Presence Of Toll-like Receptors Research Articles

Toll-like receptor-7 activation in CD8+ T cells modulates inflammatory mediators in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder of unknown etiology with aberrant immunological responses leading to inflammation, swelling and pain of the joints. CD8+ T cells have been known to be one of the major immune modulators in the progression of RA and the presence of toll-like receptors (TLRs) on these cells further accentuate their role in RA. Herein, we report an increased expression of TLR7 in the endosomes of CD8+ T cells of RA patients correlating with disease severity. The stimulation of TLR7 with Imiquimod (IMQ) in these CD8+ T cells drives the signalling cascade via NFkB and pERK activation and hence an increase in the mRNA transcripts of signature cytokines and cytolytic enzymes. However, a parallel synthesis of Tristetraprolin (TTP), an mRNA destabilizing protein prevents the translation of the mRNA transcripts, leading to a rapid degeneration of the target mRNA. We thus report that a direct TLR7 ligation by its agonist increases cytokine transcript signature but not an equivalent protein surge.

TLR9 agonist adsorbed to alum adjuvant prevents asthma-like responses induced by Blomia tropicalis mite extract.

Blomia tropicalis mite is highly prevalent in tropical and subtropical regions and it is associated with allergic diseases such as rhinitis and asthma. By using an OVA-model of allergic lung disease, we have previously shown that sensitization in the presence of toll like receptors (TLRs) agonists attenuates subsequent OVA-induced allergic responses. Here, we evaluated the effect of CpG-ODN, a specific synthetic TLR-9 agonist, on the development of experimental asthma induced by Blomia tropicalis extract, a relevant source of aeroallergens. Among different protocols of Blomia tropicalis extract sensitization, the subcutaneous sensitization in the presence of alum adjuvant induced the highest Th2 responses, including high IgE levels. Adsorption of CpG to Blomia tropicalis extract/Alum attenuated the airway hyperreactivity, the infiltration of inflammatory cells including eosinophils, and the IL-5 content in BAL. In addition, lung peribronchial inflammatory infiltrate, mucus production and IL-5-producing CD3+ CD4+ T cells were significantly reduced in the Blomia tropicalis extract/Alum+CpG group. Importantly, CpG inhibited total IgE production as well as active systemic or cutaneous anaphylaxis reactions. Inhibition of pulmonary Th2 responses was associated with increased IL-10 production but not with IFN-γ production. Notably, in IL-10-deficient mice, sensitization with OVA/Alum+CpG resulted in intense lung neutrophilia and IFN-γ production, indicating that IL-10 is necessary to inhibit subsequent Th1 immunity. Our work highlights the mechanisms of allergy attenuation by CpG and it indicates the potential use of Alum-based formulation with CpG to treat allergic processes.

CD14 dictates differential activation of mesenchymal stromal cells through AKT, NF-κB and P38 signals.

Mesenchymal stromal cells (MSCs) widely exist in many tissues and have multiple differentiation potential and immunomodulatory capacities. Recently, MSCs have become promising tools for the treatment of various degenerative disorders and autoimmune diseases. The properties of MSCs could be modified in different microenvironments. Thus, it is important to explore the factors controlling MSC function. The presence of Toll-like receptors (TLRs) in MSCs was demonstrated according to previous studies. Consistently, we also illustrated the expression of TLRs in both murine and human MSCs, and displayed that the expression patterns of TLRs in MSCs from different sources. Furthermore, we explored the role of TLR and TLR signaling pathway in MSCs. Interestingly, activation of TLR4-induced expression of cytokines and some specific genes in MSCs. However, MSCs retained much lower mRNA level compared with macrophages. We explored the expression of CD14 in MSCs from different sources, which played a vital role in TLR4 signaling pathway, and found that MSCs are almost negative for CD14. Moreover, only partial activation of TLR4 signaling pathway was observed in MSCs, with no activation of AKT, NF-κB and P38. Here, in the study we defined TLR expression, function and activation in MSCs, which is critical for designing MSC-based therapies.

