Joint NOD2/RIPK2 Signaling Regulates IL-17 Axis and Contributes to the Development of Experimental Arthritis

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1(-/-)!, NOD2(-/-), or receptor-interacting serine-threonine kinase 2(-/-) (RIPK2(-/-)) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2(-/-) and RIPK2(-/-), but not NOD1(-/-), mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1 beta, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4(+) IL-17(+) cells in the lymph node between arthritic wild-type and NOD2(-/-) mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17-dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis. The Journal of Immunology, 2012, 188: 5116-5122.

IL-5 and IL-17A are critical for the chronic IgE response and differentiation of long-lived antibody-secreting cells in inflamed tissues

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120 d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220(neg)CD43(high) phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220(high) and B220(low)), whereas the largest pool of ASC into in the BM was constituted by the B220(high)CD43(low) phenotype; and finally, terminally differentiated cells (B220(neg)CD43(high)) were only maintained in the inflamed peritoneal cavity in late phase. After 120 d a sustained production of cytokines (KC, IL-5, TNF-alpha, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220(neg) in the peritoneal cavity and negatively regulate the number of ASC B220(Pos), confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A. (C) 2012 Elsevier Ltd. All rights reserved.

The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection

OBJECTIVES: FTY720 modulates CD4(+)T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment. METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis. RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4(+) graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft. CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.

Titanium dioxide induced inflammation in the small intestine

AIM: To investigate the effects of titanium dioxide (TiO2) nanoparticles (NPTiO2) and microparticles (MPTiO2) on the inflammatory response in the small intestine of mice. METHODS: BI 57/6 male mice received distilled water suspensions containing TiO2 (100 mg/kg body weight) as NPTiO2 (66 nm), or MPTiO2 (260 nm) by gavage for 10 d, once a day; the control group received only distilled water. At the end of the treatment the duodenum, jejunum and ileum were extracted for assessment of cytokines, inflammatory cells and titanium content. The cytokines interleukin (IL)-1b, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17, IL-23, tumor necrosis factor-alpha (TNF-alpha), intracellular interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) were evaluated by enzyme-linked immunosorbent assay in segments of jejunum and ileum (mucosa and underlying muscular tissue). CD4(+) and CD8(+) T cells, natural killer cells, and dendritic cells were evaluated in duodenum, jejunum and ileum samples fixed in 10% formalin by immunohistochemistry. The titanium content was determined by inductively coupled plasma atomic emission spectrometry. RESULTS: We found increased levels of T CD4(+) cells (cells/mm(2)) in duodenum: NP 1240 +/- 139.4, MP 1070 +/- 154.7 vs 458 +/- 50.39 (P < 0.01); jejunum: NP 908.4 +/- 130.3, MP 813.8 +/- 103.8 vs 526.6 +/- 61.43 (P < 0.05); and ileum: NP 818.60 +/- 123.0, MP 640.1 +/- 32.75 vs 466.9 +/- 22.4 (P < 0.05). In comparison to the control group, the groups receiving TiO2 showed a statistically significant increase in the levels of the inflammatory cytokines IL-12, IL-4, IL-23, TNF-alpha, IFN-gamma and TGF-beta. The cytokine production was more pronounced in the ileum (mean SE): IL-12: NP 33.98 +/- 11.76, MP 74.11 +/- 25.65 vs 19.06 +/- 3.92 (P < 0.05); IL-4: NP 17.36 +/- 9.96, MP 22.94 +/- 7.47 vs 2.19 +/- 0.65 (P < 0.05); IL-23: NP 157.20 +/- 75.80, MP 134.50 +/- 38.31 vs 22.34 +/- 5.81 (P < 0.05); TNF alpha: NP 3.71 +/- 1.33, MP 5.44 +/- 1.67 vs 0.99 +/- 019 (P < 0.05); IFN gamma: NP 15.85 +/- 9.99, MP 34.08 +/- 11.44 vs 2.81 +/- 0.69 (P < 0.05); and TGF-alpha: NP 780.70 +/- 318.50, MP 1409.00 +/- 502.20 vs 205.50 +/- 63.93 (P < 0.05). CONCLUSION: Our findings indicate that TiO2 particles induce a Th1-mediated inflammatory response in the small bowel in mice. (C) 2012 Baishideng. All rights reserved.

Decreased RNA expression of interleukin 17A in skin of leprosy

Interleukin-17A (IL-17A) is a proinflamatory cytokine that plays an important role in fighting pathogens at mucosal interfaces, by summoning neutrophils and upregulating cytoplasmatic antimicrobial peptides. So far, the presence of IL-17A in leprosy has not been demonstrated. The expression of IL-17A and related cytokines (IL-6 and IL-23p19) was addressed through RNA extraction and cDNA quantitative amplification in macerated biopsies of active lesions of 48 leprosy patients and 20 fragments of normal skin of individuals. Blood levels of IL-17A, IL-23p19 and IL-6 were determined by ELISA. We found an abrogated mRNA IL-17A response in all biopsies of leprosy patients, as compared with controls. Circulating IL-17A and IL-23p19 were undetectable in both patients and controls, but IL-6 was higher in lepromatous patients. Although at low levels, IL-17A mRNA in lepromatous patients had an inverse linear correlation with bacillary burden. Low expression of IL-17A in patients may be a constitutive genetic feature of leprosy patients or a circumstantial event induced by the local presence of the pathogen, as an escape mechanism.

