Literature Review

Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders

Article· Literature ReviewinSeminars in Immunology 21(4):242-53 · August 2009with 149 Reads
Abstract
Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are two major forms of innate immune sensors, which provide immediate responses against pathogenic invasion or tissue injury. Activation of these sensors induces the recruitment of innate immune cells such as macrophages and neutrophils, initiates tissue repair processes, and results in adaptive immune activation. Abnormalities in any of these innate sensor-mediated processes may cause excessive inflammation due to either hyper responsive innate immune signaling or sustained compensatory adaptive immune activation. Recent gene association studies appear to reveal strong associations of NLR gene mutations and development of several idiopathic inflammatory disorders. In contrast, TLR polymorphisms are less often associated with inflammatory disorders. Nevertheless, TLRs are up-regulated in the affected tissue of most inflammatory disorders, suggesting TLR signaling is involved in the pathogenesis of chronic and/or idiopathic inflammatory disorders. NLR signaling results in the formation of a molecular scaffold complex (termed an inflammasome) and orchestrates with TLRs to induce IL-1beta and IL-18, both of which are important mediators in the majority of inflammatory disorders. Therefore, understanding the roles of TLRs and NLRs in the pathogenesis of chronic and idiopathic inflammatory disorders may provide novel targets for the prevention and/or treatment of many common and uncommon diseases involving inflammation.
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    Innate immunity has been extended to respond environmental pathogen other than microbial components. Here we explore a novel pollen/TLR4 innate immunity in allergic inflammation. In experimental allergic conjunctivitis induced by short ragweed (SRW) pollen, typical allergic signs, stimulated IL-33/ST2 signaling and overproduced Th2 cytokine were observed in ocular surface, cervical lymph nodes and isolated CD4⁺ T cells of BALB/c mice. These clinical, cellular and molecular changes were significantly reduced/eliminated in TLR4 deficient (Tlr4-d) or MyD88 knockout (MyD88−/−) mice. Aqueous SRW extract (SRWe) directly stimulated IL-33 mRNA and protein expression by corneal epithelium and conjunctiva in wild type, but not in Tlr4-d or MyD88−/− mice with topical challenge. Furthermore, SRWe-stimulated IL-33 production was blocked by TLR4 antibody and NF-kB inhibitor in mouse and human corneal epithelial cells. These findings for the first time uncovered a novel mechanism by which SRW pollen initiates TLR4-dependent IL-33/ST2 signaling that triggers Th2-dominant allergic inflammation.
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    Background: The immune host response related to bacterial and viral infections in the airways and lung of COPD patients is unclear. Objectives: To investigate the expression of anti-bacterial and anti-viral antigens in bronchial biopsies and lung parenchyma of stable COPD patients in relation to bacterial load. Methods: Immunohistochemical (IHC) and qRT-PCR-expression of TLR2-3-4-7-8-9, NOD1, NOD2, MYD88, TRIF, TIRAP, pIRAK1, IRAK4, IRF3, pIRF3, IRF7, pIRF7, RIG1, MDA5, LGP2, MAVS, STING, DAI, IFNα and IFNβ was measured in bronchial mucosa in patients with mild/moderate (n=16), severe/very severe (n=18) stable COPD, control smokers (n=12) and control non-smokers (n=12). Selected relevant antigens were measured by IHC in peripheral lung from COPD (n=12) and control smokers (n=12) and in the bronchoalveolar lavage (BAL) supernatant. Total bacterial load (16S), P. aeruginosa, H. influenzae, M. catarralis, S. pneumonie were measured by qRT-PCR in bronchial biopsies of COPD and control subjects. Results: In bronchial biopsies TLR4+ and NOD1+ epithelial cells were increased in severe/very severe COPD patients compared to both control groups as did NOD1+ cells in lamina propria. qRT-PCR showed increased MDA5 and STING mRNA levels in severe/very severe COPD compared to control smokers. Total bacterial load (16S/mm³) and P. aeruginosa/mm³ were reduced in severe/very severe COPD compared to both control groups. In peripheral lung tissue and in the BAL supernatant TLR4, NOD1, IFNα and IFNβ were similarly expressed in COPD patients and control subjects. Conclusions: Increased expression of TLR4 and NOD1 in the bronchial mucosa may be relevant in the progression of COPD.
  • Article
    Nucleotide-binding and oligomerization domain like receptors (NLR) are pattern recognition receptors used to provide rapid immune response by detecting intracellular pathogen associated molecules. Loss of NLR activity is implicated in genetic disorders, disruption of adaptive immunity, and chronic inflammation. One NLR protein, NOD2, is frequently mutated in Crohn's disease (CD), which is an inflammatory disease of the gastrointestinal tract. Three commonly occurring CD associated NOD2 mutations, R702W, G908R and L1007fs, are clustered near the regulatory domain, leucine rich region (LRR), and lowers the activity of NOD2 in response to muramyl dipeptide (MDP). Since LRR is also the ligand binding domain, this suggests that the mutations either affect the binding of MDP or how the molecule responds to ligand binding. To model the role of R702 in ligand-dependent activation of NOD2, we used homology modeling to map the residue R702 to the interface between the oligomerization domain and LRR. We show that a peptide derived from NOD2(697-718) binds LRR in vitro, and co-expressing or importing the peptide into HEK293 expressing NOD2, there is an increase in the MDP dependent NOD2 activity. The study thus suggests that the R702W mutation interferes with the conformational changes needed for MDP binding and activation. This article is protected by copyright. All rights reserved.
  • Purpose of review: To highlight recent advances in the understanding of nutritional immunology and in the development of novel therapeutics for inflammatory bowel disease (IBD). Recent findings: We highlight the variety of factors that contribute to the interaction of the immune system and nutrition including the microbiome and the nervous system stimulation of the gut. We describe the potential for therapeutic development in IBD. Further, we review the cellular metabolic effects on immune activation and promising therapeutic targets. Finally, we show how the progression of understanding the role of lanthionine synthetase C-like 2 has encompassed both nutritional and therapeutic advances and led to the development of novel oral small molecule therapeutics for IBD. Summary: Nutritional immunology and drug development research centered around immunoregulatory pathways can provide safer and more effective drugs while accelerating the path to cures.
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    Hepatic ischemia/reperfusion injury (IRI) is tissue damage resulting from return of the blood supply to the tissue after a period of ischemia or lack of oxygen. Much of the morbidity associated with liver transplantation and major hepatic resections is, in part, due to IRI. Both innate immunity and autophagy play important roles in hepatic IRI. With regard to innate immunity, one factor that plays a key role is NOD1, an intracellular pattern recognition receptor. NOD1 has recently been shown to be associated with autophagy, but the mechanisms involved with this process remain obscure. This relationship between NOD1 and autophagy prompted us to examine the role and potential mechanisms of NOD1 in regulating autophagy as related to hepatic IRI. We found that NOD1 was upregulated during hepatic IRI and was associated with an activation of the autophagic signaling pathway. Moreover, levels of Atg5, a critical protein associated with autophagy, were decreased when NOD1 was inhibited by NOD1 small interfering RNA. We conclude that NOD1 appears to exert a pivotal role in hepatic IRI by activating autophagy to aggravate hepatic IRI, and Atg5 was required for this process. The identification of this novel pathway, that links expression levels of NOD1 with Atg5‐mediated autophagy, may provide new insights for the generation of novel protective therapies directed against hepatic IRI. NOD1 appeared to exert a pivotal role in hepatic ischemia/reperfusion injury (IRI), by activating autophagy to aggravated hepatic IRI, and Atg5 was required for this process. The identification of this novel pathway, that links expression levels of nod1 with Atg5‐mediated autophagy, may provide new insights for the generation of novel protective therapies directed against hepatic IRI.
  • Article
    Background Even though bacteria trigger inflammation, most of the tissue destruction in periodontitis is due to the host inflammatory response. In addition to immunological events that drive development of early periodontitis, numerous environmental factors like genetics and smoking play a role. We investigated whether the carriage of selected single nucleotide polymorphisms (SNP) of toll‐like receptors (TLR), NOD‐like receptors (NLR) and RIG‐I‐like receptors (RLR) was associated with the diagnosis of early periodontitis in a case‐control study. Methods Adolescents with positive (n = 87) and negative (n = 73) diagnosis for periodontitis had blood samples taken. All participants were genotyped for 42 SNP in the genes encoding TLR1‐10, NOD1‐2, DDX58, and IFIH1 using multiplex assays. Associations between SNP and periodontitis diagnosis were tested. Results TLR1‐rs5743611 showed protective effect for periodontitis (CC vs. GG and GC, P = 0.01, OR 0.10, 95%CI 0.01‐0.78). Carriage of the TLR4‐rs7873784 was associated with higher odds for periodontitis (GG vs. CC and GC, P = 0.05, OR 2.30, 95% CI 1.00‐5.63; GG vs. GC, P = 0.05, OR 2.46, 95% CI 1.01‐5.99). In male participants, reduced susceptibility to periodontitis was observed in carriers of TLR7‐rs3853839 (CC vs. GG and CG, P = 0.02, OR 0.30, 95% CI 0.11‐0.85) and TLR8‐rs3764879 (CC vs. GG and CG, P = 0.02, OR 0.31, 95% CI 0.12‐0.82). Associations were maintained after adjustments for sex, smoking habits and mother´s education. Conclusions This study demonstrated an association between TLR1‐rs5743611, TLR4‐rs7873784, TLR7‐rs3853839 and TLR8‐rs3764879 and susceptibility to periodontitis in adolescents. This article is protected by copyright. All rights reserved
  • Article
    Obligately intracytosolic rickettsiae that cycle between arthropod and vertebrate hosts cause human diseases with a spectrum of severity, primarily by targeting microvascular endothelial cells, resulting in endothelial dysfunction. Endothelial cells and mononuclear phagocytes have important roles in the intracellular killing of rickettsiae upon activation by the effector molecules of innate and adaptive immunity. In overwhelming infection, immuno-suppressive effects contribute to the severity of illness. Rickettsia-host cell interactions involve host cell receptors for rickettsial ligands that mediate cell adhesion and, in some instances, trigger induced phagocytosis. Rickettsiae interact with host cell actin to effect both cellular entry and intracellular actin-based mobility. The interaction of rickettsiae with the host cell also involves rickettsial evasion of host defense mechanisms and exploitation of the intracellular environment. Signal transduction events exemplify these effects. An intriguing frontier is the array of rickettsial noncoding RNA molecules and their potential effects on the pathogenesis and transmission of rickettsial diseases. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease Volume 14 is January 24, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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    Trauma remains a leading cause of death worldwide. Hemorrhagic shock and direct injury to vital organs are responsible for early mortality whereas most delayed deaths are secondary to complex pathophysiological processes. These processes result from imbalanced systemic reactions to the multiple aggressions associated with trauma. Trauma results in the uncontrolled local and systemic release of endogenous mediators acting as danger signals [damage-associated molecular patterns (DAMPs)]. Their recognition by the innate immune system triggers a pro-inflammatory immune response paradoxically associated with concomitant immunosuppression. These responses, ranging in intensity from inappropriate to overwhelming, promote the propagation of injuries to remote organs, leading to multiple organ failure and death. Some of the numerous DAMPs released after trauma trigger the assembly of intracellular multiprotein complexes named inflammasomes. Once activated by a ligand, inflammasomes lead to the activation of a caspase. Activated caspases allow the release of mature forms of interleukin-1β and interleukin-18 and trigger a specific pro-inflammatory cell death termed pyroptosis. Accumulating data suggest that inflammasomes, mainly NLRP3, NLRP1, and AIM2, are involved in the generation of tissue damage and immune dysfunction after trauma. Following trauma-induced DAMP(s) recognition, inflammasomes participate in multiple ways in the development of exaggerated systemic and organ-specific inflammatory response, contributing to organ damage. Inflammasomes are involved in the innate responses to traumatic brain injury and contribute to the development of acute respiratory distress syndrome. Inflammasomes may also play a role in post-trauma immunosuppression mediated by dysregulated monocyte functions. Characterizing the involvement of inflammasomes in the pathogenesis of post-trauma syndrome is a key issue as they may be potential therapeutic targets. This review summarizes the current knowledge on the roles of inflammasomes in trauma.