The role of platelets in inflammation and immunity

In recent decades, significant progress has been made in understanding the mechanisms of platelet function and platelet hemostasis correction. Platelets are considered as the most important participants of both normal, and pathological thrombotic process characteristic of the most different diseases and states. In the present review pathophysiological mechanisms of platelet synthesis of various mediators with paracrine effects, which can influence the function of other cells, are consecrated. The physiology of platelets was considered in detail. The leading role of platelets in pathogenesis of the majority of diseases of cardiovascular system as modulators of inflammatory reactions of the immune response which are considered as the leading mechanism of development of atherosclerosis was shown. The ability of platelets to encode inflammatory proteins allowing them to influence adaptive immunity functions was discussed. The role of platelets as a key component of the innate immune system was presented, which is confirmed by the presence of Tolllike receptors (TLR) and glycoproteins, such as integrin αIIbβ3, glycoprotein Ib-IX and FcγRIIa, involved in interaction with bacterial cells. The pathogenesis of the formation of platelet-leukocyte aggregates due to the rapid reversible interaction of P-selectin (CD62P) on the platelet surface with ligand-1 glycoprotein P-selectin (PSGL-1) on the plasma of leukocytes and the mechanism of extracellular neutrophil traps (NETs), as well as the influence of platelets on the function of lymphocytes was presented. The role of platelets in cancer progression, metastasis and thrombosis is considered, and the interrelation of thrombosis and metastasis in malignant diseases was analyzed. The efficiency of the use of antithrombotic drugs in the prevention of thrombosis and, as a consequence, in the prevention of cardiovascular diseases and cancer was discussed.

Early pregnancy-related changes in toll-like receptors expression in ovine trophoblasts and peripheral blood leukocytes

In the present study, we aimed to 1) demonstrate the presence of all 10 toll-like receptors (TLRs) in ovine trophoblasts, and 2) investigate the expression profiles of TLR1–10 mRNAs in peripheral blood leukocytes (PBLs) in ewes during early pregnancy. For those purposes, ovine trophoblasts (n = 6) were collected from pregnant ewes on day 13. PBLs were collected from non-pregnant (n = 6) and pregnant ewes (n = 17) on days of mating (d) 0 and 18. TLR mRNAs in ovine trophoblasts were visualized by free-floating in situ hybridization (ISH). To assess the expression profiles of TLR1–10 in PBLs, total RNA was isolated and transcribed to cDNA. TLR1–10 mRNA levels were determined by real-time PCR in triplicate. The Relative Expression Software Tool (REST 2009) was used for statistical analysis. We detected mRNAs for TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR10 but not for TLR1, TLR3, and TLR9 in trophoblasts. TLR2, TLR5, TLR6, TLR7, TLR8, and TLR10 mRNAs were expressed by all trophoblasts, whereas TLR4 mRNA and protein in trophoblasts were more limited. In PBLs, TLR expression did not differ between day 0 and day 18 in non-pregnant ewes; however, ewes in early pregnancy exhibited significantly upregulated expression of TLR2 (2.3-fold), TLR4 (3.1-fold), TLR6 (1.7-fold), and TLR8 (2.2-fold) on day 18 compared with day 0. In contrast, TLR10 was downregulated (2-fold) on day 18 by pregnancy. Similar results were also obtained for TLR2, TLR4, TLR6, TLR8 and TLR10 from the comparison between day 18 non –pregnant and day 18 pregnant groups. According to these results, the presence of TLRs in early ovine trophoblasts suggests that these cells play an immunological role at the maternal–fetal interface. The results also suggest that tight regulation of some components of TLRs in PBLs due to embryo- and/or pregnancy-related factors is necessary for successful establishment of early pregnancy in ewes.

Toll-like Receptors in the Vascular System: Sensing the Dangers Within.

Toll-like receptors (TLRs) are components of the innate immune system that respond to exogenous infectious ligands (pathogen-associated molecular patterns, PAMPs) and endogenous molecules that are released during host tissue injury/death (damage-associated molecular patterns, DAMPs). Interaction of TLRs with their ligands leads to activation of downstream signaling pathways that induce an immune response by producing inflammatory cytokines, type I interferons (IFN), and other inflammatory mediators. TLR activation affects vascular function and remodeling, and these molecular events prime antigen-specific adaptive immune responses. Despite the presence of TLRs in vascular cells, the exact mechanisms whereby TLR signaling affects the function of vascular tissues are largely unknown. Cardiovascular diseases are considered chronic inflammatory conditions, and accumulating data show that TLRs and the innate immune system play a determinant role in the initiation and development of cardiovascular diseases. This evidence unfolds a possibility that targeting TLRs and the innate immune system may be a novel therapeutic goal for these conditions. TLR inhibitors and agonists are already in clinical trials for inflammatory conditions such as asthma, cancer, and autoimmune diseases, but their study in the context of cardiovascular diseases is in its infancy. In this article, we review the current knowledge of TLR signaling in the cardiovascular system with an emphasis on atherosclerosis, hypertension, and cerebrovascular injury. Furthermore, we address the therapeutic potential of TLR as pharmacological targets in cardiovascular disease and consider intriguing research questions for future study.

