Rapid CD4+ T‐cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9

Toll‐like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as‐MyD88 or (TRIF TIR‐domain‐containing adapter‐inducing interferon‐β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin‐specific T‐cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR‐signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin‐specific CD4⁺ T‐cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin‐specific T‐cell responses.     

Hepatitis C core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation

BACKGROUND AND AIMS: Recent evidence suggests that toll-like receptors (TLRs) recognize certain viruses. We reported that hepatitis C virus (HCV) core and nonstructural 3 (NS3) proteins activate inflammatory pathways in monocytes. The aim of this study was to investigate the role of TLRs in innate immune cell activation by core and NS3 proteins. METHODS: Human monocytes, human embryonic kidney cells transfected with TLR2, and peritoneal macrophages from TLR2, MyD88 knockout, and wild-type mice were studied to determine intracellular signaling and proinflammatory cytokine induction by HCV proteins. RESULTS: HCV core and NS3 proteins triggered inflammatory cell activation via the pattern recognition receptor TLR2 and failed to activate macrophages from TLR2 or MyD88-deficient mice. HCV core and NS3 induced interleukin (IL)-1 receptor-associated kinase (IRAK) activity, phosphorylation of p38, extracellular regulated (ERK), and c-jun N-terminal (JNK) kinases and induced AP-1 activation. Activation of nuclear factor-kappaB by core and NS3 was associated with increased IkappaBalpha phosphorylation. TLR2-mediated cell activation was dependent on the conformation of core and NS3 proteins and required sequences in the regions of aa 2-122 in core and aa 1450-1643 in NS3. Although cellular uptake of core and NS3 proteins was independent of TLR2 expression, cell activation required TLR2. HCV core protein and TLR2 showed intracellular colocalization. The hyper-elevated TNF-alpha induction by TLR2 ligands in monocytes of HCV-infected patients was not due to increased TLR2 expression. CONCLUSIONS: HCV core and NS3 proteins trigger inflammatory pathways via TLR2 that may affect viral recognition and contribute to activation of the innate immune system.     

Pivotal role of NOD2 in inflammatory processes affecting atherosclerosis and periodontal bone loss

The purpose of this study was to elucidate the role of nucleotide binding oligomerization domain-containing protein 2 (NOD2) signaling in atherosclerosis and periodontal bone loss using an Apolipoprotein E^−/− (ApoE^−/−) mouse model based on the proposed role of NOD2 in inflammation. NOD2^−/−ApoE^−/− and ApoE^−/− mice fed a standard chow diet were given an oral gavage of Porphyromonas gingivalis for 15 wk. NOD2^−/−ApoE^−/− mice exhibited significant increases in inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and the heart compared with ApoE^−/− mice. In contrast, ApoE^−/− mice injected i.p. with Muramyl DiPeptide (MDP) to stimulate NOD2 and given an oral gavage of P. gingivalis displayed a reduction of serum inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and aortic sinus compared with ApoE^−/− mice orally challenged but injected with saline. A reduction in body weight gain was observed in ApoE^−/− mice fed a high-fat diet (HFD) and injected with MDP compared with ApoE^−/− mice fed a high-fat diet but injected with saline. MDP treatment of bone marrow-derived macrophages incubated with P. gingivalis increased mRNA expressions of NOD2, Toll-like receptor 2, myeloid differentiation primary response gene 88, and receptor-interacting protein-2 but reduced the expressions of inhibitor of NF-κB kinase-β, NF-κB, c-Jun N-terminal kinase 3, and TNF-α protein levels compared with saline control, highlighting pathways involved in MDP antiinflammatory effects. MDP activation of NOD2 should be considered in the treatment of inflammatory processes affecting atherosclerosis, periodontal bone loss ,and possibly, diet-induced weight gain.The nucleotide binding and oligomerization domain 2 protein (NOD2) is an intracellular protein containing leucine-rich repeats similar to the repeats found in Toll-like receptors (TLRs) that are capable of sensing bacteria-derived muramyl dipeptide (MDP), and it was initially described as a susceptibility gene for Crohn disease and intestinal inflammatory diseases (1–3). NOD2 is expressed in various cell subsets, including myeloid cells (particularly macrophages, neutrophils, and dendritic cells), as well as Paneth cells in the small intestine (4), and it was found to process inflammatory signals (5). Immune cells express receptors that recognize a broad range of molecular patterns foreign to the mammalian host but commonly found on pathogens. These molecules trigger immune responses through interactions with members of the toll-like receptor family (TLRs) at the cell membrane and NACHT, neuronal apoptosis inhibitor protein (NAIP), CIITA, HET-E and TP-1 domain–Leucine-rich repeat (LRR) proteins (NLRs) in the cytosol (6, 7). Cells expressing NOD2 can activate NF-κB after intracellular recognition of MDP (8, 9). The recognition of MDP is mediated through the LRR domain of NOD2, leading to downstream signaling through interaction between the caspase recruitment domain (CARD) of Receptor-interacting serine/threonine-protein kinase 2 (RIP2) and the CARDs of NOD2. In vitro, NOD2 has been found to be involved in bacterial clearance (10). NOD2-deficient mice display increased susceptibility to Staphylococcus aureus because of, in part, defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden (11). However, stimulation of NOD2 with MDP was found to enhance host antibacterial function in vitro (12).Atherosclerosis is a chronic inflammatory condition that can lead to an acute clinical event by plaque rupture and thrombosis. It is a multifactorial disease characterized by the accumulation of cells from both the innate and acquired immune system within the intima of the arterial wall (13). Triglyceride-rich lipoproteins and free fatty acids are important factors involved in fatty streak formation and advanced atherosclerosis (14). Microorganisms have also been implicated as aggravating factors in atherosclerosis (15). In atherosclerosis, normal homeostatic functions of the endothelium are altered, promoting an inflammatory response that results in an increased expression of adhesion molecules. This increased expression leads to the recruitment of leukocytes, including monocytes, that penetrate the intima, predisposing the vessel wall to lipid deposits (13). Reportedly, mast cells also contribute to coronary plaque progression and diet-induced obesity and diabetes through the secretion of vasoactive mediators, cytokines, and proteinases (16).Evidence is accumulating that distant bacterial infection is involved in the pathophysiology of local chronic inflammatory processes underlying atherosclerosis (17). The transfer of bacteria into the blood or lymph system from barrier organ surfaces has been suggested as a possible mechanism of atherosclerosis. Advanced gum infection (periodontitis) is known to induce local inflammation, often leading to gingival ulcerations and local vascular changes, which have the potential to increase the incidence and severity of transient bacteremia. Porphyromonas gingivalis, an important microorganism associated with periodontitis, has been identified in atherosclerotic plaques of patients suffering from atherosclerosis, suggesting that this pathogen might be critical for atheroma formation (18). Indeed, we showed that endothelial dysfunction associated with repetitive exposure by P. gingivalis can exacerbate the development of atherosclerosis (19).NOD2 expression and unique functions have also been described in other cell types, including adipocytes, gingival, pulp and periodontal fibroblasts, oral epithelial cells, and vascular endothelial cells (20–25). However, the precise role of NOD2 in chronic inflammatory diseases remains unclear.We showed previously that, in Apolipoprotein E^+/− (ApoE^+/−) mice, TLR2 deficiency reduces pathogen-associated atherosclerosis (26). In this report, we tested the role of NOD2 in two chronic inflammatory diseases, atherosclerosis and alveolar bone loss, by capitalizing on our model of P. gingivalis-associated atherosclerosis in ApoE^−/− mice.     