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    Alzheimer's disease (AD), a neurodegenerative disorder, is marked by the accumulation of amyloid-β (Aβ) and neuroinflammation which promote the development of AD. Geniposide, the main ingredient isolated from Chinese herbal medicine Gardenia jasminoides Ellis, has a variety of pharmacological functions such as anti-apoptosis and anti-inflammatory activity. Hence, we estimated the inflammatory cytotoxicity caused by Aβ25–35 and the neuroprotective effects of geniposide in HT22 cells. In this research, following incubation with Aβ25–35 (40 μM, 24 h) in HT22 cells, the methylthiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) release assays showed that the cell survival rate was significantly decreased. In contrast, the reactive oxygen species (ROS) assay indicated that Aβ25–35 enhanced ROS accumulation and apoptosis showed in both hoechst 33342 staining and annexin V-FITC/PI double staining. And then, immunofluorescence test revealed that Aβ25–35 promoted p65 to transfer into the nucleus indicating p65 was activated by Aβ25–35. Moreover, western blot analysis proved that Aβ25–35 increased the expression of nitric oxide species (iNOS), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-1β (IL-1β). Simultaneously, Aβ25–35 also promoted the expression of toll-like receptor 4 (TLR4), p-p65 and p-IκB-α accompanied with the increase in the level of beta-secretase 1 (BACE1) and caspase-3 which further supported Aβ25–35 induced apoptosis and inflammation. Fortunately, this up-regulation was reversed by geniposide. In conclusion, our data suggest that geniposide can alleviate Aβ25–35-induced inflammatory response to protect neurons, which is possibly involved with the inhibition of the TLR4/NF-κB pathway in HT22 cells. Geniposide may be the latent treatment for AD induced by neuroinflammation and apoptosis.
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    The use of antibiotics as growth promoters in feed has been fully or partially banned in several countries. The objective of this study was to investigate the effects of benzoic acid (A), bacillus coagulans (B) and oregano oil (O) combined supplementation on growth performance and intestinal barrier in piglets challenged with enterotoxigenic Escherichia coli (ETEC). Thirty piglets were randomly assigned to 6 treatments: (1) nonchallenged control (CON); (2) ETEC-challenged control (ETEC); (3) antibiotics + ETEC (AT); (4) A + B + ETEC (AB); (5) A + O + ETEC (AO); (6) A + B + O + ETEC (ABO). On day 22, piglets were orally challenged with ETEC or saline. The trial lasted 26 days. Dietary AO and ABO inhibited the reduction of growth performance and the elevation of diarrhoea incidence in piglets induced by ETEC ( P <0.05). AB, AO, and ABO prevented the elevation of serum TNF- α and LPS concentrations in piglets induced by ETEC ( P <0.05). ABO alleviated the elevation of TNF- α and IL-1 β concentrations and the reduction of sIgA level in jejunal mucosa induced by ETEC ( P <0.05). Furthermore, ABO upregulated mRNA expressions of Claudin-1 and Mucin2 ( P <0.05), downregulated mRNA abundances of TLR4 and NOD2 signaling pathways related genes in jejunal mucosa ( P <0.05), and improved the microbiota in jejunal and cecal digesta ( P <0.05) compared with ETEC group. These results indicated that benzoic acid, bacillus coagulans, and oregano oil combined supplementation could improve growth performance and alleviate diarrhoea of piglets challenged with ETEC via improving intestinal mucosal barrier integrity, which was possibly associated with the improvement of intestinal microbiota and immune status. The combination of 3000 g/t benzoic acid + 400 g/t bacillus coagulans + 400 g/t oregano oil showed better effects than other treatments in improving growth performance and intestinal health of piglets, which could be used as a viable substitute for antibiotic.
  • Article
    Rimulus cinnamon is the dried twig of Cinnamomum cassia Presl. It is widely used in China for the treatment of inflammatory processes, amenorrhea, and other diseases. We aimed to study the protective effects of ethyl acetate extracts of R. cinnamon (EAE) on systemic inflammation and lung injury in endotoxin-poisoned mice. EAE was administered 5 d prior to lipopolysaccharide (LPS) challenge with 15 mg/kg LPS. The administration of EAE increased the levels of interferon-γ (IFN-γ) and decreased the levels of interleukin-18 (IL-18) and tumor necrosis factor-α (TNF-α) in the serum. Additionally, EAE relieved the pathological changes in the tissues of the lungs and spleen, and significantly reduced the number of neutrophils in the lung tissues. In addition, treatment with EAE decreased the mRNA expression of the NLR family, pyrin domain-containing protein 3 (NLRP3), caspase-1, and interleukin-1β (IL-1β) in the lungs, as well as the expression of NLRP3, caspase-1 (p20), and pro-IL-1β proteins. These results demonstrated the promising anti-inflammatory effects of EAE in endotoxin-poisoned mice. Furthermore, EAE could alleviate the lung injury of endotoxin-poisoned mice by antagonizing the activation of the NLRP3 inflammasome.
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    Maintenance of health involves a synchronized network of catabolic and anabolic signals among organs/tissues/cells that requires differential bioenergetics from mitochondria and glycolysis (biological laws or biorhythms). We defined biological circadian rhythms as Yin (tumoricidal) and Yang (tumorigenic) arms of acute inflammation (effective immunity) involving immune and non-immune systems. Role of pathogens in altering immunity and inducing diseases and cancer has been documented for over a century. However, in 1955s decision makers in cancer/medical establishment allowed public (current baby boomers) to consume million doses of virus-contaminated polio vaccines. The risk of cancer incidence and mortality sharply rose from 5% (rate of hereditary/genetic or innate disease) in 1900s, to its current scary status of 33% or 50% among women and men, respectively. Despite better hygiene, modern detection technologies and discovery of antibiotics, baby boomers and subsequent 2-3 generations are sicker than previous generations at same age. American health status ranks last among other developed nations while America invests highest amount of resources for healthcare. In this perspective we present evidence that cancer is an induced disease of twentieth century, facilitated by a great deception of cancer/medical establishment for huge corporate profits. Unlike popularized opinions that cancer is 100, 200 or 1000 diseases, we demonstrate that cancer is only one disease; the severe disturbances in biorhythms (differential bioenergetics) or loss of balance in Yin and Yang of effective immunity. Cancer projects that are promoted and funded by decision makers are reductionist approaches, wrong and unethical and resulted in loss of millions of precious lives and financial toxicity to society. Public vaccination with pathogen-specific vaccines (e.g., flu, hepatitis, HPV, meningitis, measles) weakens, not promotes, immunity. Results of irresponsible projects on cancer sciences or vaccines are increased population of drug-dependent sick society. Outcome failure rates of claimed 'targeted' drugs, 'precision' or 'personalized' medicine are 90% (± 5) for solid tumors. We demonstrate that aging, frequent exposures to environmental hazards, infections and pathogen-specific vaccines and ingredients are 'antigen overload' for immune system, skewing the Yin and Yang response profiles and leading to induction of 'mild', 'moderate' or 'severe' immune disorders. Induction of decoy or pattern recognition receptors (e.g., PRRs), such as IRAK-M or IL-1dRs ('designer' molecules) and associated genomic instability and over-expression of growth promoting factors (e.g., pyruvate kinases, mTOR and PI3Ks, histamine, PGE2, VEGF) could lead to immune tolerance, facilitating cancer cells to hijack anabolic machinery of immunity (Yang) for their increased growth requirements. Expression of constituent embryonic factors would negatively regulate differentiation of tumor cells through epithelial-mesenchymal-transition and create "dual negative feedback loop" that influence tissue metabolism under hypoxic conditions. It is further hypothesized that induction of tolerance creates 'dark energy' and increased entropy and temperature in cancer microenvironment allowing disorderly cancer proliferation and mitosis along with increased glucose metabolism via Crabtree and Pasteur Effects, under mitophagy and ribophagy, conditions that are toxic to host survival. Effective translational medicine into treatment requires systematic and logical studies of complex interactions of tumor cells with host environment that dictate clinical outcomes. Promoting effective immunity (biological circadian rhythms) are fundamental steps in correcting host differential bioenergetics and controlling cancer growth, preventing or delaying onset of diseases and maintaining public health. The author urges independent professionals and policy makers to take a closer look at cancer dilemma and stop the 'scientific/medical ponzi schemes' of a powerful group that control a drug-dependent sick society before all hopes for promoting public health evaporate.
  • Chapter
    The gastrointestinal (GI) tract executes a range of functions. GI response to invaders constituting the immunomodulatory function of the gut is crucial to life. This function is mainly carried out by the gut-associated lymphoid tissue (GALT), the largest component of the mucosa-associated lymphoid tissue. This function of the gut is of immense importance not only to the GI tract, but also to the entire organism. The GI tract lymphoid tissues are influenced by several environmental signals which may predispose the organism to the development of diseases including cancers, lymphoma, infections, chronic inflammation, and autoimmune diseases. This chapter is concerned with structure–function relationship of the lymphoid tissues of the GI tract and associated pathological conditions. The molecular mechanisms of functioning of GALT in normal and diseases are also discussed.
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  • Article
    Lactobacillus strains, a major group of lactic acid bacteria, are representative food microorganisms that have many potential beneficial effects via their interactions with immune and intestinal epithelial cells. However, little is known about the effect of Lactobacillus strains on atopic dermatitis via keratinocytes, which comprise the physical barrier of the skin. In this study, we report that Lactobacillus strains have a significant suppressive effect on tumour necrosis factor (TNF)-α-induced expression and production of thymus and activation-regulated chemokine (TARC), a T helper 2 cell chemokine responsible for atopic dermatitis, in human keratinocytes. An RNA interference study showed that the effect of Lactobacillus reuteri strain Japan Collection of Microorganisms (JCM) 1112, the most suppressive strain, depended on the presence of Toll-like receptor 2 and the induction of A20 (also known as TNF-α-induced protein 3) and cylindromatosis in HaCaT cells. Topical application of a water-soluble extract of homogenised JCM 1112 cells significantly suppressed the development of house dust mite-induced atopic skin lesions and TARC expression at the lesion sites in NC/Nga mice. Our study provides new insights into the use of Lactobacillus strains as suppressive agents against keratinocyte-involved atopic inflammation of the skin.
