Impaired Toll-like receptor 8-mediated IL-6 and TNF-alpha production in antigen-presenting cells from patients with X-linked agammaglobulinemia

The critical role of Bruton tyrosine kinase (Btk) in B cells has been documented by the block of B-cell development in X-linked agammaglobulinemia (XLA). Less is known about Btk function in myeloid cells. Several pieces of evidence indicate that Btk is a component of Toll-like receptor (TLR) signaling. We analyzed whether Btk deficiency in XLA is associated with an impaired dendritic cell (DC) compartment or defective TLR signaling. We analyzed the expression of TLRs 1 to 9 on myeloid DCs generated from XLA patients and evaluated their response to activation by specific TLR agonists. We show that XLA patients have normal numbers of circulating DCs. Btk-deficient DCs have no defect in response to stimulation of TLRs 1/2, 2/6, 3, 4, and 5 but display a profound impairment of IL-6 and TNF-alpha production in response to stimulation by TLR-8 cognate agonist, ssRNA. These findings may provide an explanation for the susceptibility to enteroviral infections in XLA patients.     

Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degradation

Toll-like receptors (TLRs) play a crucial role in several innate immune responses by regulating autophagy, but little is known about how TLR signaling controls autophagy. Here we demonstrate that p62/SQSTM1 is required for TLR4-mediated autophagy, which we show as selective autophagy of aggresome-like induced structures (ALIS). Treatment with LPS or Escherichia coli induced LC3(+) dot-like structures, and their assembly, but not lysosomal degradation, occurred independently of classic autophagic machinery. Microscopic and ultrastructural analyses showed that p62 is a component of the induced LC3(+) dots and these TLR4-induced p62(+) structures resemble ALIS. The levels of p62 mRNA and protein were increased in TLR4-activated cells and knockdown of p62 suppressed the ALIS formation and LC3-II conversion. The accumulation of p62 and ALIS required activation of Nrf2 by reactive oxygen species-p38 axis-dependent TLR4/MyD88 signaling, suggesting a link between innate immune and oxidative-stress responses. These findings indicate that TLR4-driven induction of p62 plays an essential role in the formation and the autophagic degradation of ALIS, which might be critical for regulating host defense.     

Toll-like receptor 2 deficiency improves insulin sensitivity and hepatic insulin signalling in the mouse

Aims/hypothesis  

Substantial evidence suggests a link between elevated inflammation and development of insulin resistance. Toll-like receptor 2 (TLR2) recognises a large number of lipid-containing molecules and transduces inflammatory signalling in a variety of cell types, including insulin-responsive cells. Considering the contribution of the fatty acid composition in TLR2-depedent signalling, we hypothesised that the inflammatory signals transduced by TLR2 contribute to insulin resistance.     

Prognostic value of serum IL-10 and soluble IL-2 receptor levels in aggressive non-Hodgkin's lymphoma

Summary We investigated the prognostic significance of interleukin-10 (IL-10) and soluble interleuckin-2 receptor (sIl-2r) levles in the pretreatment serum of 105 individuals with newly-diagnosed aggressive non-Hodgkin's lymphoma (NHL). Commercially available enzyem-linked immunoassay kits were used for cytokine and receptor measurements. Detectable levels of IL-10 were found in 42 (40%) patients at diagnosis, with no correlation with clinico-haematological parameters, but in no control samples (P < 0.001).
Pretreatment concentrations of sIL-2r were markedly increased in individuals with NHL when compared to controls (2614 ± 893 U/ml v 219 ± 65U/ml, P < 0.001), patients with stage III/IV presenting higher values than those with stage II disase (3885 ± 1196U/ml v 1732 ± 646U/ml, P < 0.001). No single parameter was associated with the achiveement of complete remission, but the combination of elevated IL-10 and of sIL-2r greater than 3000U/ml selected a subset of patients with a high probability of failing induction therapy (P < 0.001). Lifetable analysis also indicated thatj patients with these characteristics have a significantly shorter event-free survival. In a multivariate analysis the combination of IL-10 with sIL-2r was found to have greater predictive strength than the combination of IL-10 with β₂-micro-globulin. We conclude that IL-10 and sIL-2r measurements can be expected to improve existing methods of risk assignment in aggressive NHL.     

Interleukin 10 deficiency attenuates induction of anti-TSH receptor antibodies and hyperthyroidism in a mouse Graves' model

Experimental Graves'-like hyperthyroidism can be induced in susceptible mouse strains by repetitive immunizations with recombinant adenovirus expressing the human full-length TSH receptor (TSHR) or its A-subunit. Previous studies have shown that splenocytes from immunized mice produce interferon (IFN)-γ and interleukin (IL) 10 in response to antigen stimulation in an in vitro T cell recall assay. Although IFN-γ is now well known to be essential for disease induction, the role(s) played by IL10 are unknown. Therefore, this study was conducted to clarify the significance of endogenous IL10 in the pathogenesis of experimental Graves' disease using IL10 deficient (IL10(-/-)) mice. Our results show that T cell response was augmented when estimated by their antigen-specific secretion of the key cytokine IFN-γ, but B cell function was dampened, that is, anti-TSHR antibody titers were decreased in IL10(-/-) mice, resulting in a lower incidence of Graves' hyperthyroidism (54% in IL10(+/+) vs 25% in IL10(-/-)). Thus, in addition to IFN-γ, these data clarified the role of IL10 for optimizing anti-TSHR antibody induction and eliciting Graves' hyperthyroidism in our Graves' mouse model.     