Mucins and toll-like receptors: kith and kin in infection and cancer

Inflammation is underlying biological phenomenon common in infection and cancer. Mucins are glycoproteins which establish a physical barrier for undesirable entry of foreign materials through epithelial surfaces. A deregulated expression and an anomalous glycosylation pattern of mucins are known in large number of cancers. TLRs are class of receptors which recognize the molecular patterns of invading pathogens and activate complex inflammatory pathways to clear them. Aberrant expression of TLRs is observed in many cancers. A highly orchestrated action of mucins and TLRs is well evolved host defence mechanism; however, a link between the two in other non-infectious conditions has received less attention. Here we present an overview as to how mucins and TLRs give protection to the host and are deregulated during carcinogenesis. Further, we propose the possible mechanisms of cross-regulation between them in pathogenesis of cancer. As both mucins and TLRs are therapeutically important class of molecules, an understanding of the underlying molecular mechanisms connecting the two will open new avenues for the therapeutic targeting of cancer.     

Toll样受体2在关节置换术后小鼠表皮葡萄球菌感染中的研究

目的探讨Toll样受体2(TLR2)在关节置换术后感染中的作用。方法构建昆明小鼠的简易右膝关节置换术后表皮葡萄球菌感染模型。将60只动物随机分为假体置换术后感染组(A组)、假体置换术后无感染组(B组)、关节内注射细菌组(C组)和关节内注射生理盐水的对照组(D组)。于操作后的第1、3、7天眼球取血0.2mL,流式细胞术分析TLR2在外周血白细胞的表达;取血后处死并取右膝关节及周围组织作细菌培养。对各组各时相TLR2的表达水平结果进行统计分析,组间同一指标比较采用单因素ANOVA方差分析,同组内同一指标术前术后变化水平比较采用自身配对t检验。结果 A组、C组的组织培养均为阳性,B、D组的组织培养均为阴性。操作后第1天和第3天,A/B组TLR2的阳性表达率比较有统计学意义,P0.05。至第7天,TLR2的表达在各组间的比较差异均无统计学意义。A组和C组对比差异无统计学意义,P0.05。A组第3天的阳性表达率明显低于第1天,P0.05。结论假体植入不影响TLR2的表达,TLR2可能是关节置换术后感染的一项敏感指标。     