  • Article
    Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death and disability worldwide by 2030; with cigarette smoking (active or passive) being one of the chief cause of its occurrence. Cigarette smoke exposure has been found to result in excessive inflammation and tissue injury, which might lead to COPD, although the exact pathophysiology of the disease remains elusive. While previous studies have demonstrated the role of membrane-bound Toll-like receptors (TLRs) in cigarette smoke (CS)-induced inflammation, scant information is available about the role of cytosolic NOD-like receptors (NLRs) in regulating CS-mediated inflammatory responses. Thus, we investigated the role of NLRP10 and NLRP12 in regulating inflammatory responses in human alveolar type II epithelial cells (A549) and human monocytic cells (THP-1) in response to a challenge with cigarette smoke extract (CSE). We observed CSE-mediated increase in caspase-1 activity; production of IL-1β and IL-18; and expression of NLRP10 and NLRP12 in A549 and THP-1 cells. Interestingly, immunofluorescence imaging results demonstrated an increase in the membrane recruitment of NLRP10 and NLRP12 proteins in CSE-challenged A549 cells. We also observed an increase in the expression of lipid raft proteins (caveolin-1, caveolin-2, and flotillin-1) and an induction of lipid raft assembly following CSE-exposure in A549 cells. Lipid rafts are cholesterol-rich membrane microdomains well known to act as harbours for signalling molecules. Here we demonstrate the recruitment of NLRP10 and NLRP12 in lipid raft entities as well as the interaction of NLRP12 with the lipid raft protein caveolin-1 in CSE-challenged A549 cells. Furthermore, enrichment of lipid raft entities with poly-unsaturated fatty acids (PUFA) rescued A549 cells from CSE-mediated membrane recruitment of NLRP10 and NLRP12, and also from inflammatory responses and inflammasome activation. Enrichment of membrane microdomains with PUFA was able to reverse filipin (chemical agent used for disrupting lipid rafts)-mediated enhanced inflammation in CSE-challenged A549 cells. Overall, our findings unveil a novel mechanism by identifying an important role of membrane microdomains (lipid rafts) in regulating CSE-induced inflammation and NLRP10/NLRP12-dependent signalling in A549 cells.
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    Enterovirus 71 (EV71) is a global infectious disease that affects millions of people. The virus is the main etiological agent for hand, foot, and mouth disease with outbreaks and epidemics being reported globally. Infection can cause severe neurological, cardiac, and respiratory problems in children under the age of 5. Despite on-going efforts, little is known about the pathogenesis of EV71, how the host immune system responds to the virus and the molecular mechanisms behind these responses. Moreover, current animal models remain limited, because they do not recapitulate similar disease patterns and symptoms observed in humans. In this review the role of the host–viral interactions of EV71 are discussed together with the various models available to examine: how EV71 utilizes its proteins to cleave host factors and proteins, aiding virus replication; how EV71 uses its own viral proteins to disrupt host immune responses and aid in its immune evasion. These discoveries along with others, such as the EV71 crystal structure, have provided possible targets for treatment and drug interventions.
  • Article
    Functional maturation of liver sinusoidal endothelial cells (LSECs) induced by a NOD1 ligand (diaminopimelic acid [DAP]) during viral infection has not been well defined. Thus, we investigated the role of DAP-stimulated LSEC maturation during hepatitis B virus (HBV) infection and its potential mechanism in a hydrodynamic injection (HI) mouse model. Primary LSECs were isolated from wild-type C57BL/6 mice and stimulated with DAP in vitro and in vivo and assessed for the expression of surface markers as well as for their ability to promote T cell responses via flow cytometry. The effects of LSEC maturation on HBV replication and expression and the role of LSECs in the regulation of other immune cells were also investigated. Pretreatment of LSECs with DAP induced T cell activation in vitro. HI-administered DAP induced LSEC maturation and subsequently enhanced T cell responses, which was accompanied by an increased production of intrahepatic cytokines, chemokines, and T cell markers in the liver. The HI of DAP significantly reduced the HBsAg and HBV DNA levels in the mice. Importantly, the DAP-induced anti-HBV effect was impaired in the LSEC-depleted mice, which indicated that LSEC activation and T cell recruitment into the liver were essential for the antiviral function mediated by DAP application. Taken together, the results showed that the Ag-presenting ability of LSECs was enhanced by DAP application, which resulted in enhanced T cell responses and inhibited HBV replication in a mouse model.
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    In recent studies, UFL1 (ubiquitin-like modifier 1 ligating enzyme 1) has been identified as a significant regulator of NF- κ B signaling and cellular stress response, yet its physiological function in LPS-stimulated bovine mammary epithelial cells (BMECs) remains unknown. In this study, we investigated the modulating effect of UFL1 on the regulation of LPS-induced inflammation and cell damage, with a focus on apoptosis, ER stress, autophagy, oxidative stress, and the TLR4/NF- κ B signaling pathway. The results showed that UFL1 depletion aggravated the LPS-induced inflammatory response and cell damage by positively regulating the TLR4/NF- κ B pathway (increased the expression of TLR4, NF- κ B P65 in nuclear, and phospho-I κ B α ), exacerbating LPS-induced ER stress (increased the expression of CHOP, Hsp70, and GRP78), apoptosis (increased the expression of Bax/Bcl-2 and activity of caspase-3), autophagy (increased LC3-II and decreased P62 expression), and oxidative stress (decreased SOD and CAT levels and increased MDA levels). Overexpression of UFL1 suppressed the activation of the TLR4/NF- κ B pathway and relieved the LPS-induced ER stress, apoptosis, autophagy, and oxidative stress, thereby alleviating the inflammatory response and cell damage. Collectively, UFL1 may play an important role during the inflammatory response and thereby acts as a potential therapeutic target for bovine mastitis.
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  • Article
    Sensitisation of the respiratory tract to chemicals resulting in respiratory allergy and allergic asthma is an important occupational health problem, and presents toxicologists with no shortage of challenges. A major issue is that there are no validated or, even widely recognised, methods available for the identification and characterisation of chemical respiratory allergens, or for distinguishing respiratory allergens from contact allergens. The first objective here has been review what is known (and what is not known) of the mechanisms through which chemicals induce sensitisation of the respiratory tract, and to use this information to construct a hybrid Adverse Outcome Pathway (AOP) that combines consideration of both skin and respiratory sensitisation. The intention then has been to use the construction of this hybrid AOP to identify areas of commonality/confluence, and areas of departure/divergence, between skin sensitisation and sensitisation of the respiratory tract. The hybrid AOP not only provides a mechanistic understanding of how the processes of skin and respiratory sensitisation differ, buy also a means of identifying areas of uncertainty about chemical respiratory allergy that benefit from a further investment in research.
  • Chapter
    The outcomes of two centuries of repeated failed cancer research and therapy show that cancer has been made as an imaginary problem, a black box, not to be solved by the decision makers in the medical/cancer establishment for huge profit. In this chapter the ‘Other Side of Political Darkness’ in cancer science will be discussed by attempting to systematically analyze and integrate available data and demonstrate that growth of cancer cells is the results of severe, aggressive, and progressive aggregation of immune response alterations (immune tsunami or cancer tsunami) or accumulation of delayed hypersensitivity responses in tissues. Cancer cell growth is proposed to initiate as ‘mild’ (often sub-clinical) altered immune dynamics (immune disorder) and progress innately, genetically or be induced primarily in immune-responsive tissues. Severe altered immune-responses could also damage the immune-privileged tissues by shifting the architectural integrities and barriers of these oxidative-sensitive tissues to behave as immune-responsive and promote growth.
  • Article
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    Background: The expression and localization of transforming growth factor-beta (TGF-β) pathway proteins in different compartments of the lower airways of stable COPD patients is unclear. Objective: To determine TGF-β pathway protein expression in patients with stable COPD. Methods: The expression and localization of TGF-β pathway components was measured in the bronchial mucosa and peripheral lung of patients with stable COPD (n=44), control smokers with normal lung function (n=24) and control non-smoking subjects (n=11) using immunohistochemistry. Results: TGF-β1, TGF-β3 and CCN2 expression were significantly decreased in the bronchiolar epithelium, with TGF-β1 also decreased in alveolar macrophages, of stable COPD patients compared to control smokers with normal lung function. TGF-β3 expression was increased in the bronchial lamina propria of both control smokers with normal lung function and mild/moderate stable COPD compared to control non-smokers and correlated significantly with pack-years. However, TGF-β3+ cells decreased in severe/very severe COPD compared to control smokers. LTBP-1 expression was increased in the bronchial lamina propria in stable COPD of all severities compared with control smokers with normal lung function. BAMBI expression in the bronchial mucosa was significantly increased in stable COPD of all severities compared to control subjects. No other significant differences were observed between groups for all the other molecules studied either in the bronchial mucosa and peripheral lung. Conclusion: Expression of TGF-βs and their regulatory proteins is distinct within different lower airway compartments in stable COPD. Selective reduction in TGF-β1 and enhanced BAMBI expression may be associated with the increase in autoimmunity in COPD.
  • Chapter
    Many systemic diseases are featured by muscle atrophy. Cellular proteins are modified by covalent attachment to a small protein known as ubiquitin (Ub) through ubiquitination. This ubiquitination process serves as signal for protein turnover that leads to rapid muscle mass lack. This process is carried out through an enzymatic cascade, which includes three groups of enzymes termed ubiquitin E1 (activating enzyme), ubiquitin E2 (conjugating enzyme), and ubiquitin E3 (ligase). There are several ways of ubiquitin conjugation driving to ubiquitination of specific proteins through ubiquitin-proteasome system (UPS). A lot of UPS genes stated to be included in skeletal muscle atrophy. These genes do their effects by modifying different processes which affect muscle mass including myofibrillar protein degradation, myogenesis inhibition, and even modulation of autophagy as well as upstream regulatory pathways.