Activation of cytokines corroborate with development of inflammation and autoimmunity in thromboangiitis obliterans patients

Thromboangiitis obliterans (TAO) is a segmental inflammatory occlusive disorder that affects the arm and leg arteries of young smokers. The immune system seems to play a critical role in the aetiology of TAO; however, knowledge of the aspects involved in the progression of vascular tissue inflammation and, consequently, the evolution of this disease is still limited. This study was carried out to investigate the cytokine levels of tumour necrosis factor (TNF)-a, interleukin (IL)-1 beta, IL-4, IL-17 and IL-23 in the plasma of TAO patients presenting with acute clinical manifestations. The study included 20 TAO patients (n = 10 women; n = 10 men) aged 3859 years under clinical follow-up, classified into two groups: (i) TAO former smokers (n = 11) and (ii) TAO active smokers (n = 9); the control groups included normal volunteer non-smokers (n = 10, active smokers (n = 10) and former smokers (n = 10). Patients' plasma samples were measured using the sandwich enzyme-linked immunosorbent assay. Statistical analyses were performed using the non-parametric MannWhitney U-test, with parameters significant at P < 0.05. The activities of all cytokines were different in groups of TAO patients when compared with normal controls, and decreased for control smokers. Increased levels of TNF-a, IL-1 beta, IL-4, IL-17 and IL-23 were significant in patients with TAO when compared to the controls (P < 0.005, all parameters). The results presented here indicate an increased production of cytokines in TAO, possibly contributing to the inflammatory response observed in the patients' vascular levels. In addition, the increased levels of IL-17 and IL-23 suggest that the disturbance of TAO is involved with mechanisms of autoimmunity. Thus, the discovery of IL-17 and its association with inflammation and autoimmune pathology has reshaped our viewpoint regarding the pathogenesis of TAO, which was based previously on the T helper type 1 (Th1)Th2 paradigm.

Influence of Mid-Trimester Amniotic Fluid on Endogenous and Lipopolysaccharide-Mediated Responses of Mononuclear Lymphoid Cells

Problem We evaluated the influence of amniotic fluid (AF) on immune mediator production by mononuclear leukocytes. Method of study Thirty mid-gestation AFs were incubated with peripheral blood mononuclear cells (PBMCs) in the presence or absence of lipopolysaccharide (LPS). Supernatants were tested for interleukin (IL) -6, 10, 12, 23, tumor necrosis factor-alpha (TNF-alpha) and monocyte chemotactic protein (MCP)-1. Results Endogenous mediator production was minimal or non-detectable. AF stimulated endogenous MCP-1, IL-6 and TNF-alpha release. In the presence of LPS, production of MCP-1 and IL-10 by PBMCs was enhanced eightto ninefold by AF. Release of IL-6 and IL-23 was enhanced less than twofold by the addition of AF while TNF-alpha production was unchanged. AF-stimulated mediator production was similar irrespective of pregnancy outcome. Conclusion Selective AF stimulation of LPS-mediated MCP-1 and IL-10 release may be a mechanism to promote antibody production and the influx of phagocytic cells to engulf pathogens while downregulating the production of pro-inflammatory cytokines.

PPAR-gamma agonists, mainly 15d-PGJ(2), reduce eosinophil recruitment following allergen challenge

We evaluate the immunomodulation of Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists 15d-PGJ(2) and rosiglitazone (RGZ) in a model of chronic eosinophilia. 15d-PGJ(2) and RGZ significantly reduce eosinophil migration into the peritoneal cavity and down-regulate the eosinopoiesis. The synthesis of IL-5 was decreased after the treatment with 15d-PGJ(2) and RGZ corroborating with the eosinophil migration inhibition. However, IgE was decreased only after the administration of 15d-PGJ(2) in part due to B-cell inhibition. We also observed a decrease in the synthesis of IL-33, IL-17 and IL-23, suggesting that besides the modulation of Th2 pattern, there is a modulation via IL-23 and IL-17 suggesting a role of these cytokines in the eosinophil recruitment. In fact IL-17(-1-) mice failed to develop an eosinophilic response. Altogether, the results showed that PPAR-gamma agonists mainly 15d-PGJ(2), have therapeutic efficacy in eosinophil-induced diseases with an alternative mechanism of control, via IL-23/IL-17 and IL-33. (C) 2011 Elsevier Inc. All rights reserved.