congresos y reuniones científicas
Deficiency of TNF receptor p55 impacts on dendritic cell population of lymph nodes in Yersinia enterocolitica-induced reactive arthritis.
Congreso; Inmunocolombia 2015. 11° Congress of the Latin American association of Immunology -ALAI. 10° Colombian Congress of Allergy, Asthma and Immunology-ACCAI; 2015
Yersinia enterocolitica (Ye) is a Gram-negative bacterium that causes intestinal infection which could be complicated with reactive arthritis (ReA). This rheumatic disease is an aseptic synovitis that belongs to spondyloarthritis and follows a gastrointestinal or genitourinary infection. Ye O:3 is the most frequent serotype associated with human arthritis. After oral infection, Ye travel to the terminal ileum, where they invade the Peyer?s patches, replicate extracellularly and may eventually disseminate to mensenteric lymph nodes (MLN) and then to deeper tissues including liver, spleen, and lung. After infection, macrophages and dendritic cells (DCs) are crucial to the immune response to Ye infection. TNF has been considered a critical cytokine during the protective host response against Ye. TNFR p55 (TNFR1; CD120a) is implicated in most TNF effects. In previous studies we showed that TNFR p55-deficient (TNFRp55-/-) mice develop severe chronic arthritis in contrast with their wild-type (WT) counterparts; however, the mechanisms underlying these pathogenic effects remain uncertain. Previously, we demonstrated that Th1 and Th17 effector cells may act in concert to sustain the inflammatory response in Ye-induced ReA in TNFRp55-/-. Higher IL-12/23 p40, common chain that is shared by the Th1-promoting cytokine IL-12 and the Th17-promoting cytokine IL-23 explained these data. IL-12 is a pro-inflammatory cytokine produced by DCs, macrophages and B cells in response to microbial pathogens. We detected an unleashed pro-inflammatory response by Ye LPS stimulation in TNFRp55-deficient macrophage. However, the role for DCs in ReA induced by Ye in TNFRp55-/- mice is unknown. DCs migrate from peripheral tissues to secondary lymphoid organs, where they present antigens to T cells to initiate a specific immune response. Whether DCs subpopulations contribute to the link between gut inflammation and arthritis in Ye-induced ReA has not been studied. Within the broad classification of DCs as CD11c+ cells, CD11b+CD8a- and CD11b-CD8a+ subpopulations have been identified. The CD8+ rather than CD11b+cells produce IL-12 after stimulation in vitro or in vivo with inflammatory stimuli and is the predominant DC population producing IFN-g in an IL-12-dependent manner in vitro. These DC subpopulations have not been analyzed in ReA. The purpose of the present work was to study the role of DCs in Ye-induced ReA in TNFRp55-deficient mice. Therefore, first we compared total number and frequency of DCs and their subpopulations in MLN and in regional lymph nodes (RLN) at different days after oral Ye infection. Moreover, the main cellular source of IL-12p40 was analyzed. TNFRp55-/- mice (C57BL/6) were kindly provided by the Max von Pettenkofer Institute (Munich, Germany). C57BL/6 WT mice were purchased from the Animal Facilities of the National University of La Plata, Argentina. Breeding colonies were established at the Animal Facilities of the National University of San Luis, Argentina. Male mice (6-8wk old) were used for the experiments. Animal work was approved by the Animal Care and Use Committee of National University of San Luis.Mice were starved for 2 h and then were infected orogastrically with 1?5 x 108 yersiniae in 200 ul PBS, using a gastric tube. Seven, 14 and 21 days after infection, MLN and RLN were collected. As a control, MLN and RLN from mice administrated only with PBS were analyzed. The organs were finely cut and digested for 20 min at 37 ºC in HBSS containing collagenase and DNAse I. For flow-cytometric staining, the cells were first incubated with anti-mouse CD16/32 (Fc block). The cells were phenotyped using cell surface markers (DCs as CD11c+/MHC class II+, which levels of expression difference migratory and resident DCs, subpopulation of DCs as CD11b+ or CD8a+, and macrophages/neutrophils as CD11c-/CD11b+). For intracellular cytokine staining, the cells were stimulated with PMA and ionomycin or with commercial Escherichia coli LPS in presence of brefendin A. After surface staining, the cells were fixed and permeabilized and incubated with PE-anti-IL-12p40 and the appropriate isotype control. Data were acquired on a FACSCalibur flow cytometer and analyzed with FlowJo software. Statistical difference of mean values was assessed by the nonparametric Mann-Whitney test or one-way analysis of variance (ANOVA) followed by Tukey?s comparison test using GraphPad Prism 5 software. A p value less than 0.05 was considered as statistically significant. We observed in Ye-infected TNFRp55-/- mice significant increase of inflammatory cells in both MLN and RLN only at day 14 after infection (p<0.001, compared with WT mice), this time corresponded with arthritis onset. When we analyzed DCs, we found that Ye-infected TNFRp55-/- mice showed significantly higher total number of both migratory (CD11c+MHChi) and resident (CD11c+MHCint) DCs in MLN and RLN (p<0.001 compared with WT mice). Resident DCs frequency was also augmented in knockout mice (p<0.05, compared with WT mice). Moreover, both CD11b+ and CD8+ DC subpopulations were increased in this group of mice (p<0.05, compared with WT). Other inflammatory cells such as CD11b+CD11c-(macrophages/neutrophils) and CD11cint CD11b+ cells were also in significantly higher amount in the lymph nodes of knockout mice (p<0.05, compared with WT). When IL-12p40 was analyzed by intracellular flow cytometry, we found that migratory DCs were the main source of IL-12p40 and that TNFRp55-/- mice showed higher frequency of IL-12p40?producing migratory DCs than WT mice (p<0.01). Moreover, DCs from TNFRp55-/- mice exhibited higher IL-12 mean fluorescence intensity (MFI) compared with DCs of their WT counterparts (p<0.05).In conclusion, the changes in the number of DCs on arthritis onset provide evidence that DCs may contribute to arthritis develop in TNFRp55-/- mice after Ye infection, and suggest that TNFRp55 deficiency impact on DCs in joint draining lymph nodes. The increase of migratory and resident DCs in both MLN and RLN support a mucosal-joint connection. Furthermore, since migratory DCs were the main source of IL-12p40 in Ye-induced ReA in TNFRp55-/- mice, desregulation of the number or function of this DC population may result in the unbalanced immune responses observed in this ReA murine model. The specific mechanism by which TNFRp55 deficiency increase the number of DCs in MLN and RLN remain to be determined and are currently under investigation.