  • Article
    Background Treatment of active ulcerative colitis is associated with incomplete efficacy, adverse events, and loss of response. Toll-like receptor-9 mediates innate and adaptive immune response toward intestinal microorganisms. The oral synthetic oligonucleotide toll-like receptor-9 modulator has demonstrated anti-inflammatory properties in colitis murine models and a satisfactory safety profile in humans. AimTo evaluate the efficacy and safety of BL-7040 (a Toll-like receptor-9 modulator) in patients with moderately active ulcerative colitis. Methods Moderately active ulcerative colitis patients were included in this multicenter, open-label phase IIa trial. Concomitant mesalamine and steroids at a stable dose were allowed. Clinical outcome was evaluated using the Mayo score, histology, and mucosal cytokine levels. Side effects were registered. ResultsSixteen out of 22 patients completed a 5-week treatment course and 2-week follow-up. Six patients discontinued the study, three of them due to adverse events. Clinical remission was observed in two patients (12.5 %), and clinical response as well as mucosal healing were achieved in half (50 %) of the patients, while all others remained stable. Furthermore, mucosal neutrophil (p = 0.002) and mucosal interleukin-6 levels (p = 0.046) were significantly reduced in responders compared to non-responders. Toll-like receptor-9 was well tolerated with only one unrelated to study drug serious adverse event (hemoglobin decrease) and 29 mild-to-moderate adverse events. Conclusions Oral administration of the Toll-like receptor-9 agonist BL-7040 appeared efficacious, safe and well tolerated in patients with moderately active ulcerative colitis.
Literature Review
  • Article
    Precise regulation of major histocompatibility complex class II (MHC-II) gene expression plays a crucial role in the control of the immune response. A major breakthrough in the elucidation of the molecular mechanisms involved in MHC-II regulation has recently come from the study of patients that suffer from a primary immunodeficiency resulting from regulatory defects in MHC-II expression. A genetic complementation cloning approach has led to the isolation of CIITA and RFX5, two essential MHC-II gene transactivators. CIITA and RFX5 are mutated in these patients, and the wild-type genes are capable of correcting their defect in MHC-II expression. The identification of these regulatory factors has furthered our understanding of the molecular mechanisms that regulate MHC-II genes. CIITA was found to be a non-DNA binding transactivator that functions as a molecular switch controlling both constitutive and inducible MHC-II expression. The finding that RFX5 is a subunit of the nuclear RFX-complex has confirmed that a deficiency in the binding of this complex is indeed the molecular basis for MHC-II deficiency in the majority of patients. Furthermore, the study of RFX has demonstrated that MHC-II promoter activity is dependent on the binding of higher-order complexes that are formed by highly specific cooperative binding interactions between certain MHC-II promoter-binding proteins. Two of these proteins belong to families of which the other members, although capable of binding to the same DNA motifs, are probably not directly involved in the control of MHC-II expression. Finally, the facts that CIITA and RFX5 are both essential and highly specific for MHC-II genes make possible novel strategies designed to achieve immunomodulation via transcriptional intervention.
  • Article
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    NOD-like receptors (NLRs) are a family of intracellular sensors of microbial- or danger-associated molecular patterns. Here, we report the identification of NLRX1, which is a new member of the NLR family that localizes to the mitochondria. NLRX1 alone failed to trigger most of the common signalling pathways, including nuclear factor-κB (NF)-κB- and type I interferon-dependent cascades, but could potently trigger the generation of reactive oxygen species (ROS). Importantly, NLRX1 synergistically potentiated ROS production induced by tumour necrosis factor α, Shigella infection and double-stranded RNA, resulting in amplified NF-κB-dependent and JUN amino-terminal kinases-dependent signalling. Together, these results identify NLRX1 as a NLR that contributes to the link between ROS generation at the mitochondria and innate immune responses.
  • Article
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    Originally published as Nature 401, 811–815; 1999Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses1. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors2, 3; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence4, 5, 6. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria7, 8, 9, 10, 11. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.
  • Article
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    Several cytokines are involved in the complex processes ultimately leading to autoimmune diseases. In a preceding review, we have already discussed the role of the IL-12 and -17 families of cytokines. This review is focused on IL-15 and -18. Both these molecules have pro-inflammatory activity and act on many cell types and because of their broad spectrum of activity they play an important role in autoimmunity and disease pathogenesis. Their biological activity is ultimately regulated by the signalling cascades set into motion within their target cells. In this second review, we will, once again, describe the signal transduction pathways activated by these two cytokines and focus on how this relates to the pathogenesis of autoimmune diseases. We will also describe some of the therapeutic approaches that are being investigated to curtail the pro-inflammatory activities of these two molecules.
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  • Article
    Background: The first major Crohn's disease (CD) susceptibility gene, NOD2, implicates the innate intestinal immune system and other pattern recognition receptors in the pathogenesis of this chronic, debilitating disorder. These include the Toll-like receptors, specifically TLR4 and TLR5. A variant in the TLR4 gene (A299G) has demonstrated variable association with CD. We aimed to investigate the relationship between TLR4 A299G and TLR5 N392ST, and an Australian inflammatory bowel disease cohort, and to explore the strength of association between TLR4 A299G and CD using global meta-analysis. Methods: Cases (CD = 619, ulcerative colitis = 300) and controls (n = 360) were genotyped for TLR4 A299G, TLR5 N392ST, and the 4 major NOD2 mutations. Data were interrogated for case-control analysis prior to and after stratification by NOD2 genotype. Genotype-phenotype relationships were also sought. Meta-analysis was conducted via RevMan. Results: The TLR4 A299G variant allele showed a significant association with CD compared to controls (P = 0.04) and a novel NOD2 haplotype was identified which strengthened this (P = 0.003). Furthermore, we identified that TLR4 A299G was associated with CD limited to the colon (P = 0.02). In the presence of the novel NOD2 haplotype, TLR4 A299G was more strongly associated with colonic disease (P < 0.001) and nonstricturing disease (P = 0.009). A meta-analysis of 11 CD cohorts identified a 1.5-fold increase in risk for the variant TLR4 A299G allele (P < 0.00001). Conclusions: TLR 4 A299G appears to be a significant risk factor for CD, in particular colonic, nonstricturing disease. Furthermore, we identified a novel NOD2 haplotype that strengthens the relationship between TLR4 A299G and these phenotypes.
  • Article
    Background: Mimetics of bacterial DNA, given orally or subcutaneously, protect mice from experimental colitis via a toll-like receptor (TLR)-9-dependent mechanism. The goal of the study was to define whether synthetic viral RNA, polyinosinic acid:cytidylic acid [poly(I:C)], which is also a potent immunomodulator, might also affect murine colitis and, if so, define whether such effects were mediated by TLR3, which is one of at least 4 known receptors for this viral RNA analog. Methods: Mice (C57BL6, IL-10KO, or TLR3 KO) were administered 1.5% dextran sodium sulfate (DSS) in drinking water for 7 days. Two hours before treatment with DSS, mice were given phosphate-buffered saline (PBS) or poly(I:C) 20 mug subcutaneously (s.c.), or 100 mug intragastrically (i.g.). Results: In wildtype mice s.c. administration of poly(I:C) dramatically protected against DSS-induced colitis as assessed by every parameter analyzed, which included body weight, rectal bleeding, colonic myeloperoxidase, histopathology, serum keratinocyte-derived chemokine, serum amyloid A, and lipocalin-2. In contrast, i.g. administration of poly(I:C) offered no protection in this colitis model nor did its administration activate the innate immune system as assessed by serologic parameters. Subcutaneous poly(I:C) protected against DSS-induced colitis equally well in C57BL6 and IL-10KO mice, indicating that this antiinflammatory cytokine is not required for such protection. Protection against colitis given by poly(I:C) treatment was ablated in TLR3 KO, indicating that the protective action of this viral RNA analog was mediated by this receptor. Conclusions: Activation of TLR3 on cells that are accessible by systemic, but not oral, administration of synthetic viral RNA results in protection against the acute inflammation that can ensue upon damage of the gut epithelium. Thus, this viral RNA analog, which is under clinical trials for other inflammatory disorders (e.g., lupus), may also have therapeutic value for inflammatory bowel disease.
  • Article
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    Sudden exposure of human populations to chemicals, pathogens, or radiation has the potential to result in substantial morbidity. A potential means of rapidly protecting such populations might be to activate innate host defense pathways, which can provide broad protection against a variety of insults. However, innate immune activators can, by themselves, result in severe inflammatory pathology, which in large part is driven by hemopoietic-derived cytokines such as TNF-alpha. We reasoned that, because it preferentially activates epithelial cells, the TLR5 agonist flagellin might not induce severe inflammatory pathology and yet be an ideal agent to provide such non-specific protection, particularly at the mucosal surfaces that serve as a front line of host defense. In accordance, we observed that systemic treatment of mice with purified flagellin did not induce the serologic, histopathologic, and clinical hallmarks of inflammation that are induced by LPS but yet protected mice against chemicals, pathogens, and ionizing radiation. Flagellin-elicited radioprotection required TLR5, the TLR signaling adaptor MyD88, and was effective if given between 2 h before to 4 h after exposure to irradiation. Flagellin-elicited radioprotection was, in part, mediated via effects on cells in bone marrow but yet rescued mortality without a pronounced rescue of radiation-induced anemia or leukopenia. Thus, systemic administration of flagellin may be a relatively safe means of providing temporary non-specific protection against a variety of challenges.
  • Article
    Familial cold autoinflammatory syndrome (FCAS) is an autosomal dominant disorder characterised by recurrent episodes of rash, arthralgia, and fever after cold exposure. The genetic basis of this disease has been elucidated. Cryopyrin, the protein that is altered in FCAS, is one of the adaptor proteins that activate caspase 1, resulting in release of interleukin 1. An experimental cold challenge protocol was developed to study the acute inflammatory mechanisms occurring after a general cold exposure in FCAS patients and to investigate the effects of pretreatment with an antagonist of interleukin 1 receptor (IL-1Ra). ELISA, real-time PCR, and immunohistochemistry were used to measure cytokine responses. After cold challenge, untreated patients with FCAS developed rash, fever, and arthralgias within 1-4 h. Significant increases in serum concentrations of interleukin 6 and white-blood-cell counts were seen 4-8 h after cold challenge. Serum concentrations of interleukin 1 and cytokine mRNA in peripheral-blood leucocytes were not raised, but amounts of interleukin 1 protein and mRNA were high in affected skin. IL-1Ra administered before cold challenge blocked symptoms and increases in white-blood-cell counts and serum interleukin 6. The ability of IL-1Ra to prevent the clinical features and haematological and biochemical changes in patients with FCAS indicates a central role for interleukin 1beta in this disorder. Involvement of cryopyrin in activation of caspase 1 and NF-kappaB signalling suggests that it might have a role in many chronic inflammatory diseases. These findings support a new therapy for a disorder with no previously known acceptable treatment. They also offer insights into the role of interleukin 1beta in more common inflammatory diseases.
  • Article
    Chronic obstructive pulmonary disease (COPD) is a global health problem. Being a progressive disease characterized by inflammation, it deteriorates pulmonary functioning. Research has focused on airway inflammation, oxidative stress, and remodelling of the airways. Macrophages, neutrophils and T cells are thought to be important key players. A number of new research topics received special attention in the last years. The combined use of inhaled corticosteroids and long-acting β2-adrenoceptor agonists produces better control of symptoms and lung function than that of the use of either compound alone. Furthermore, collagen breakdown products might be involved in the recruitment and activation of inflammatory cells by which the process of airway remodelling becomes self-sustaining. Also, TLR (Toll-like receptor)-based signalling pathways seem to be involved in the pathogenesis of COPD. These new findings may lead to new therapeutic strategies to stop the process of inflammation and self-destruction in the airways of COPD patients.