慢性乙型肝炎与Toll样受体2的关系

目的探讨CHB与Toll样受体2（TLR2）的相关性。方法采用Elivision二步法检测CHB、慢性重型肝炎患者及健康对照组肝组织TLR2的表达。采用直接免疫荧光流式细胞术检测TLR2在肝癌细胞株HepG2、HepG2-X及HepG2．2．15上表达的平均荧光强度（MFI）及阳性细胞率。结果TLR2在CHB及慢性重型肝炎肝组织上的表达明显高于健康对照组，差异有统计学意义，P〈0．01，主要表达于肝细胞质及部分胞膜。在CHB中，TLR2的表达强度与肝组织炎症活动度分级（G）呈显著正相关（r=0．597，P〈0．01），各级炎症患者肝组织TLR2阳性表达强度患者的血清TBil值，差异有统计学意义（P〈0．05）。在慢性乙型重型肝炎患者中，TLR2主要呈小灶性表达。TLR2在肝癌细胞株HepG2．2．15上表达的平均荧光强度为10．7±2．8，阳性细胞率为16．3％±7．0％；HepG2的平均荧光强度为1．0±0．3，阳性细胞率为0．4％±0．1％，两组比较t值分别为11．92和7．92，P值均〈0．0l。而HepG2和HepG2-X之间，差异无统计学意义。结论CHB患者肝组织中TLR2的表达明显升高，与肝组织的炎症活动密切相关。     

Serum amyloid A induces G-CSF expression and neutrophilia via Toll-like receptor 2

The acute-phase protein serum amyloid A (SAA) is commonly considered a marker for inflammatory diseases; however, its precise role in inflammation and infection, which often result in neutrophilia, remains ambiguous. In this study, we demonstrate that SAA is a potent endogenous stimulator of granulocyte colony-stimulated factor (G-CSF), a principal cytokine-regulating granulocytosis. This effect of SAA is dependent on Toll-like receptor 2 (TLR2). Our data demonstrate that, in mouse macrophages, both G-CSF mRNA and protein were significantly increased after SAA stimulation. The induction of G-CSF was blocked by an anti-TLR2 antibody and markedly decreased in the TLR2-deficient macrophages. SAA stimulation results in the activation of nuclear factor-kappaB and binding activity to the CK-1 element of the G-CSF promoter region. In vitro reconstitution experiments also support that TLR2 mediates SAA-induced G-CSF expression. In addition, SAA-induced secretion of G-CSF was sensitive to heat and proteinase K treatment, yet insensitive to polymyxin B treatment, indicating that the induction is a direct effect of SAA. Finally, our in vivo studies confirmed that SAA treatment results in a significant increase in plasma G-CSF and neutrophilia, whereas these responses are ablated in G-CSF- or TLR2-deficient mice.     

Genetic and environmental context determines the course of colitis developing in IL-10-deficient mice

This review summarizes how interleukin-10 (IL-10)-deficient mice have permitted new insight into the complex interaction between genes and environment underlying pathogenesis of inflammatory bowel disease (IBD). The C57BL/6J strain develops only mild typhlocolitis in response to IL-10 deficiency. In contrast, C3H/HeJBir represents an unrelated inbred strain with high IBD susceptibility. Ability to identify quantitative trait loci segregating for susceptibility when the two IL-10-deficient stocks were intercrossed depended both on genome "context" (F2 versus reciprocal backcrosses) and on the physical environment. These findings are discussed in the context of recent advances in understanding the complex genetic basis for IBD in humans.     

Up-regulation of A 2B adenosine receptor in A 2A adenosine receptor knockout mouse coronary artery