Neisseria meningitidiselicits a pro-inflammatory response involving IκBζ in a human blood-cerebrospinal fluid barrier model

BackgroundThe human-specific, Gram-negative bacterium Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis worldwide. The blood-cerebrospinal fluid barrier (BCSFB), which is constituted by the epithelial cells of the choroid plexus (CP), has been suggested as one of the potential entry sites of Nm into the CSF and can contribute to the inflammatory response during infectious diseases of the brain. Toll-like receptors (TLRs) are involved in mediating signal transduction caused by the pathogens.MethodsUsing a recently established in vitro model of the human BCSFB based on human malignant CP papilloma (HIBCPP) cells we investigated the cellular response of HIBCPP cells challenged with the meningitis-causing Nm strain, MC58, employing transcriptome and RT-PCR analysis, cytokine bead array, and enzyme-linked immunosorbent assay (ELISA). In comparison, we analyzed the answer to the closely related unencapsulated carrier isolate Nm α14. The presence of TLRs in HIBCPP and their role during signal transduction caused by Nm was studied by RT-PCR and the use of specific agonists and mutant bacteria.ResultsWe observed a stronger transcriptional response after infection with strain MC58, in particular with its capsule-deficient mutant MC58siaD−, which correlated with bacterial invasion levels. Expression evaluation and Gene Set Enrichment Analysis pointed to a NFκB-mediated pro-inflammatory immune response involving up-regulation of the transcription factor IκBζ. Infected cells secreted significant levels of pro-inflammatory chemokines and cytokines, including, among others, IL8, CXCL1-3, and the IκBζ target gene product IL6. The expression profile of pattern recognition receptors in HIBCPP cells and the response to specific agonists indicates that TLR2/TLR6, rather than TLR4 or TLR2/TLR1, is involved in the cellular reaction following Nm infection.ConclusionsOur data show that Nm can initiate a pro-inflammatory response in human CP epithelial cells probably involving TLR2/TLR6 signaling and the transcriptional regulator IκBζ.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0163-x) contains supplementary material, which is available to authorized users.

Gene discovery through transcriptome sequencing for the invasive mussel Limnoperna fortunei.

The success of the Asian bivalve Limnoperna fortunei as an invader in South America is related to its high acclimation capability. It can inhabit waters with a wide range of temperatures and salinity and handle long-term periods of air exposure. We describe the transcriptome of L. fortunei aiming to give a first insight into the phenotypic plasticity that allows non-native taxa to become established and widespread. We sequenced 95,219 reads from five main tissues of the mussel L. fortunei using Roche’s 454 and assembled them to form a set of 84,063 unigenes (contigs and singletons) representing partial or complete gene sequences. We annotated 24,816 unigenes using a BLAST sequence similarity search against a NCBI nr database. Unigenes were divided into 20 eggNOG functional categories and 292 KEGG metabolic pathways. From the total unigenes, 1,351 represented putative full-length genes of which 73.2% were functionally annotated. We described the first partial and complete gene sequences in order to start understanding bivalve invasiveness. An expansion of the hsp70 gene family, seen also in other bivalves, is present in L. fortunei and could be involved in its adaptation to extreme environments, e.g. during intertidal periods. The presence of toll-like receptors gives a first insight into an immune system that could be more complex than previously assumed and may be involved in the prevention of disease and extinction when population densities are high. Finally, the apparent lack of special adaptations to extremely low O2 levels is a target worth pursuing for the development of a molecular control approach.