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  • Article
    Ketamine may affect the host immunity. Interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) are pivotal cytokines produced by macrophages. This study aimed to evaluate the effects of ketamine on the regulation of inflammatory cytokine gene expression, especially IL-1 beta, in lipopolysaccharide (LPS)-activated murine macrophage-like Raw 264.7 cells and its possible signal-transducing mechanisms. Administration of Raw 264.7 cells with a therapeutic concentration of ketamine (100 microM), LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. Exposure to 100 microM ketamine decreased the binding affinity of LPS and LPS-binding protein but did not affect LPS-induced RNA and protein synthesis of TLR4. Treatment with LPS significantly increased IL-1 beta, IL-6, and TNF-alpha gene expressions in Raw 264.7 cells. Ketamine at a clinically relevant concentration did not affect the synthesis of these inflammatory cytokines, but significantly decreased LPS-caused increases in these cytokines. Immunoblot analyses, an electrophoretic mobility shift assay, and a reporter luciferase activity assay revealed that ketamine significantly decreased LPS-induced translocation and DNA binding activity of nuclear factor-kappa B (NF kappaB). Administration of LPS sequentially increased the phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK. However, a therapeutic concentration of ketamine alleviated such augmentations. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA reduced cellular TLR4 amounts and ameliorated LPS-induced RAS activation and IL-1 beta synthesis. Co-treatment with ketamine and TLR4 siRNA synergistically ameliorated LPS-caused enhancement of IL-1 beta production. Results of this study show that a therapeutic concentration of ketamine can inhibit gene expression of IL-1 beta possibly through suppressing TLR4-mediated signal-transducing phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK and subsequent translocation and transactivation of NF kappaB.
  • Article
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    Recent data suggest functional interactions between NOD2 and other receptors of the innate immune system modulating inflammatory responses. Here we analyzed the role of Toll-like receptor 9 (TLR-9) gene variants with respect to susceptibility to inflammatory bowel disease (IBD) and tested for genetic interactions with NOD2 and other susceptibility genes for Crohn's disease (CD). The single-nucleotide polymorphisms (SNPs) -1237T/C (rs5743836) and 2848A/G (rs352140=p.Pro545Pro) in TLR9, the main CD-associated variants within the genes for NOD2, IL23R, ATG16L1, and variants in the IBD5 locus and in the DLG5 gene were assessed in 956 patients with IBD (606 CD and 350 ulcerative colitis) and in 792 healthy controls. The associations with disease susceptibility and phenotype, and epistatic gene-gene interactions, were analyzed. The TLR9 -1237T/C polymorphism showed significant interactions with NOD2 mutations. The frequency of -1237C was significantly higher in CD patients with at least one NOD2 mutation (P=0.004 vs. controls, odds ratio (OR) 1.60, 95% confidence interval (CI) (1.15-2.21)) and further increased in CD patients with two mutated NOD2 alleles (P=0.002 vs. controls, OR 2.37, 95% CI (1.35-4.15)). Significant gene-gene interactions were also observed for the TLR9 polymorphism -1237T/C with IL23R variants (most significantly with rs1004819, P=0.0007), with a particular high frequency of -1237C in CD patients carrying CD-protective IL23R variants. Epistatic interactions of the TLR9 -1237T/C SNP were also noted with the DLG5 113G/A variant (P=0.0007). Our results provide evidence for genetic interactions between polymorphisms in TLR9 and CD-associated variants in NOD2, IL23R, and DLG5, differentially modulating CD susceptibility.
  • Article
    In many patients with atopic dermatitis (AD), the disease is complicated by their enhanced susceptibility to bacterial skin infections, especially with Staphylococcus aureus. The pattern recognition receptor toll-like receptor (TLR)-2 recognizes components of S. aureus, for example, lipoteichoic acid (LTA) and peptidoglycan (PGN) and, therefore, might be crucial in the pathogenesis and flare-ups of AD. To investigate TLR-2 expression and cytokine secretion in macrophages from patients with AD compared to healthy controls upon TLR-2 stimulation with PGN, LTA and Pam3Cys. Macrophages were cultivated from highly purified peripheral blood monocytes of AD patients and nonatopic healthy controls and stimulated with PGN, LTA and Pam3Cys in a time and dose-dependent manner. Afterwards, TLR-2 expression and cytokine secretion were measured on protein and mRNA level. TLR-1 and TLR-6 expression were investigated on the mRNA level. Immunohistochemical stainings from punch biopsies were performed to investigate TLR-2 expression in skin macrophages. We could clearly show that macrophages from patients with AD expressed significantly less TLR-2, whereas the expression pattern of TLR-1 and TLR-6 were not altered. Macrophages had a reduced capacity to produce pro-inflammatory cytokines such as IL-6, IL-8 and IL-1beta after stimulation with TLR-2 ligands. Our findings clearly show an impaired TLR-2 expression and functional differences of TLR-2-mediated effects on macrophages of AD patients compared to healthy controls which might contribute to the enhanced susceptibility to skin infections with S. aureus in AD.
  • Article
    Allergic asthma is a complicated genetic disorder caused by interaction of the acquired and innate immune responses. Acquired immune responses to protein antigens could induce type 2 T lymphocyte-driven responses and result in atopic asthma. Recent studies demonstrated that endotoxin, LPS and air pollution-induced innate immunity induce asthma through Toll-like receptors (TLR). However, the definite mechanism of LPS-induced asthma is still not known. Here, we investigated the effects of different doses of LPS in a mouse model of allergic asthma to define the molecular mechanism of LPS-induced asthma. We found that low doses of LPS in OVA induced significant inflammatory infiltration in lung tissue of asthmatic mice. Histologic studies demonstrated that lungs of these asthmatic mice were characterized by the recruitment of both eosinophils and neutrophils, increased airway mucus secretion and the elevated levels of Th2 cytokines. A high dose of LPS in OVA can induce a Th1 associated response, histologically characterized by neutrophil recruitment, the absence of airway mucus secretion and an increase of IFN-gamma production. Regardless of high or low dose of LPS, TLR4 in alveolar macrophages (AM) was up-regulated in lungs of asthmatic mice. Our data demonstrated that the dose of LPS exposure determines the type of inflammatory response and a low dose of LPS together with OVA augments the antigen-induced lung inflammation in asthma. This study demonstrates that the TLR4 signaling pathway plays a vital role in the development of asthma and indicates the tight connection between endotoxin exposure and asthma prevalence in the clinic.
  • Article
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    Dysregulated innate immune responses to commensal bacteria contribute to the development of inflammatory bowel disease (IBD). TLR4 is overexpressed in the intestinal mucosa of IBD patients and may contribute to uncontrolled inflammation. However, TLR4 is also an important mediator of intestinal repair. The aim of this study is to examine the effect of a TLR4 antagonist on inflammation and intestinal repair in two murine models of IBD. Colitis was induced in C57BL/6J mice with dextran sodium sulfate (DSS) or by transferring CD45Rb(hi) T cells into RAG1-/- mice. An antibody (Ab) against the TLR4/MD-2 complex or isotype control Ab was administered intraperitoneally during DSS treatment, recovery from DSS colitis, or induction of colitis in RAG1-/- mice. Colitis severity was assessed by disease activity index (DAI) and histology. The effect of the Ab on the inflammatory infiltrate was determined by cell isolation and immunohistochemistry. Mucosal expression of inflammatory mediators was analyzed by real-time PCR and ELISA. Blocking TLR4 at the beginning of DSS administration delayed the development of colitis with significantly lower DAI scores. Anti-TLR4 Ab treatment decreased macrophage and dendritic cell infiltrate and reduced mucosal expression of CCL2, CCL20, TNF-alpha, and IL-6. Anti-TLR4 Ab treatment during recovery from DSS colitis resulted in defective mucosal healing with lower expression of COX-2, PGE(2), and amphiregulin. In contrast, TLR4 blockade had minimal efficacy in ameliorating inflammation in the adoptive transfer model of chronic colitis. Our findings suggest that anti-TLR4 therapy may decrease inflammation in IBD but may also interfere with colonic mucosal healing.
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    Goblet cells (GC) facilitate mucosal protection and epithelial barrier repair, yet the innate immune mechanisms that selectively drive GC functions have not been defined. The aim of this study was to determine whether Toll-like receptor (TLR) 2 and modulation of GC-derived trefoil factor (TFF) 3 are functionally linked in the intestine. GC modulation was assessed using quantitative real-time polymerase chain reaction analysis (qRT-PCR), Western blotting, and confocal microscopy. Dextran sulfate sodium (DSS) colitis was induced in wild-type, TFF3(-/-), and TLR2(-/-) mice. Recombinant TLR2 ligand or TFF3 peptide were orally administered after DSS termination. Caco-2 cells overexpressing full-length TLR2 or mutant TLR2-R753Q were tested for TFF3 synthesis and functional-related effects in a wounding assay. Data from in vitro (Ls174T) and ex vivo models of murine and human GC reveal that TLR2 activation selectively induces synthesis of TFF3. In vivo studies using TFF3(-/-) or TLR2(-/-) mice demonstrate the ability for oral treatment with a TLR2 agonist to confer antiapoptotic protection of the intestinal mucosa against inflammatory stress-induced damage through TFF3. Recombinant TFF3 rescues TLR2-deficient mice from increased morbidity and mortality during acute colonic injury. Severe ulcerative colitis (UC) has recently been found to be associated with the R753Q polymorphism of the TLR2 gene. The relevance of the observed functional effect of TLR2 in regulating GC is confirmed by the finding that the UC-associated TLR2-R753Q variant is functionally deficient in the ability to induce TFF3 synthesis, thus leading to impaired wound healing. These data demonstrate a novel function of TLR2 in intestinal GC that links products of commensal bacteria to innate immune protection of the host via TFF3.
