock-out of downstream adaptor proteins resulted in a significantly decreased firm endothelial c-kit+ cell adhesion only for MyD88 but not for TRIF. Conclusions: Artificially applied c-kit+ cells are capable to interact with the target organ endothelium following I-R injury. C-kit+ cell-endothelial cell interaction following I-R injury depends on TLR associated signaling, with a special relevance being demonstrated here for the MyD88 pathway. Our findings suggest, that therapeutic blockade of TLR signaling following I-R injury for anti-inflammatory purposes might interfere with progenitor cell based therapy protocols.

Abstract 12161: Toll-like Receptor 9 Plays a Pivotal Role in Angiotensin II-induced Atherosclerosis

Background: Chronic vascular inflammation causes atherosclerosis. Toll-like receptor (TLR) 9 recognizes bacterial unmethylated DNA and plays a role in innate defense, whereas it can also provoke inflammation in response to fragmented DNA released from damaged mammalian cells. However, the role of TLR9 in the development of atherosclerosis remains unknown. Here, we investigated whether genetic deletion of TLR9 attenuates atherosclerosis in apolipoprotein E knockout (ApoE KO) mice. Methods and Results: We generated TLR9/ApoE double knockout (dKO) mice by crossing ApoE KO mice and TLR9 KO mice, and infused angiotensin II (Ang II) (1000 ng/kg/min) subcutaneously for 28 days using osmotic pump. There were no differences in blood pressure and plasma lipid levels between ApoE KO mice and TLR9/ApoE dKO mice. Ang II infusion significantly increased plasma levels of single-stranded DNA and double-stranded DNA, endogenous ligands of TLR9, in both strains of mice (P<0.001, respectively). TLR9/ApoE dKO mice showed less atherosclerotic lesion progression in the aortic arch and less lipid accumulation in atherosclerotic plaques in the aortic root compared with ApoE KO mice (P<0.05, respectively). TLR9/ApoE dKO mice also showed less gene expression of inflammatory molecules (e.g., ICAM-1, MCP-1) in the abdominal aorta. The result of cell sorting experiment using atherosclerotic aorta obtained from Ang II-infused ApoE KO mice demonstrated that macrophage population mainly expressed TLR9 in this tissue. We, therefore, performed in vitro experiments using thioglycolate-stimulated peritoneal macrophages. CpG ODN, agonistic oligonucleotide for TLR9, markedly promoted the expression of inflammatory molecules (e.g., ICAM-1 and MCP-1) in wild-type macrophages but not in TLR9 KO macrophages. Furthermore, conditioned medium (CM) of human umbilical vein endothelial cells (HUVECs) treated with Ang II promoted gene expression of ICAM-1 and TNF-α in THP-1 macrophages compared with CM of non-treated HUVECs. Conclusion: Our results suggested that Ang II infusion activates the TLR9 signaling in macrophages, leading to the promotion of vascular inflammation and atherosclerosis. TLR9 may serve as a potential therapeutic target for atherosclerotic diseases.

Candida glabrata induced infection of rat tracheal epithelial cells is mediated by TLR-2 induced activation of NF-κB

An increasing number of reports identified Candida glabrata (C. glabrata) as the important causative agent of invasive pulmonary fungal infection. However, little is known about immune responses to C. glabrata in rat tracheal epithelial cell (RTEC). Here, the effect of C. glabrata on RTEC and the role of TLR-2 and NF-κB in the immune response were investigated by treatment with TLR-2 siRNA and NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), respectively. Our results showed that the knockdown of TLR-2 and pretreatment of PDTC led to inhibition of cell proliferation by C. glabrata, further enhanced cells in G0/G1 phases, and promoted C. glabrata -induced apoptosis. C. glabrata infection induced the expression or secretion of TLR-2, NF-κB, TNF-α, and IL-6, and its effect was inhibited by knockdown of TLR-2. Pretreatment with PDTC inhibited the C. glabrata -induced expression of TLR2, and also inhibited the expression of p65 subunit of NF-κB in the first 4 h. Although the expression of p65 subunit at 6 h was elevated compared to baseline, the C. glabrata -induced expression of TNF-α and IL-6 remained attenuated by PDTC pretreatment. Therefore, C. glabrata recognized the TLR-2 in rat tracheal epithelial cell (RTEC), and then activated the transcription factor NF-κB and further promoted the secretion of TNF-α and IL-6 to contribute to the immune response and inflammation.

Toll-like receptor 2 activators modulate oral tolerance in mice.

