Deficiency of TNF receptor p55 impacts on dendritic cell population of lymph nodes in Yersinia enterocolitica-induced reactive arthritis

SILVA JE; DAVE MN; MAYORDOMO AC; CARGNELUTTI E; GORLINO C; DI GENARO MS

Medellin

Congreso; IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología; 2015

Latin American Association of Immunology

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 Peyers 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 Tukeys 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