  • Article
    Therapeutic vaccination against cancer-associated antigens represents an attractive option for cancer therapy in view of its potential efficacy, the possibility of long-lasting immunity against the cancer, and safety profile. Cancer patients are however usually immunosuppressed and most cancer-associated antigens are 'self-antigens', making it a significant challenge to vaccinate patients against a cancer-associated antigen. Various immunostimulant adjuvants are therefore under investigation in an effort to boost the immune system to overcome the tolerance to tumour associated self-antigens and the ambient immunosuppressory influence of cytokines and regulatory T-cells (Tregs). Many adjuvants appear to be specific ligands for toll-like receptors (TLR) which are potent activators of innate immune responses [Kwissa M, Kasturi SP, Pulendran B. Expert Rev Vaccines. The Science of Adjuvants 2007 6(October(5)):673-84] [1], activating dendritic cell (DC) maturation and inflammatory cytokine secretion, inducing an increase in the cross talk between the innate and adaptive immune systems, and thereby driving the expansion of CTLs that can destroy cancer cells. However, recently some TLR agonists such as CpG have been shown to generate parallel immunosuppressive and inflammatory responses in innate immune cells capable of induction and expansion of Tregs [Conroy H, Marshall NA, Mills KH. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 2008 27(January 7(2)):168-80 [2]; Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 2008;27(2):218-24] [3]. In this context it is noteworthy that poly(I:C), a synthetic double-stranded RNA polymer and a TLR3 agonist, has been shown in mouse models to be capable of enhancing the T cell response to nonimmunogenic self-antigen linked dendritic cell based vaccine strategy, and stimulating diabetic insulitis in the presence of Tregs [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65-72] [4]. Ampligen poly(I:C(12)U) is a GMP-grade synthetic analogue of poly(I:C) and therefore suitable for human use. Its effects in a preclinical setting have been recently tested to be similar to poly(I:C) [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65-72]. In particular, it potently induces in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. In addition human monocyte derived DC primed with tumour lysate and matured with Ampligen are capable of generating Th1 specific anti-cancer responses in peripheral blood T-cells derived from cancer patients in the presence of ascites medium containing immunosuppressory cytokines. In this review the key findings are summarised and discussed with a view to offering a rationale for the use of Ampligen as a potentially safe adjuvant capable of overcoming the tumour-related immune tolerance mechanisms in a clinical setting.
  • Article
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    We used a candidate gene approach to identify a set of SNPs, located in a predicted regulatory region on chromosome 1q44 downstream of NLRP3 (previously known as CIAS1 and NALP3) that are associated with Crohn's disease. The associations were consistently replicated in four sample sets from individuals of European descent. In the combined analysis of all samples (710 father-mother-child trios, 239 cases and 107 controls), these SNPs were strongly associated with risk of Crohn's disease (P(combined) = 3.49 x 10(-9), odds ratio = 1.78, confidence interval = 1.47-2.16 for rs10733113), reaching a level consistent with the stringent significance thresholds imposed by whole-genome association studies. In addition, we observed significant associations between SNPs in the associated regions and NLRP3 expression and IL-1beta production. Mutations in NLRP3 are known to be responsible for three rare autoinflammatory disorders. These results suggest that the NLRP3 region is also implicated in the susceptibility of more common inflammatory diseases such as Crohn's disease.
  • Article
    Both genetic predispositions and environmental factors contribute to the development of autoimmunity. Toll-like receptors (TLR) are a family of pattern recognition receptors (PRRs), and their stimulus by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) is an important prerequisite for the induction of various autoimmune diseases. However, activation of specific TLRs can not only induce but also inhibit autoimmune diseases in certain mouse models. The contribution of individual TLRs to the induction of autoimmunity or tolerance involves hematopoietic as well as nonhematopoietic cells expressing combinations of different TLRs. The intercellular and intracellular orchestration of signals from different TLRs, other PRRs, and membrane-standing receptors dictates activating or inhibitory responses. Here, we summarize TLR-dependent tolerance mechanisms in B cells and intestinal epithelial cells and TLR-mediated activation mechanisms leading to the induction of Th17 T cell differentiation in different autoimmune diseases and in inflammatory bowel diseases. Understanding the opposing mechanisms of TLRs for the induction and suppression of autoimmune processes in specific diseases will help to develop novel therapies to treat autoimmunity.
  • Article
    Background We investigated the expression profile of toll-like receptors (TLRs) and TLR ligand-activated production profile of asthma-related inflammatory cytokines in asthmatic patients. The expression of TLR1–8 on monocytes, CD4+ T helper lymphocytes, CD8+ T cytotoxic lymphocytes, CD19+ B lymphocytes, and dendritic cells, and ex vivo production of cytokines from peripheral blood mononuclear cells activated by TLR ligands were measured by flow cytometry. Discussion Ex vivo productions of TNF-α, IL-10, and IL-1β by TLR4 and TLR5 ligand LPS and flagellin were significantly lower in asthmatic patients (all P < 0.05). Expression of TLR4 and TLR5 was also found to be significantly lower in asthmatic patients when compared to that of control subjects (all P < 0.05). Therefore, the decreased activation of TLR4 and TLR5 in asthmatic patients might contribute to the immunopathological mechanisms of asthma by reducing the release of Th1 and anti-inflammatory cytokines.
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    Toll like receptors (TLRs) are a family of conserved pattern recognition receptors that recognizes specific microbial patterns and allow the cell to distinguish between self and non-self materials. The very property of the TLRs to link innate and adaptive immunity offers a novel prospect to develop vaccines engaging TLR signaling. The presence of TLR ligands as adjuvant in conjunction with the vaccine is shown to increase the efficacy and response to the immunization with a particular antigen. For infectious as well as for noninfectious diseases, TLR activation have been used in both established and experimental vaccines. The choice of the TLR agonist to be used, the subsequent efficacy and the safety profile of the vaccine is thus a crucial step in vaccine development. Recent studies also suggest the involvement of other non-TLR immune receptors to control vaccine immunogenicity. Here we focus on the findings dealing with TLR ligands as adjuvant and discuss the importance of these studies to develop an optimal vaccine.
  • Article
    The anti-phospholipid syndrome (APS) is a systemic autoimmune disease characterized by an adaptive immune response against self phospholipid (PL)--binding proteins. Although APS is considered as an autoantibody-mediated disease, there is now evidence that anti-phospholipid antibodies (aPL) are necessary but not sufficient to trigger some of the clinical manifestations of the syndrome. For example, mediators of the innate immunity are recognized to be additional second hits able to induce the thrombotic events in the presence of aPL. Finally, environmental agents - in particular infectious ones - were reported to act as triggers for the production of autoantibodies cross-reacting with PL-binding proteins as well as inflammatory stimuli that potentiate the aPL thrombogenic effect. Altogether, these findings suggest a role for the innate immunity in APS pathogenesis. Toll-like receptors (TLR) are receptors that induce prompt inflammatory responses and mediate functional activation in immune effector cells. There is evidence that aPL, and in particular anti-beta(2) glycoprotein I (beta(2)GPI) antibodies, may activate endothelial cells and monocytes through TLR-4-dependent signalling. Whether or not TLR may behave as surface receptors for beta(2)GPI is still matter of research. Drugs or molecules able to interfere with TLR involvement may represent new therapeutic approaches for APS.
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    Graves' disease (GD) is a systemic autoimmune disease which targets the thyroid, orbit, and skin. Thyroid-associated ophthalmopathy (TAO) refers specifically to the orbital and periorbital manifestations of GD. Several important concepts have emerged from our enhanced understanding of the molecular mechanisms of the disease. Considerable debate remains concerning the specific identity and roles of inflammatory T-cell subsets, soluble and contact-mediated signalling, and autoantigens driving TAO. However B and T lymphocytes appear central in the process through production of disease mediators including activating autoantibodies to the thyrotropin receptor and insulin-like growth factor-1 receptor; cytokines including IL-1beta, IL-6, and IL-16; and chemokines including RANTES. Many of these molecules appear central to the inflammation, accumulation of extracellular matrix macromolecules, and fibrosis in the disease. Novel therapeutics targeting other autoimmune diseases may provide an opportunity for disrupting disease pathogenesis. It is imperative that agents targeting B-and T-cell functions be further evaluated in the treatment of aggressive forms of TAO utilizing multicenter clinical trials that allow adequate statistical power and sample size.
  • Article
    The body's ability to keep a steady homeostatic state is crucial to health and life. This involves providing an adequate response to a variety of challenges both physical and mental, such as microbial invasion and emotional distress. Interplay between the neuroendocrine and immune systems is essential in either case. Studies have demonstrated that toll-like receptors, or TLRs, play a regulatory role in both systems, and have been proposed as a possible link between the immune, hormonal and metabolic systems. As part of the innate immune system, these receptors control the identification by the body of microbial invaders and its immediate reaction in immune and inflammatory response. What are referred to as pattern recognition receptors are mostly expressed by cells involved in hematopoietic linkage, but an increasing number of studies have demonstrated their expression in other cell types such as neurons and endocrine cells on the hypothalamic-pituitary-adrenal (HPA) axis, thyrocytes, adipocytes and islets of Langerhans. Together with endocrine and metabolic dysregulation, immune system overreaction is often associated with infection and autoimmunity, clearly indicating TLR involvement at organ level which affects organ function. Several diseases such as autoimmune thyroid and pancreatic diseases, septic dysregulation of the HPA axis, diabetes and the metabolic syndrome have been linked to TLR activation and polymorphism. To gain insight into stress response and adaptation, we need to know more about TLRs and the specific physiological role they play in the endocrine and metabolic system and its processes.
  • Article
    Hereditary major histocompatibility complex (MHC) class II deficiency (or bare lymphocyte syndrome) is a form of severe primary immunodeficiency with a total lack of MHC class II expression. It is due to a defect in the regulation of MHC class II genes. A novel gene was isolated by complementation cloning, using an MHC class II-negative mutant cell line. This gene (CIITA) functions as a transactivator of MHC class II gene expression and restores expression of all MHC class II isotypes in mutant cells. In addition, CIITA fully corrects the MHC class II regulatory defect of cells from patients with bare lymphocyte syndrome. In this disease we have identified a splicing mutation that results in a 24 amino acid deletion in CIITA, resulting in loss of function of the transactivator. Hence, the CIITA gene is essential for MHC class II gene expression and has been shown to be responsible for hereditary MHC class II deficiency.
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    Background: CD14, the receptor for LPS, plays an important role in innate immunity. A polymorphism in the promotor for CD14, -159 C→T, has been implicated in atopy. Objective: We explored the relationship of this polymorphism with both atopic and nonatopic asthma, as well as with food allergy. Methods: Patients with asthma and food allergy were recruited along with nonatopic, nonasthmatic control subjects. The -159 C→T polymorphism was genotyped by using the PCR-based RFLP assay. Results: The -159 T allele was more common among patients with nonatopic asthma and food allergy than among control subjects (χ2 = 6.03, P = .01 and χ2 = 4.94; P = .03, respectively). Patients with food allergy had a 4-fold increased odds of having the TT genotype versus carriers of the C allele compared with control subjects (odds ratio [OR] = 3.9, 95% CI = 1.5-10.3), whereas patients with nonatopic asthma had a 3-fold increased odds of having the TT genotype (OR = 3.1 [95% CI = 1.1-9.1]). Controlling for sex differences between groups did not alter this relationship, which remained significant for patients with food allergy (OR = 3.7 [95% CI = 1.4-10.1]) or nonatopic asthma (OR = 2.7 [95% CI = 0.9-8.0]). We performed a stratified analysis, limited to white patients, to reduce population stratification. The relationship with the TT genotype was stronger in white patients with nonatopic asthma (OR = 4.4 [95% CI = 1.3-14.8]) and patients with food allergy (OR = 5.1 [95% CI = 1.6-16.2]), even adjusting for sex differences (OR = 3.9 [95% CI = 1.1-13.5] and OR = 4.6 [95% CI = 1.4-14.8], respectively). Conclusions: The TT genotype of -159 C→T CD14 is associated with nonatopic asthma and food allergy, particularly in white subjects. Thus CD14 is a candidate gene specifically for nonatopic asthma and not for asthma in general. This indicates that atopic and nonatopic asthma might be distinct conditions in their genetic predisposition, despite the fact that they are very similar once they have been established.