SummaryBackgroundToll‐like receptor 2 (TLR2) is a widely expressed pattern recognition receptor critical for innate immunity. TLR2 is also a key regulator of mucosal immunity implicated in the development of allergic disease. TLR2 activators are found in many common foods, but the role of TLR2 in oral tolerance and allergic sensitization to foods is not well understood.ObjectiveThe purpose of this study was to evaluate the impacts of TLR2 expression and TLR2 activation on oral tolerance to food antigens in a murine model.MethodsMice were fed ovalbumin (OVA) or peanut butter with or without the addition of low doses of TLR2 activators Pam3 CSK 4 or FSL‐1. Oral tolerance was assessed by analysing antibody responses after a systemic antigen challenge. OVA‐specific Tregs were assessed in the Peyer's patches, mesenteric lymph nodes, and spleen in wild‐type and TLR2−/− mice. Low‐dose Pam3 CSK 4 was also tested as an oral adjuvant.ResultsOral tolerance was successfully induced in both wild‐type and TLR2−/− recipient mice, with an associated regulatory T‐cell response. Oral TLR2 activation, with low‐dose Pam3 CSK 4 or FSL‐1, during oral antigen exposure was found to alter oral tolerance and was associated with the development of substantial IgE and IgA responses to foods upon systemic challenge. Low‐dose oral Pam3 CSK 4 treatment also selectively enhanced antigen‐specific IgA responses to oral antigen exposure.Conclusions and Clinical Relevance TLR2 is not necessary for oral tolerance induction, but oral TLR2 activation modulates humoral IgE and IgA responses during tolerance development. Low‐dose Pam3 CSK 4 is also an effective oral adjuvant that selectively enhances IgA production. These observations are pertinent to the optimization of oral allergen immunotherapy and oral vaccine development.

Host antioxidant enzymes and TLR-2 neutralization modulate intracellular survival of Staphylococcus aureus: Evidence of the effect of redox balance on host pathogen relationship during acute staphylococcal infection

Staphylococcus aureus is an important pathogen in bone disease and innate immune recognition receptor, TLR-2 is reported to be crucial for inflammatory bone loss. Role of TLR-2 in bacterial clearance and cytokine response to S. aureus infection in murine bone marrow macrophages has been reported but the role of host derived ROS in host–pathogen relationship still remains an obvious question. In the present study, blocking of SOD and catalase in TLR-2 neutralized fresh bone marrow cells (FBMC) with Diethyldithiocarbamic acid (DDC) and 3-Amino-1,2,4-triazole (ATZ), separately, during acute S. aureus infection, produces moderate level of ROS and limits inflammation as compared with only TLR-2 non-neutralized condition and leads to decreased bacterial count compared with only TLR-2 neutralized condition. In summary, host SOD and catalase modulates ROS generation, cytokine levels and TLR-2 expression in FBMCs during acute S. aureus infection which might be useful in the alleviation of S. aureus infection and bone loss.

Toll-like receptor and airway remodeling in asthma

Bronchial asthma is one of the most common chronic respiratory diseases in children.Airway remodeling that makes asthma difficult to control is an important pathological progress of asthma.Toll-like receptors (TLR), a kind of pattern recognition receptors, are expressed in a variety of human airway structure cells, such as airway epithelial cells, smooth muscle cells, mucus gland cells, etc.It is reported that TLRs play important roles in the regulation of airway remodeling in asthma.This article briefly summarizes the role of TLR in airway remodeling of asthma. Key words: Asthma; TLR; Airway remodeling

Flagellin influences the expression of a variety of important cytokines and chemokines without affecting the immune status of umbilical cord mesenchymal stem cells.

Multipotent differentiation ability, low expression of co‑stimulatory molecules and immunosuppressive effects render mesenchymal stem cells (MSCs) the best candidate for use in cell‑based therapy. At present, MSCs are widely used in clinical trials, which have, however, failed to demonstrate any long‑term therapeutic benefits of MSCs. Therefore, to improve the therapeutic potential of MSCs, it is important to understand the underlying mechanisms of the rapid disappearance of MSCs after transplantation. Toll‑like receptors (TLRs) have important roles in the induction of the immune response and the regulation of biological functions. The present study employed TLR5 agonist flagellin in order to assess whether the activation of the TLR5 pathway alters the immune status and influences the differentiation ability of MSCs isolated from umbilical cords (UC). Assessment of the proliferation of peripheral blood mononuclear cells co‑cultured with UCMSCs as well as their lactate dehydrogenase release indicated that flagellin stimulation failed to increase the immunogenicity of UCMSCs. Flow cytometric and histological cell differentiation analyses also confirmed that TLR5 activation neither enhanced the expression of co‑stimulatory molecules (CD80, CD86 and human leukocyte antigen‑E), nor influenced the differentiation ability of UCMSCs. However, polymerase chain reaction analysis indicated the induction of pro‑inflammatory molecules by flagellin, which suggested that the induced immune‑associated molecules may mediate biological functions of UCMSCs other than those assessed by the present study. Future studies should focus on the role of TLR5 in regulating other functions of UCMSCs.