Polymorphisms of toll-like receptors 2, 3 and 4 in patients that do and do not enter labour spontaneously at term

To assess the association of polymorphisms of toll-like receptors (TLRs) 2, 3 and 4 with the delay in onset of labour at term pregnancies, patients delivering at > 37 weeks and without pre-eclampsia, IUGR or a history of preterm delivery were prospectively evaluated. TLR2 Arg753Gln, TLR3 (c.1377C/T) and TLR4 Asp299Gly and Thr399Ile polymorphisms were genotyped by using PCR-RFLP. Patients labouring spontaneously before the 41st week were compared with those who did not labour spontaneously until this week in terms of baseline characteristics, TLR 2, 3 and 4 polymorphisms. The same comparisons were also performed by using a 40th week cut-off. Chi-square test, two-sample t-test or Mann–Whitney U tests were used for comparisons, as appropriate. In total, 79 patients delivering after 37 weeks were evaluated. All had CC genotype for TLR2 Arg753Gln and TLR4 Thr399Ile. There were no significant differences for TLR4 Asp299Gly GA and TLR3 (c.1377C/T) polymorphisms between patients spontaneously entering or not entering labour until the 41st week; the same was true when the 40th week cut-off was used. Delay in onset of labour at term pregnant women does not seem to be affected by the presence of TLR 2, 3 or 4 polymorphisms. Further studies are needed.

Molecular and biochemical expression of TLRs in human amniotic membrane: a comparative study of fresh and cryopreserved specimens

To assess the expression of toll-like receptors (TLRs) in human amniotic membrane (AM) specimens and compare this expression with those of AMs undergoing the standard preservation procedure (handling) for ocular surgery. Human fresh (n = 10; five spontaneous and five cesarean) or handled (n = 5) AMs were analyzed for TLR gene and protein expression. Two pieces were obtained from each specimen, and subjected to molecular or biochemical analysis. Relative real-time PCR and SDS-PAGE were carried out according to standard procedures. The REST-ANOVA coupled analysis was used to compare the molecular and biochemical data. The fresh membranes expressed all the TLRs (TLR1-10), with different gene expression as detected/evidenced by the Ct values, the intra-fresh group analysis showing that there was a variation of TLR expression whichvaried within the fresh membranes. The handled AMs retained the TLR expression after standard processing and preservation, but with a particular pattern which included a high TLR3/TLR4 and low TLR6 expression, when compared to the fresh membranes. The molecular data were confirmed by Western blot analysis. AM is routinely used in several ophthalmic surgical procedures, and notwithstanding its preservation procedure, AM is reported to favour wound healing and exert anti-angiogenic, anti-inflammatory, anti-scarring as well as anti-bacterial activities. The presence of TLRs in handled AM would imply that TLRs might be preserved in AMs used in ocular surgery. The findings herein described provide additional data concerning the presence of TLRs in cryopreserved AM, and suggest a possible contribution of AM in ocular surgery, via the innate immune response.

Hymenolepis diminuta: Analysis of the expression of Toll-like receptor genes (TLR2 and TLR4) in the small and large intestines of rats

Toll receptors play a critical role in the rapid activation of innate immune responses to a variety of pathogens. In mammals, Toll-like receptors (TLR) have been found in both immune related cells and other cells. At present little is known about the participation of TLR in host defense mechanisms during parasitic infections. The aim of this study was to determine the expression of TLR2 and TLR4 genes in rat intestines during experimental hymenolepidosis. There is difference in expression of TLR2 and TLR4 genes in the colon and jejunum in uninfected rats: in the colon, mRNA of the examined TLR is present in much higher amounts than the jejunum, while the protein of the TLR also had a segmented specific distribution. In the jejunum isolated rats infected with Hymeolepis diminuta 6 and 8days post infection (dpi), mRNA for TLR4 and TLR2 were significantly more strongly expressed in comparison with the uninfected controls. In the colon, a statistically significantly increased expression of TLR4 gene was observed only at 6dpi, and at 8dpi for the TLR2 gene. Moreover, we observed that during inflammation, the immunopositive cell number and the intensity of immunohistochemical staining (indicating the presence of TLR within intestinal epithelial cells), increased together with the duration of the infection period.

Hepatitis C Virus Core Protein Induces Homotolerance and Cross‐Tolerance to Toll‐Like Receptor Ligands by Activation of Toll‐Like Receptor 2