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    Interstitial fibrosis resulting from workplace exposure to asbestos and crystalline silica persists throughout the world despite knowledge of the causes and effective means for prevention. Asbestosis and silicosis occurrence is predictable among people overexposed to dusts in various industries and occupations such as mining, construction, manufacturing, and building maintenance. Asbestosis and silicosis are incurable and may be progressive even after dust exposure has ceased, therefore early recognition and supportive interventions are important. Although current disease is a result of past exposures, effective control of current workplace exposures is the only way to prevent continued occurrence of these potentially debilitating diseases. Physicians can contribute to this effort through accurate diagnosis and disease reporting.
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    The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning of a class of putative human receptors with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors--named TLRs 1-5--are probably the direct homologs of the fly molecule and, as such, could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role--akin to Toll in the dorsoventralization of the Drosophila embryo--as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence in situ hybridization and sequence-tagged site database analyses, we also show that the cognate Tlr genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors and MyD88 factors, and plant disease-resistance proteins recognizes a parallel beta/alpha fold with an acidic active site; a similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria.
  • Article
    To evaluate the efficacy and safety of interleukin-1 receptor antagonist (IL-1Ra) in patients with rheumatoid arthritis (RA). Patients with active and severe RA (disease duration <8 years) were recruited into a 24-week, double-blind, randomized, placebo-controlled, multicenter study. Doses of nonsteroidal antiinflammatory drugs and/or oral corticosteroids (< or =10 mg prednisolone daily) remained constant throughout the study. Any disease-modifying antirheumatic drugs that were being administered were discontinued at least 6 weeks prior to enrollment. Patients were randomized to 1 of 4 treatment groups: placebo or a single, self-administered subcutaneous injection of IL-1Ra at a daily dose of 30 mg, 75 mg, or 150 mg. A total of 472 patients were recruited. At enrollment, the mean age, sex ratio, disease duration, and percentage of patients with rheumatoid factor and erosions were similar in the 4 treatment groups. The clinical parameters of disease activity were similar in each treatment group and were consistent with active and severe RA. At 24 weeks, of the patients who received 150 mg/day IL-1Ra, 43% met the American College of Rheumatology criteria for response (the primary efficacy measure), 44% met the Paulus criteria, and statistically significant improvements were seen in the number of swollen joints, number of tender joints, investigator's assessment of disease activity, patient's assessment of disease activity, pain score on a visual analog scale, duration of morning stiffness, Health Assessment Questionnaire score, C-reactive protein level, and erythrocyte sedimentation rate. In addition, the rate of radiologic progression in the patients receiving IL-1Ra was significantly less than in the placebo group at 24 weeks, as evidenced by the Larsen score and the erosive joint count. IL-1Ra was well tolerated and no serious adverse events were observed. An injection-site reaction was the most frequently observed adverse event, and this resulted in a 5% rate of withdrawal from the study among those receiving IL-1Ra at 150 mg/day. This study confirmed both the efficacy and the safety of IL-1Ra in a large cohort of patients with active and severe RA. IL-1Ra is the first biologic agent to demonstrate a beneficial effect on the rate of joint erosion.
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    The life-threatening complications of sepsis in humans are elicited by infection with Gram-negative as well as Gram-positive bacteria. Recently, lipopolysaccharide (LPS), a major biologically active agent of Gram-negative bacteria, was shown to mediate cellular activation by a member of the human Toll-like receptor family, Toll-like receptor (TLR) 2. Here we investigate the mechanism of cellular activation by soluble peptidoglycan (sPGN) and lipoteichoic acid (LTA), main stimulatory components of Gram-positive bacteria. Like LPS, sPGN and LTA bind to the glycosylphosphatidylinositol-anchored membrane protein CD14 and induce activation of the transcription factor NF-κB in host cells like macrophages. We show that whole Gram-positive bacteria, sPGN and LTA induce the activation of NF-κB in HEK293 cells expressing TLR2 but not in cells expressing TLR1 or TLR4. The sPGN- and LTA-induced NF-κB activation was not inhibited by polymyxin B, an antibiotic that binds and neutralizes LPS. Coexpression together with membrane CD14 enhances sPGN signal transmission through TLR2. In contrast to LPS signaling, activation of TLR2 by sPGN and LTA does not require serum. These findings identify TLR2 as a signal transducer for sPGN and LTA in addition to LPS.
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    Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.
  • Article
    Toll-like receptor (TLR) 2 and TLR4 are implicated in the recognition of various bacterial cell wall components, such as lipopolysaccharide (LPS). To investigate in vivo roles of TLR2, we generated TLR2-deficient mice. In contrast to LPS unresponsiveness in TLR4-deficient mice, TLR2-deficient mice responded to LPS to the same extent as wild-type mice. TLR2-deficient macrophages were hyporesponsive to several Gram-positive bacterial cell walls as well as Staphylococcus aureus peptidoglycan. TLR4-deficient macrophages lacked the response to Gram-positive lipoteichoic acids. These results demonstrate that TLR2 and TLR4 recognize different bacterial cell wall components in vivo and TLR2 plays a major role in Gram-positive bacterial recognition.
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    Human heat shock protein 60 (hsp60) elicits a potent proinflammatory response in cells of the innate immune system and therefore has been proposed as a danger signal of stressed or damaged cells. We report here that macrophages of C3H/HeJ mice, carrying a mutant Toll-like-receptor (Tlr) 4 are nonresponsive to hsp60. Both the induction of TNF-alpha and NO formation were found dependent on a functional Tlr4 whereas stimulation of macrophages by CpG DNA was Tlr4 independent. We conclude that Tlr4 mediates hsp60 signaling. This is the first report of a putative endogenous ligand of the Tlr4 complex.
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    Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice but not in humans. Although Taxol is structurally unrelated to LPS, Taxol and LPS are presumed to share a receptor or signaling molecule. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS responsiveness on TLR4. To determine whether TLR4.MD-2 complex mediates a Taxol-induced signal, we constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion, and then examined whether Taxol induced NFkappaB activation in these transfectants. Noticeable NFkappaB activation by Taxol was detected in Ba/F3 expressing mouse TLR4 and mouse MD-2 but not in the other transfectants. Coexpression of human TLR4 and human MD-2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4. MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFkappaB activation via TLR4.MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFkappaB activation via TLR4.MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4.MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice.
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    Recent data suggest that IL-15 plays an important role in the pathogenesis of rheumatoid arthritis. In the present study, we hypothesized that elevated in the joints of rheumatoid arthritis, but not osteoarthritis, patients, IL-15 may exert its proinflammatory properties via the induction of IL-17, a cytokine known to stimulate synoviocytes to release several mediators of inflammation including IL-6, IL-8, GM-CSF and PGE2. To test this hypothesis, we first measured the levels of IL-17 and IL-15 using specific ELISA and found that synovial fluids of patients with rheumatoid arthritis, but not with osteoarthritis, contain high levels of these cytokines. A strong correlation between IL-15 and IL-17 levels in synovial fluids was observed. Among tested factors, LPS and TNF-alpha failed, IL-15 and IL-2 were equipotent, and PMA + ionomycin was far more efficient in the induction of IL-17 secretion by PBMCs isolated from healthy blood donors. Interestingly, synovial fluid cells, in contrast to PBMCs isolated from patients with rheumatoid arthritis, but not osteoarthritis, respond to PMA + ionomycin with much lower, comparable to IL-15-triggered IL-17 secretion. Moreover, PMA + ionomycin-triggered IL-17 secretion is completely or partially blocked in the presence of low doses of cyclosporin A or high doses of methylprednisolone, respectively. IL-15-triggered IL-17 secretion by PBMCs was completely inhibited by these drugs. Thus, our results suggest for the first time that IL-15 may represent a physiological trigger that via cyclosporin A and steroid sensitive pathways leads to the overproduction of IL-17 in the joints of rheumatoid arthritis patients.
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    The interleukin-1 (IL-1) receptor/Toll-like receptor (TLR) superfamily is a recently defined and expanding group of receptors that participate in host responses to injury and infection. The superfamily is defined by the Toll/IL-1 receptor (TIR) domain, which occurs in the cytosolic region of family members, and is further subdivided into two groups based on homology to either the Type I IL-1 receptor or Drosophila Toll receptor extracellular domain. The former group includes the receptor for the important Th1 cytokine IL-18, and T1/ST2, which may have a role in Th2 cell function. The latter group includes six mammalian TLRs, including TLR2 and TLR4, that largely mediate the host response to gram-positive and gram-negative bacteria, respectively. Whether bacterial products are actual ligands for TLRs, or whether they generate ligands via as yet unidentified pattern recognition receptors, has yet to be determined. Signaling pathways activated via the TIR domain trigger the activation of downstream kinases, and transcription factors such as NF-kappaB, and involve the adaptor protein MyD88, which itself contains a TIR domain.
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    Endotoxin (lipopolysaccharide (LPS), 100 ng/ml) and muramyl dipeptide (MDP 100 ng/ml), two immunomodulatory bacterial cell wall products, were incubated with human whole blood, and the expression of receptors involved in antigen presentation, costimulation, and cell activation was investigated by use of flow cytometry. On monocytes, LPS and MDP increased surface expression of human leukocyte antigen-DR (HLA-DR), CD18, CD54 (intercellular adhesion molecule-1, ICAM-1), and CD86 (B7-2). On lymphocytes, LPS but not MDP increased HLA-DR expression after 18 h. The expression of CD28, CD49d/CD29, and CD106 (vascular cell adhesion molecule-1, VCAM-1) remained unchanged on both monocytes and lymphocytes. The early increase (1-6 h) of CD18 and ICAM-1 expression led us to hypothesize that CD18-dependent costimulatory signals were involved in the later (6 h) increase of monocyte HLA-DR expression. However, blocking studies using monoclonal antibodies against CD18 (IB4, 15 microg/ml) demonstrated that the LPS- and MDP-induced increase of HLA-DR and ICAM-1 expression on monocytes was not mediated through CD18. LPS induced the expression of the early activation marker CD69 by a CD14-dependent but CD18-independent mechanism, whereas MDP did not induce CD69 expression. Analysis of leukocyte subsets demonstrated that CD4(+) T-cells, CD8(+) T-cell, CD19(+) B-cells, CD56(+) natural killer (NK)-cells, and CD14(+) monocytes increased the expression of CD69 after stimulation with LPS. Collectively, these data demonstrate a stronger immunomodulatory effect of LPS compared with MDP which may, in part, explain the established difference of toxicity between these two bacterial cell wall products.