Decreased TLR2 signal expression in peripheral blood mononuclear cell from patients with cryptococcal meningitis.

Toll-like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT-PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti-TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre-incubation of PBMCs from healthy donors with anti-TLR2 led to reduced expression of IFN-γ and IL-12p70, but not of IL-4 and IL-10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response.

Toll-like receptor 8 signaling facilitates West Nile virus infection in mice (VIR9P.1161)

Abstract West Nile virus (WNV) is a neurotropic virus that can cause encephalitis, meningitis or possible death in humans. Human and mouse Toll-like receptor 7 (h/mTLR7) and human Toll-like receptor 8 (hTLR8) detect viral single-stranded (ss)RNA motifs. Importantly, mTLR7 recognizes WNV, as TLR7-/- mice are susceptible to lethal WNV infection. However, the role of TLR8 in WNV pathogenesis is not clear. In this study, we found that mice deficient in TLR8 (TLR8-/-) infected with WNV (2,000 plaque forming units) had improved percentage survival (65%) compared to wild-type (WT) controls (25%), which was coupled to reduced viral burden in the brains of TLR8-/- mice. These data suggest that TLR8 signaling facilities WNV infection in mice. Further mechanistic studies showed that expression of TLR7 and the interferon-induced anti-viral gene Isg-56 (IFIT-1) were significantly increased in TLR8-/- mice whole-brain samples and mixed neuronal cultures following WNV infection, compared to WT controls. These results indicate that TLR8 signaling may down-regulate TLR7 and Isg-56 expression, thus promoting WNV-induced neuroinvasive disease.

TLR2 and TLR9 contribute to alcohol-mediated liver injury through induction of CXCL1 and neutrophil infiltration.

Although previous studies reported the involvement of the TLR4-TRIF pathway in alcohol-induced liver injury, the role of TLR2 and TLR9 signaling in alcohol-mediated neutrophil infiltration and liver injury has not been elucidated. Since alcohol binge drinking is recognized to induce more severe form of alcohol liver disease, we used a chronic-binge ethanol-feeding model as a mouse model for early stage of alcoholic hepatitis. Whereas a chronic-binge ethanol feeding induced alcohol-mediated liver injury in wild-type mice, TLR2- and TLR9-deficient mice showed reduced liver injury. Induction of neutrophil-recruiting chemokines, including Cxcl1, Cxcl2, and Cxcl5, and hepatic neutrophil infiltration were increased in wild-type mice, but not in TLR2- and TLR9-deficient mice. In vivo depletion of Kupffer cells (KCs) by liposomal clodronate reduced liver injury and the expression of Il1b, but not Cxcl1, Cxcl2, and Cxcl5, suggesting that KCs are partly associated with liver injury, but not neutrophil recruitment, in a chronic-binge ethanol-feeding model. Notably, hepatocytes and hepatic stellate cells (HSCs) produce high amounts of CXCL1 in ethanol-treated mice. The treatment with TLR2 and TLR9 ligands synergistically upregulated CXCL1 expression in hepatocytes. Moreover, the inhibitors for CXCR2, a receptor for CXCL1, and MyD88 suppressed neutrophil infiltration and liver injury induced by chronic-binge ethanol treatment. Consistent with the above findings, hepatic CXCL1 expression was highly upregulated in patients with alcoholic hepatitis. In a chronic-binge ethanol-feeding model, the TLR2 and TLR9-dependent MyD88-dependent pathway mediates CXCL1 production in hepatocytes and HSCs; the CXCL1 then promotes neutrophil infiltration into the liver via CXCR2, resulting in the development of alcohol-mediated liver injury.

The role of toll-like receptor 9 in chronic stress-induced apoptosis in macrophage.

Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.

The role of Toll-like receptor 3 and 4 in regulating the function of mesenchymal stem cells isolated from umbilical cord.