Panax ginseng C.A. Mayer G115 modulates pro-inflammatory cytokine production in mice throughout the increase of macrophage toll-like receptor 4 expression during physical stress

The effect of Panax ginseng C.A. Meyer G115 on inflammatory cytokine production and toll-like receptor 4 (TLR4) RNA expression was examined in mice during 4 weeks of swimming stress. Mice were assigned to four groups: (1) control (no exercise); (2) control-G115 (25 mg/kg/day p.o.); (3) stress (kept swimming for 60 min daily); and (4) stress-G115 (25 mg/kg/day p.o. and kept swimming for 60 min daily). Peritoneal macrophages were collected at rest each week. RNA was extracted and processed for real-time PCR. An aliquot of macrophages was lipopolysaccharide (LPS)-stimulated for TNF-alpha and IL-1beta production. Different expression patterns between untreated and treated groups, and between TLR2 and TLR4 were found. High levels of TLR4 expression in the control-G115 group were detectable significantly at the first, and at the second week (P<.01 and P<.001, respectively). In the stress group, TLR4 showed a peak at the first week (P<.001 vs. controls) and then decreased gradually. In the stress-G115 group, the levels of TLR4 expression increased gradually at the second week (P<.001 vs. controls) with a peak at the third week (P<.001). Levels of TLR4 expression at the fourth week had returned to the basal level. Levels of TLR2 expression were not affected by treatment in all groups. A significant increase of LPS-stimulated IL-1beta and TNF-alpha concentrations was present in trained animals with similar patterns of TLR4 expression. These results support the hypothesis that enhancement of the production of pro-inflammatory cytokine can be linked to an increased expression of TLR4 on macrophages. Moreover, G115 increases the expression of TLR4 and the release of cytokines with a different pattern compared to the stressed alone group.     

A time-lapse approach to examine chromium and nickel effects on wound healing in vitro

Chromium and nickel cause allergic contact dermatitis, a common biological skin response to sensitizing agents. This study used a conventional in vitro wounding model to study the impact of sensitizing agents on the innate immune response of human keratinocytes. Experiments were designed to evaluate the involvement of specific Toll-like receptors and metalloproteinases as effectors molecules downstream, at a molecular level. Further, keratinocytes were co-cultured with monocytes (THP-1 cells) to reproduce an inductive stimulus on monocytes made by metals. Human keratinocytes (HaCat) were grown on plates covered with collagen type I, chemically treated, and then mechanically injured with a sterile pipette tip. Restoration of the monolayer integrity was monitored by time-lapse video microscopy. Effector gene expression was evaluated by real-time PCR. The presence of chromium significantly dropped the rate of wound closure, while nickel-induced hyper-proliferation ended in an acceleration of the healing process, an event that does not occur in vivo. This latter outcome led to considering nickel as an unsuitable example for use in the experimental model. Focusing solely on the chromium aspect of this study, RNA profiles of selected molecular markers were generated to ascertain if the detrimental stimulus from chromium was eliminated or persisted both in keratinocytes alone and/or during co-cultures of keratinocytes and monocytes. Monocytes accelerated the process of wound repair. This in vitro experimental model highlighted the involvement of innate immunity in response to chromium and might be useful for test molecules of therapeutic interest for the treatment of skin lesions. However, the experience with nickel reveals that there are limitations to the utility of this wound model system after all.     

New DiaP277 analogue shifts DCs to tolerogenic,and modulates NF-Kβ1 to suppress autoreactive T lymphocytes in the type 1 diabetic mice

Abstract

Therapeutic efficacy of P277 against type 1 diabetes was extensively investigated and clinically evidenced. Clinical trials Phases I and II concluded promising results, while the data of P277 immunogenicity in Phase III trials represented weak responses that led to abolish medical use. But, a therapeutic performance of P277 cannot be forgotten. So, in order to exploit its therapeutic benefits and improve its immunogenicity, we developed a new analogue VP to optimize therapeutic efficacy and enhancing immunosuppressive modulations. However, new analogue was purified, and then used to immunize diabetic NOD mice to investigate antidiabetic effects through modulation of immunological status. So, DCs immune responses, relative TLRs, MyD88, and NF-Kβ1 mRNA expression on DCs and splenocytes under VP effect were tested. Circulating and intracellular cytokines were also evaluated at treated and non-treated mice. Splenic T lymphocytes proliferation (Th1 and Treg cells) were also determined. Results revealed that VP significantly down regulates DCs maturation through TLR2, TLR4, and MyD88 pathways. It also shifts DCs to a tolerogenic polarization through NF-Kβ1 pathway that mediates Th1 immunosuppression and enhances iTreg expanding in type1diabetes mice. Meanwhile, we noticed that VP significantly enhances iTreg CD25?+?FoxP3+ proliferation. In conclusion, VP showed promising immune potential to modulate immune regulatory responses and shifts DCs to suppress autoreactive Th1 cells which ameliorated immunosuppressive potency in the type1 diabetic mice.