  • Article
    Chronic arthritis is characterised by chronic joint inflammation and concurrent joint erosion and destruction. The inflammatory cytokine interleukin 1 (IL1) has been shown to be a key mediator in the autoimmune disease rheumatoid arthritis (RA). Interleukin 1 mediates bone resorption and cartilage destruction, but may not play as dominant a part in joint swelling and inflammation. Interleukin 1 receptor antagonist (IL1Ra) selectively inhibits the effects of IL1 by competing for the IL1 receptor on all surfaces of the synovium. In a randomised controlled trial in 472 patients with active disease, IL1Ra 30 mg/day, 75 mg/day or 150 mg/day given by subcutaneous injection significantly reduced the signs and symptoms of RA at 24 weeks. An American College of Rheumatology (ACR) 20% response was seen in 43% of the patients treated with 150 mg/day at 24 weeks. IL1Ra was well tolerated; injection site reactions were the most common adverse event. In another trial, in 419 patients with active RA treated concomitantly with methotrexate, there were ACR 20% responses after 24 weeks in 42% of the patients treated with 1 mg/kg/day by subcutaneous injection and in 35% of those treated with 2 mg/kg/day. I1Ra offers a unique selective, targeted mechanism of action to block the IL1 mediated effects of RA.
  • Article
    The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.
  • Article
    Initiation and perpetuation of the inflammatory intestinal responses in inflammatory bowel disease (IBD) may result from an exaggerated host defense reaction of the intestinal epithelium to endogenous lumenal bacterial flora. Intestinal epithelial cell lines constitutively express several functional Toll-like receptors (TLRs) which appear to be key regulators of the innate response system. The aim of this study was to characterize the expression pattern of TLR2, TLR3, TLR4, and TLR5 in primary intestinal epithelial cells from patients with IBD. Small intestinal and colonic biopsy specimens were collected from patients with IBD (Crohn's disease [CD], ulcerative colitis [UC]) and controls. Non-IBD specimens were assessed by immunofluorescence histochemistry using polyclonal antibodies specific for TLR2, TLR3, TLR4, and TLR5. Primary intestinal epithelial cells (IEC) of normal mucosa constitutively expressed TLR3 and TLR5, while TLR2 and TLR4 were only barely detectable. In active IBD, the expression of TLR3 and TLR4 was differentially modulated in the intestinal epithelium. TLR3 was significantly downregulated in IEC in active CD but not in UC. In contrast, TLR4 was strongly upregulated in both UC and CD. TLR2 and TLR5 expression remained unchanged in IBD. These data suggest that IBD may be associated with distinctive changes in selective TLR expression in the intestinal epithelium, implying that alterations in the innate response system may contribute to the pathogenesis of these disorders.
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    Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system, but it is not clear how a restricted family of receptors has the capacity to recognize the wide spectrum of TLR stimuli known to exist. We report here that two members of the TLR family, TLR2 and TLR6, together coordinate macrophage activation by Gram-positive bacteria and the yeast cell-wall particle, zymosan. TLR6 and TLR2 both are recruited to the macrophage phagosome, where they recognize peptidoglycan, a Gram-positive pathogen component. By contrast, TLR2 recognizes another component, bacterial lipopeptide, without TLR6. The requirement for TLR cooperation is supported by the finding that TLR2 needs a partner to activate tumor necrosis factor-alpha production in macrophages. Dimerization of the cytoplasmic domain of TLR2 does not induce tumor necrosis factor-alpha production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain does. We show that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 or TLR1, and this interaction leads to cytokine induction. Thus, the cytoplasmic tails of TLRs are not functionally equivalent, with certain TLRs requiring assembly into heteromeric complexes, whereas others are active as homomeric complexes. Finally, we show that TLR6, TLR2, and TLR1 are recruited to macrophage phagosomes that contain IgG-coated erythrocytes that do not display microbial components. The data suggest that TLRs sample the contents of the phagosome independent of the nature of the contents, and can establish a combinatorial repertoire to discriminate among the large number of pathogen-associated molecular patterns found in nature.
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    Members of the Toll-like receptor (TLR) family are components of the mammalian anti-microbial response, signaling with a domain closely related to that of IL-1 receptors. In this report the expression and function of TLR1, a TLR of unknown function, are examined. TLR1 is expressed by monocytes, as demonstrated using a novel mAb. Monocytes also express TLR2. TLR1 transfection of HeLa cells, which express neither TLR1 nor TLR2, was not sufficient to confer responsiveness to several microbial extracts. However, cotransfection of TLR1 and TLR2 resulted in enhanced signaling by HeLa cells to soluble factors released from Neisseria meningitidis relative to the response with either TLR alone. This phenomenon was also seen with high concentrations of some preparations of LPS. The N. meningitidis factors recognized by TLR1/TLR2 were not released by N. meningitidis mutant in the LpxA gene. Although LpxA is required for LPS biosynthesis, because cooperation between TLR1 and TLR2 was not seen with all LPS preparations, the microbial component(s) TLR1/2 recognizes is likely to be a complex of LPS and other molecules or a compound metabolically and chemically related to LPS. The functional IL-1R consists of a heterodimer; this report suggests a similar mechanism for TLR1 and TLR2, for certain agonists. These data further suggest that mammalian responsiveness to some bacterial products may be mediated by combinations of TLRs, suggesting a mechanism for diversifying the repertoire of Toll-mediated responses.
  • Article
    Toll-like receptor (TLR) 2 and TLR4 play important roles in the early, innate immune response to microbial challenge. TLR2 is preferentially involved in the inflammatory response to lipoteichoic acid, lipopeptides, and glycans from a variety of microbes, whereas TLR4 is essential for a complete response to LPSs. We report here that TLR2 transduces the response to phenol-soluble modulin, a factor secreted by Staphylococcus epidermidis. The TLR2-mediated response to this modulin was enhanced by TLR6 but inhibited by TLR1, indicating a functional interaction between these receptors. We also demonstrate that a response to phenol-soluble modulin mediated by TLR2 and TLR6 was more refractory to inhibition by TLR1 than one mediated by TLR2 alone.
  • Article
    DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.
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    Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDA-containing fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.
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    The bare lymphocyte syndrome (BLS) is a hereditary immunodeficiency resulting from the absence of major histocompatibility complex class II (MHCII) expression. Considering the central role of MHCII molecules in the development and activation of CD4(+) T cells, it is not surprising that the immune system of the patients is severely impaired. BLS is the prototype of a "disease of gene regulation." The affected genes encode RFXANK, RFX5, RFXAP, and CIITA, four regulatory factors that are highly specific and essential for MHCII genes. The first three are subunits of RFX, a trimeric complex that binds to all MHCII promoters. CIITA is a non-DNA-binding coactivator that functions as the master control factor for MHCII expression. The study of RFX and CIITA has made major contributions to our comprehension of the molecular mechanisms controlling MHCII genes and has made this system into a textbook model for the regulation of gene expression.
  • Article
    The MHC class II transactivator (CIITA) is a critical transcription factor that regulates genes involved in antigen presentation function. At least three functional forms of CIITA gene products are transcribed from three different promoters. The CIITA gene expressed in dendritic cells (DC-CIITA) has a unique first exon encoding an extended N-terminal region of CIITA. Here, we show that the N terminus of DC-CIITA has high homology to a caspase recruitment domain (CARD) found in components of apoptosis and nuclear factor-kappaB signaling pathways. However, DC-CIITA does not regulate cell death, nor does it induce nuclear factor-kappaB activity. Instead, DC-CIITA is transcriptionally a more potent activator of the MHC class II gene than the form expressed in B cells. A single amino acid substitution in the CARD of DC-CIITA, predicted to disrupt CARD-CARD interactions, diminished the transactivation potential of DC-CIITA. These results indicate that the CARD in the context of CIITA serves as a regulatory domain for transcriptional activity and may function to selectively enhance MHC class II gene expression in dendritic cells.
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    Recently Toll-like receptors (TLRs) have been found to be involved in cellular activation by microbial products, including lipopolysaccharide, lipoproteins, and peptidoglycan. Although for these ligands the specific transmembrane signal transducers TLR-4, TLR-2, or TLR-2 and -6 have now been identified, the molecular basis of recognition of lipoteichoic acids (LTAs) and related glycolipids has not been completely understood. In order to determine the role of TLRs in immune cell activation by these stimuli, experiments involving TLR-2-negative cell lines, TLR-expression plasmids, macrophages from TLR-4-deficient C3H/HeJ-mice, and inhibitory TLR-4/MD-2 antibodies were performed. Glycolipids from Treponema maltophilum and Treponema brennaborense, as well as highly purified LTAs from Staphylococcus aureus and Bacillus subtilis exhibited TLR-2 dependence in nuclear factor kappaB activation and cytokine induction; however, T. brennaborense additionally appeared to signal via TLR-4. Fractionation of the T. brennaborense glycolipids by hydrophobic interaction chromatography and subsequent cell stimulation experiments revealed two peaks of activity, one exhibiting TLR-2-, and a second TLR-4-dependence. Furthermore, we show involvement of the signaling molecules MyD88 and NIK in cell stimulation by LTAs and glycolipids by dominant negative overexpression experiments. In summary, the results presented here indicate that TLR-2 is the main receptor for Treponema glycolipid and LTA-mediated inflammatory response.
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    CIITA is the master regulator of class II major histocompatibility complex gene expression. We present evidence that CIITA can self-associate via two domains: the C terminus (amino acids 700 to 1130) and the GTP-binding domain (amino acids 336 to 702). Heterotypic and homotypic interactions are observed between these two regions. Deletions within the GTP-binding domain that reduce GTP-binding and transactivation function also reduce self-association. In addition, two leucine residues in the C-terminal leucine-rich repeat region are critical for self-association as well as function. This study reveals for the first time a complex pattern of CIITA self-association. These interactions are discussed with regard to the apoptosis signaling proteins, Apaf-1 and Nod1, which share domain arrangements similar to those of CIITA.
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    Crohn's disease is a chronic inflammatory disorder of the gastrointestinal tract, which is thought to result from the effect of environmental factors in a genetically predisposed host. A gene location in the pericentromeric region of chromosome 16, IBD1, that contributes to susceptibility to Crohn's disease has been established through multiple linkage studies, but the specific gene(s) has not been identified. NOD2, a gene that encodes a protein with homology to plant disease resistance gene products is located in the peak region of linkage on chromosome 16 (ref. 7). Here we show, by using the transmission disequilibium test and case-control analysis, that a frameshift mutation caused by a cytosine insertion, 3020insC, which is expected to encode a truncated NOD2 protein, is associated with Crohn's disease. Wild-type NOD2 activates nuclear factor NF-kappaB, making it responsive to bacterial lipopolysaccharides; however, this induction was deficient in mutant NOD2. These results implicate NOD2 in susceptibility to Crohn's disease, and suggest a link between an innate immune response to bacterial components and development of disease.
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