Mesenchymal stem cells (MSCs) have been applied to cell-based therapy due to their multiple differentiation ability, the low expression of co-stimulatory molecules and immunosuppressive properties. Despite their immunomodulatory role, the issue of the survival and permanence of MSCs at the site of injury has not yet been fully resolved. Therefore, in order to improve the therapeutic potential of MSCs, it is important to study the mechanisms mediating the relative instability of MSCs in clinical trials. The Toll-like receptors (TLRs) are an important component of innate and adaptive immune responses. In this study, we demonstrate that the activation of two TLRs, namely TLR3 and TLR4, in human umbilical cord-derived MSCs (UCMSCs) induces the expression of inflammatory markers. In addition, as shown by our results, TLR3 upregulated the expression of stem cell markers, while TLR4 downregulated their expression. The upregulation in the expression of the inflammatory markers did not alter the immune status of the UCMSCs or mediate the immune attack of the MSCs by allogeneic immune cells. We found that the activation of TLR3 inhibited the differentiation of UCMSCs into osteocytes, while that of TLR4 increased this differentiation to a certain extent. Taken together, the results of this study provide a new role for TLR3 and TLR4 as regulators of the biological functions of UCMSCs.

Toll-like receptor-associated keratitis and strategies for its management.

Keratitis is an inflammatory condition, characterized by involvement of corneal tissues. Most recurrent challenge of keratitis is infection. Bacteria, virus, fungus and parasitic organism have potential to cause infection. TLR are an important class of protein which has a major role in innate immune response to combat with pathogens. In last past years, extensive research efforts have provided considerable abundance information regarding the role of TLR in various types of keratitis. This paper focuses to review the recent literature illustrating amoebic, bacterial, fungal and viral keratitis associated with Toll-like receptor molecules and summarize existing thoughts on pathogenesis and treatment besides future probabilities for prevention against TLR-associated keratitis.

Toll-like receptor 2 and 6 interdependency in the erosive stage of Staphylococcus aureus induced septic arthritis mediated by IFN-γ and IL-6 – A possible involvement of IL-17 in the progression of the disease

Staphylococcus aureus induced septic arthritis has emerged as a potent disabling and life threatening disease; hence combating this malady has become an imperative need of medical science. Role of TLR-2 in innate recognition of S. aureus and activation of inflammatory cascade by the interplay of some proinflammatory cytokines, resulting in joint inflammation has been established. Variation in the reports suggesting both functional dependency and independency of TLR-2 on its heterodimeric partner TLR-6 in response to ligands exists, thus this study was postulated to observe the expression pattern of TLR-6 in synovial tissue and lymphoid organs after inducing septic arthritis by S. aureus in Swiss albino mouse model and the instigated cytokine profile could affirm its plausible role in SA. The functional relation of TLR-2 and 6 was verified by simulating an in vitro study design on synovial mononuclear cells, blocking TLR-2 and 6, and it was found that they are required to co-express for generating cytokine, NO and H2O2 on infection. IFN-γ, IL-6 and IL-17 were identified to play a distinguished role in SA from their secretion pattern in both in vivo and in vitro study. IFN-γ and IL-6 remained high throughout the infection possibly by the shift of response from Th1 to Th2 and Th17 and contribute in various converging pathways of inflammation. IL-17 increased with the onset of the disease but reduced on the late period. Hence IFN-γ, IL-6, IL-17 along with TLR-6 can be a potent target for therapeutic approach because of their significant contribution in SA.

Toll-like receptors (TLR) 2, 3, and 4 gene polymorphisms in critically ill patients

Considering that TLR2, TLR3 and TLR4 play very important roles in inflammatory processes, the question arises whether the presence of polymorphisms in these genes is associated with susceptibility to sepsis. The aim of this study was to examine the association of TLR2, TLR3 and TLR4 polymorphisms with clinical characteristics and outcome of critically ill patients. A follow-up study was conducted on 121 critically ill Caucasian Serbian patients. Five polymor- phisms in TLRs, TLR2 (rs5743708), TLR3 (rs3775291, rs5743312) and TLR4 (rs4986790, rs4986791) were genotyped by real-time PCR. Investigated polymorphisms in TLR2 and TLR4 were not associated with the clinical characteristics and outcome of critically ill patients. TLR3 rs3775291 polymorphism was associated with patient's outcome (p=0.018). Pa- tients with sepsis and a TLR3 rs3775291-mutated genotype had a four-fold higher mortality rate compared to wild type and heterozygous carriers. Multivariate regression analysis showed that age, sex and TLR3 rs3775291 polymorphism are independent variables of the outcome of critically ill patients. For the first time, our preliminary findings indicate the role of TLR3 with MyD88 independent signaling and its polymorphism (TLR3 rs3775291) in sepsis and survival in critically ill Serbian patients.