In this study, we looked into possible compensatory changes of other adenosine receptors (ARs) in A_2A genetic knockout mice (A2AKO) as well as the functional role of nitric oxide (NO) in A_2A AR-mediated vasodilation. Gene expression of ARs from coronary arteries of A_2A AR wild type mice (A2AWT) and A2AKO was studied using real-time PCR. Functional studies were carried out in isolated heart and isolated coronary artery preparations. A_2B AR was found to be 4.5 fold higher in A2AKO than in A2AWT, while A_2A AR expression was absent in A2AKO. There was no difference in A₁ and A₃ ARs between WT and KO animals. The concentration-relaxation curve for adenosine-5′-N-ethylcarboxamide (NECA, non-selective AR agonist) in isolated coronary arterial rings in A2AKO was shifted to the left when compared to A2AWT. The concentration-response curve for A_2B selective agonist (BAY 60-6583) was also shifted to the left in A2AKO hearts. L-NAME, a non-specific NO synthase inhibitor, did not affect baseline coronary flow (CF) until the concentration reached 10 µM in A2AWT (76.32 ± 11.35% from baseline, n = 5). In A2AKO, the CF decreased significantly by L-NAME only at a higher concentration (100 µM, 93.32 ± 5.8% from baseline, n = 5). L-NMA (1 µM, n = 4), another non-specific NO synthase inhibitor, also demonstrated similar results in decreasing CF (59.66 ± 3.23% from baseline in A2AWT, while 81.76 ± 8.91% in A2AKO). It was further demonstrated that the increase in CF by 100 µM NECA was significantly blunted with 10 µM L-NAME (377.08 ± 25.23% to 305.41 ± 30.73%, n = 9) in A2AWT but not in A2AKO (153.66 ± 22.7% to 143.88 ± 36.65%, n = 5). Similar results were also found using 50 nM of CGS-21680 instead of NECA in A2AWT (346 ± 22.85 to 277 ± 31.39, n = 6). No change in CF to CGS-21680 was noted in A_2AAKO. Our data demonstrate, for the first time, that coronary A_2B AR was up-regulated in mice deficient in A_2A AR. We also provide direct evidence supporting a role for NO in A_2A AR-mediated coronary vasodilation. The data further support the role for A_2A AR in the regulation of basal coronary tone through the release of NO.     

Comparative assessment of virulence of recombinant vaccinia viruses expressing IL-2 and IL-15 in immunodeficient mice

IL-2 and -15 belong to the four alpha-helix bundle family of cytokines and display a spectrum of overlapping immune functions because of shared signal transducing receptor components of the IL-2 receptor complex. However, recent evidence suggests a nonredundant unique role for IL-15 in the establishment and perhaps maintenance of peripheral natural killer (NK) cell populations in vivo. To explore the contribution of locally released IL-15 on peripheral NK-cell-mediated innate immune responses, we generated a recombinant vaccinia virus that expresses IL-15 and evaluated the course of vaccinial disease in athymic nude mice. Coexpression of IL-15 resulted in the attenuation of virulence of vaccinia virus, and mice inoculated with 10(5) plaque-forming units or less resolved the infection successfully. In contrast, mice inoculated with a similar dose of the control vaccinia virus failed to eliminate the virus and died of generalized vaccinial disease. Enhanced expression of IL-12 and IFN-gamma as well as induction of chemokines were evident in the mice inoculated with IL-15-expressing vaccinia virus in addition to an increase in NK cells in the spleen. However, in this model system, the degree of attenuation in viral virulence attained with coexpression of IL-15 was much less than that achieved with coexpression of IL-2, suggesting that the peripheral NK-cell-mediated events are more responsive to IL-2 than to IL-15.     

Tumor necrosis factor alpha blockade treatment down-modulates the increased systemic and local expression of Toll-like receptor 2 and Toll-like receptor 4 in spondylarthropathy

OBJECTIVE: Abnormal host defense against pathogens has been implicated in the pathogenesis of spondylarthropathy (SpA), a disease characterized by abundant synovial infiltration with innate immune cells. Given the role of Toll-like receptors (TLRs) in activation of innate inflammation and the occurrence of TLR-dependent infections after tumor necrosis factor alpha (TNFalpha) blockade treatment, the present study was undertaken to analyze TLRs and their modulation by TNFalpha blockade in SpA. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from SpA and rheumatoid arthritis (RA) patients during infliximab therapy, and from healthy controls. TLR-2 and TLR-4 expression and TNFalpha production upon lipopolysaccharide (LPS) stimulation were analyzed by flow cytometry on different monocyte subsets. Synovial biopsy specimens from 23 SpA patients before and after infliximab or etanercept treatment, from 15 RA patients, and from 18 osteoarthritis (OA) patients were analyzed by immunohistochemistry. RESULTS: Expression of TLR-4, but not TLR-2, was increased on PBMCs from patients with SpA, whereas both TLRs were increased in RA patients. TLR expression was particularly increased on the CD163+ macrophage subset. Infliximab reduced TLR-2 and TLR-4 expression on monocytes of SpA and RA patients, leading to lower levels than in controls and to impaired TNFalpha production upon LPS stimulation. In inflamed synovium, the expression of both TLRs and of CD163 was significantly higher in patients with SpA than in those with RA or OA. Paralleling the systemic effect, TLRs in synovium were down-regulated following treatment with infliximab as well as etanercept, indicating a class effect of TNFalpha blockers. CONCLUSION: Inflammation in SpA is characterized by increased TLR-2 and TLR-4 expression, which is sharply reduced by TNFalpha blockade. These findings suggest a potential role of innate immunity-mediated inflammation in SpA and provide an additional clue regarding the mechanism of action as well as the potential side effects of TNFalpha blockade.     

Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R.