Hepatitis C virus (HCV) activates host innate immune responses mediated by retinoic acid inducing gene-I (RIG-I) and Toll-like receptors (TLRs). Although the nonstructural protein 3/4A (NS3/4A) of HCV disrupts interferon responses by inhibiting RIG-I signaling, the effects of TLR activation by HCV-associated proteins on host innate immune responses are poorly understood. Proinflammatory cytokine responses to various TLR ligands in human antigen-presenting cells (APCs) were examined either with or without prestimulation by HCV core protein. TLR2 activation by the HCV core protein leads to a decrease in interleukin 6 (IL-6) production by human APCs after subsequent stimulation with TLR2 (homotolerance) ligands and TLR4 (cross-tolerance) ligands. This hyporesponsiveness induced by preexposure to the HCV core protein was partially mediated by the negative regulation of nuclear factor-kappaB activation by the induction of IRAK-M. TLR ligand-induced IL-6 production was significantly reduced in peripheral blood monocytes isolated from HCV-infected patients, compared with those of healthy control subjects. Alloantigen presentation by monocytes isolated from HCV-infected patients results in impaired production of interleukin 17 by naive CD4(+) T cells in the presence of TLR ligands. Chronic stimulation of APCs with HCV core protein is associated with hyporesponsiveness in TLR-mediated innate immunity.

Toll‐like receptors and aseptic loosening of hip endoprosthesis—a potential to respond against danger signals?

Bacterial remnants and subclinical biofilms residing on prosthesis surfaces have been speculated to play a role in hip implant loosening by opsonizing otherwise relatively inert wear particles. The innate immune system recognizes these microbial pathogen-associated molecular patterns (PAMPs) using Toll-like receptors (TLRs). Our objective was to evaluate the possible presence of TLRs in aseptic synovial membrane-like interface tissue. Bacterial culture-negative, aseptic (n = 4) periprosthetic synovial membrane-like tissue was compared to osteoarthritis synovial membrane (n = 5) for the presence of cells positive for all known human functional TLRs, stained using specific antibodies by immunohistochemistry, and evaluated using morphometry. In comparison to osteoarthtritic synovium, the number of TLR-positive cells was found to be increased in the aseptic setting, reflecting the considerable macrophage infiltration to the tissues investigated. Thus aseptic periprosthetic tissue seems to be very reactive to PAMPs. It has been recently recognized that TLR do not only respond to traditional PAMPs, but also to endogenous alarmings or danger signals released from necrotic and activated cells. Alarming-TLR interaction in the periprosthetic tissue might be a novel mechanism of aseptic loosening of endoprosthesis.

Toll-Like Receptors on Human Mesenchymal Stem Cells Drive Their Migration and Immunomodulating Responses

Adult human bone marrow-derived mesenchymal stem cells (hMSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. To achieve the desired clinical outcomes for this strategy requires a better understanding of the mechanisms that drive the recruitment, migration, and engraftment of hMSCs to the targeted tissues. It is known that hMSCs are recruited to sites of stress or inflammation to fulfill their repair function. It is recognized that toll-like receptors (TLRs) mediate stress responses of other bone marrow-derived cells. This study explored the role of TLRs in mediating stress responses of hMSCs. Accordingly, the presence of TLRs in hMSCs was initially established by reverse transcription-polymerase chain reaction assays. Flow cytometry and fluorescence immunocytochemical analyses confirmed these findings. The stimulation of hMSCs with TLR agonists led to the activation of downstream signaling pathways, including nuclear factor kappaB, AKT, and MAPK. Consequently, activation of these pathways triggered the induction and secretion of cytokines, chemokines, and related TLR gene products as established from cDNA array, immunoassay, and cytokine antibody array analyses. Interestingly, the unique patterns of affected genes, cytokines, and chemokines measured identify these receptors as critical players in the clinically established immunomodulation observed for hMSCs. Lastly, hMSC migration was promoted by TLR ligand exposure as demonstrated by transwell migration assays. Conversely, disruption of TLRs by neutralizing TLR antibodies compromised hMSC migration. This study defines a novel TLR-driven stress and immune modulating response for hMSCs that is critical to consider in the design of stem cell-based therapies.

Immunolocalization of Dendritic Cells and Pattern Recognition Receptors in Chronic Rhinosinusitis