A Toll-Like Receptor 3-Agonist as Promising Candidate in Multiple Sclerosis Treatment

Perturbations in immune processes play an important role in multiple sclerosis (MS), an autoimmune disorder where specific innate immune pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) have recently been shown to play a major role in the initiation disease, the triggering of relapses, and regulation of CNS damage. The abnormal immune response in MS has been shown to be dependent on genetic background, despite environmental factors, including pathogens capable of overstimulating innate immune response through TLRs, appear to contribute to the development of autoreactive T cells that in turn cause myelin damage. However, whereas the upregulation of most TLRs plays a detrimental role in MS pathogenesis, recent studies suggest an ameliorative role of TLR3 in the onset and progression of MS and experimental autoimmune encephalomyelitis (EAE), a murine model of MS. TLR3 activation appears to protect from the disease, mainly through induction of interferon (IFN)β. Therefore, TLR3 stimulation with synthetic immunomodulators could represent a potential alternative approach in MS therapy. Among the investigational compounds TLR3-targeting, the mismatched double-stranded RNA molecule Ampligen®, can offer promise in the treatment of relapsing MS patients. Ampligen® is currently in phase III clinical trial in the treatment of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME), an illness that shows remarkable levels of similarity with MS. The aim of this paper is to provide a brief overview about Ampligen® historical development, clinical pharmacology, clinical trials, and safety data, and to discuss about its potential role in MS treatment in the context of existing therapeutic options.

Toll-like receptors in systemic lupus erythematosus: potential for personalized treatment.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the loss of tolerance to self-nuclear antigens. The symptoms of SLE, progression of pathology and the array of autoantibodies present in the serum differ significantly from patient to patient, which calls for a personalized approach to treatment. SLE is polygenic and strongly influenced by gender, ethnicity, and environmental factors. Data from genome-wide association studies suggests that polymorphisms in as many as 100 genes contribute to SLE susceptibility. Recent research has focused on genes associated with Toll-like receptors (TLRs), type I interferons, immune regulation pathways, and immune-complex clearance. TLR7 and TLR9 have been extensively studied using lupus-prone mouse models. In multiple systems overexpression of TLR7 drives disease progression but interestingly, a loss of TLR9 results in an almost identical phenotype. While TLR7 overexpression has been linked to human SLE, the possible role of TLR9 in human disease remains elusive. In the present review, we focus on TLR polymorphisms and TLR expression in SLE patients and discuss their potential as biomarkers for individualized treatment.

Abstract 15312: Genetic Deletion of Toll-like Receptor 9 Accelerates Blood Flow Recovery after Hindlimb Ischemia

Background: Peripheral artery disease causes significant functional disability and results in impaired quality of life. Toll-like receptor (TLR)-2, 3 and 4 are suggested to participate in blood flow recovery in ischemic limb by modulating inflammation and angiogenesis, however, the role of TLR9 remains unknown. TLR9 recognizes bacterial unmethylated DNA and plays a role in innate defense, although it can also provoke inflammation in response to fragmented DNA released from regenerated mammalian cells. This study tested the hypothesis that genetic deletion of TLR9 accelerates blood flow recovery after femoral artery ligation by inhibiting inflammation and improving endothelial cell function. Methods and Results: Unilateral femoral artery ligation was performed in TLR9-deficient (TLR9KO) mice and wild type (WT) mice. Femoral artery ligation significantly increased RNA expression of TLR9 (20-times) in WT mice and plasma levels of single-stranded DNA and double-stranded DNA, endogenous ligands for TLR9, in both strains of mice compared with each sham-operated group (P<0.05). Laser Doppler perfusion imaging demonstrated that TLR9KO mice significantly improved the ratio of the blood flow in the ischemic to non-ischemic limb compared with WT mice at 2 weeks after ligation (P<0.05). TLR9KO mice showed less accumulation of macrophages and less expression of inflammatory molecules (e.g., TNF-α, MCP-1 and IL-1β in ischemic muscle compared with WT mice (P<0.05, respectively). In vitro experiments using thioglycolate-stimulated peritoneal macrophages demonstrated that CpG ODN, agonistic oligonucleotide for TLR9, promoted the expression of pro-inflammatory molecules (e.g., MCP-1 and TNF-α) in WT macrophages (P<0.05, respectively) but not in TLR9 KO macrophages. Furthermore, activation of TLR9 by CpG ODN inhibited migration and proliferation of endothelial cells as determined by scratch-wound assay and MTS assay, respectively (P<0.05). Conclusion: Our results suggested that TLR9 enhances inflammation and affects migration and proliferation of endothelial cells, leading to impaired blood flow recovery in ischemic limb. TLR9 may serve as a potential therapeutic target for ischemic limb disease.