Dendritic cell (DC) activation and antigen presentation to T cells are critical to innate and adaptive immunity. Toll-like receptors (TLRs) are known to bind pathogen-associated molecular patterns in addition to sinonasally secreted surfactant proteins (SP) such as SP-A and SP-D. TLR binding is known to activate DCs. Based on these observations, we sought to establish the presence, in sinonasal mucosa, of DC and the pattern recognition receptors (PRRs), CD14, TLR2, and TLR4. Sinonasal biopsy specimens were taken from patients with eosinophilic nonatopic nasal polyposis (n = 4), allergic fungal sinusitis (n = 1), and nondiseased patients undergoing cerebrospinal fluid leak repair or pituitary tumor resection (n = 2). Tissue samples were stained immunohistochemically for PRR (CD14, TLR2, and TLR4), mature DC marker (CD208), iDC marker (CD209), or isotype controls. Immature and mature DC were immunolocalized to the subepithelial stroma and ciliated epithelial surface, respectively. Diffuse staining of CD14 was observed throughout the stroma with additional staining in the ciliated epithelium. The TLR markers showed no staining in the ciliated epithelium. TLR2 primarily localized in stroma immediately deep to the ciliated epithelial surface. TLR4 immunolocalized to submucosal seromucinous gland ductal epithelium. Data from nondiseased patients were mixed, with one patient showing minimal staining of any of the tested cellular markers. This study indicates progressive DC activation and emigration of mature antigen-presenting cells from the epithelial surfaces of sinonasal mucosa. The presence of TLR known to bind SP-A and SP-D suggests a link between SP expression and immune response in sinonasal mucosa.

Profile of Toll-like receptor expressions and induction of nitric oxide synthesis by Toll-like receptor agonists in chicken monocytes

Toll-like receptors (TLRs) play a major role in the innate immune system for initial recognition of microbial pathogens and pathogen associated components. Nitric oxide (NO) is generated in immune cells in response to microbial stimulation and is involved in pathogenesis and control of infection. We used RT-PCR analysis to examine the TLR expression profile on chicken monocytes and demonstrated these cells express chicken TLR2, 3, 4, 6, and 7. TLR5 was not detected by the TR-PCR. We also investigated the differential induction of NO synthesis in chicken monocytes by TLR agonists, including flagellin (FGN, from Salmonella typhimurium), synthetic lipoprotein Pam3CSK4 (PAM), lipopolysaccharide (LPS, from Salmonella enteritidis), lipoteichoic acid (LTA, from Staphylococcus aureus), the synthetic double stranded RNA analog (poly I:C), the guanosine analog, loxoribine (LOX), and synthetic CpG oligodeoxydinucleotide (CpG-ODN). Our results indicate that there was a vast difference among these agonists for their ability to induce NO production. CpG-ODN and LPS were the most potent stimuli and induced significant quantities of NO in cultured monocytes, whereas LTA stimulated significant NO production only at high concentrations. Other agonists such as FGN and poly I:C stimulated very little NO, while PAM, LOX, and nCpG-ODN (control ODN) did not induce NO production. RT-PCR analysis demonstrated that LPS, LTA, and CpG-ODN induced inducible nitric oxide synthase (iNOS) expression in monocytes; whereas the other agonists did not. The presence of TLRs on chicken monocytes and the differential induction of NO production in chicken monocytes by various TLR agonists suggest the differentiation of signaling pathways downstream of individual TLRs.

Cellules dendritiques et Toll-like récepteurs dans les maladies allergiques

Bronchial airways represent a major portal of entry for pathogenic microorganisms and allergens, and they are equipped with a dual response system: innate immunity and adaptative immunity. The innate defense system is composed of phagocytic cells [(alveolar macrophages, neutrophils and dendritic cells (DCs)]. These cells express pattern recognition receptors (PRRs) that directly recognize pathogen-associated molecular patterns (PAMPs), which are shared by numerous microorganisms. Among PPRs, eleven different Toll-like receptors (TLRs) are described. DCs are also engaged in the adaptive immune response, as they are able, after antigen processing, to transfer specific information to T-lymphocytes. Moreover, through the presence of TLRs –– in particular, TLR2 and TLR4 –– on their surface, DCs are at the intersection between adaptive immunity and innate immunity. After activation by certain ligands (endotoxins such as LPS, bacterial peptidoglycans, envelope proteins), DCs are able to orientate the response to allergen either to a Th1 profile or a pro-allergenic Th2 profile. Via TLRs, PAMPs are also able to directly interact with regulatory T cells, the role of which in the natural development of tolerance towards non-self proteins is now established. Several recent studies emphasize the role of TLRs in the onset and regulation of allergic diseases: the hygiene hypothesis suggests that endotoxin exposure plays a key role in the prevention of some allergic diseases; on the other hand, polymorphisms of certain PAMPs (such as those on Chlamydia pneumoniae or RSV) might favour bronchial inflammation and could explain the development of post-infectious, nonallergic (“intrinsic”) asthma. However, it is likely that the exact nature of the endotoxins, as well as the doses inhaled and also the timing of the penetration into the respiratory tract in relation to contact with allergen could explain some of the discrepancies between experimental and clinical data. Nevertheless, it is certain that the couple TLR-ligand plays a determinant role at different steps in the development of an inflammatory allergic reaction.

Endotoxin stimulates in vivo expression of inflammatory cytokines tumor necrosis factor alpha, interleukin-1beta, -6, and high-mobility-group protein-1 in skeletal muscle.

The presence of increased levels of proinflammatory cytokines in the blood is associated with decreased muscle protein synthesis and the erosion of lean body mass in many catabolic conditions. However, little is known regarding the role of endogenous cytokine synthesis in muscle per se. The purpose of the present study was to characterize the cytokine expression profile of skeletal muscle in response to an in vivo injection of endotoxin (lipopolysaccharide, LPS). Intraperitoneal injection of a nonlethal dose of LPS (1,000 microg/kg Escherichia coli) into male rats increased the mRNA content of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta in gastrocnemius muscle as early as 1 h; IL-6 mRNA was not increased until 2 h post-LPS. Expression of TNF-alpha and IL-1beta peaked at 2 h (10- and 80-fold, respectively), whereas the increased IL-6 mRNA content (150-fold) peaked later at 4 h. The abundance of all measured cytokine mRNAs in skeletal muscle declined thereafter. The LPS-induced increase in muscle mRNA content for TNF-alpha, IL-6, and IL-1beta was dose-dependent with elevations being seen with as little as 10 microg/kg of LPS (2.5-, 8-, and 9-fold, respectively). In general, pretreatment of rats with dexamethasone attenuated but did not completely prevent the LPS-induced increase in muscle cytokine mRNA. LPS increased muscle TNF-alpha protein content approximately 2-fold and this increase was prevented by pretreatment with dexamethasone. LPS-induced increases in muscle IL-1beta and IL-6 protein were not detected. LPS also produced a 2-fold increase in the mRNA content of the high-mobility-group protein-1, a late-phase cytokine, in muscle at 12-24 h. Finally, although skeletal muscle was found to contain both the toll-like receptor (TLR)-2 and TLR4, LPS did not alter the mRNA content of TLR4 and produced a small (50%) but significant increase in TLR2 mRNA. These changes in TLRs were less dramatic than those observed for liver, spleen or cardiac muscle. Collectively these data indicate that skeletal muscle possesses many of the components of the innate immune system, including increases in both early- and late-phase cytokines and the presence of toll-like receptors.

Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation

Background & Aims: Bacterial wall products play an important role in the activation of immune and nonimmune cells of the intestinal mucosa. Toll-like receptors (TLRs) TLR2 and TLR4 have been identified as signaling receptors activated by bacterial wall components. Methods: Expression of TLRs in human intestinal mucosa obtained by endoscopy and surgery was analyzed by immunohistochemistry. Intestinal macrophages were isolated by immunomagnetic beads armed with a CD33 antibody. Reverse-transcription polymerase chain reaction was performed for TLR1–5. Results were confirmed by Northern blot and flow cytometry. Interleukin-1β messenger RNA (mRNA) was quantified by a polymerase chain reaction–enzyme-linked immunosorbent assay–kit. Results: Immunohistochemistry revealed a significant increase in the TLR2 and TLR4 antigen expression on submucosal cells in inflamed intestinal mucosa compared with non-inflamed mucosa. TLR expression was localized in intestinal macrophages by double-labeling techniques. No TLR–polymerase chain reaction product could be obtained with mRNA from CD33-positive macrophages from normal mucosa. We observed an induction of mRNA for TLR2, TLR4, and TLR5 in inflammation-associated macrophages. TLR1 and TLR3 were only detectable in blood monocytes. Monocytes reacted to lipopolysaccharide stimulation with a 3-fold and in vitro differentiated macrophages with a 16-fold increase of cellular interleukin-1β mRNA. Macrophages from normal mucosa did not respond to lipopolysaccharide showing the functional relevance of TLR expression. Conclusions: This study shows the inflammation-dependent induction of TLR2 and TLR4 expression in intestinal macrophages. The absence of TLRs abolishes the reactivity of mucosal macrophages to bacterial wall products. Presence of TLRs may thereby contribute to the inflammatory process.GASTROENTEROLOGY 2002;122:1987-2000