congresos y reuniones científicas
TNF receptor p55 signaling modulates IL-12/23p40 production 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
Reactive arthritis (ReA) is an aseptic synovitis that develops as a sequel to remote infection, often in the gastrointestinal or urogenital tract. The pathogenesis of ReA is incompletely known. In previous studies we demonstrated that TNFRp55 deficient (TNFRp55-/-) mice are more susceptible to oral Yersinia enterocolitica (Ye) O:3 infection, however, later in the infection they develop ReA. A mechanistic analysis revealed involvement of p40, a common subunit of heterodimeric IL-12 and IL-23, in the generation of augmented IFN-g and IL-17 production under TNFR p55 deficiency in this murine ReA model. Other authors have also demonstrated in other TNFR-deficient murine infectious models temporally distinct roles of TNF. Thus, the exact timing of TNF expression determined whether the cytokine played a protective or a pathogenic role. These studies also demonstrated that later in the infection, TNF was able to regulate the adaptive immune response via inhibition of IL-12 and IL-23. The biological activities of TNF are mediated by two receptors, TNFRp55 and TNFRp75. Several experiments indicate that TNFRp55 is the primary signaling receptor on most cell types through which the majority of inflammatory responses classically attributed to TNF occur. This receptor can initiate the apoptotic pathway in addition to the mitogen-activated protein kinase (MAPK) pathway, which results in NF-kB translocation competing with the apoptotic pathway branch and ensure cell survival. Recent studies have showed that TNFR1 is the main receptor responsible for IL-12p40 regulation. This anti-inflammatory effect is not due to apoptosis and is depending on the source and timing of TNF expression. However, the mechanism of temporal regulation of IL-12p40 by TNFR1 signaling is unclear as well as this modulation in Ye-induced ReA.The purpose of this work was to gain insight into a regulatory role of TNFRp55 signaling on IL-12/23p40 production in Ye-induced ReA. Therefore, we analyzed IL-12/23p40 together with TNF, IL-10, IFN-g and IL-17 after LPS stimulation of splenocytes of wild-type (WT) and TNFRp55-/-mice. Splenocytes were used to evaluate in the same cell culture the cytokines of the innate and adaptive immune responses. To examine a temporal effect, the cytokines were measured at different time after LPS stimulation. The responses were compared in splenocytes of naïve mice, and in these cells obtained from mice at day 14 after Ye infection, time that corresponded with arthritis onset in TNFRp55-/- mice. In addition, the effect of TNF on IL-12p40 production by LPS-stimulated macrophages of RAW 264.7 cell line was also evaluated.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 saline, using a gastric tube. From naïve mice or at day 14 after Ye infection, spleens were isolated, and erythrocytes were lysed in ACK buffer (pH 7.2). Cells (2 x 106 cell/well) were cultured in 24-well plates for 24h at 37°C in an humidified atmosphere of 5% CO2, in complete RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in the presence or absence of commercial Escherichia coli LPS (1 ug/ml). Signal transduction inhibitors that selectively block p38 (SB203580), ERK (PD98059) or JNK (SP600125) MAPKs were dissolved in dimethyl sulfoxide (DMSO). DMSO was used as a solvent control in all inhibitor studies. Splenocytes were treated with these inhibitors (10 uM) or DMSO for 1 h prior to LPS-stimulation and maintained in the media for the course of the experiment. Cell culture supernatants at 3, 12 and 24 h were obtained and stored at -20°C until cytokine determination.On the other hand, RAW264.7 cells were cultured in complete DMEM medium supplemented with 10 % FBS at 37 °C in a humidified incubator with 5 % CO2. The cells were preincubated with TNF followed by stimulation with IFN-g (10ng/ml) and LPS (100ng/ml) for 24 h. Supernatants were collected and assayed for cytokine production. Mouse IL-12/IL-23p40, TNF, IL-10, IL-17 and IFN-g were determined in the culture supernatants using commercial capture ELISA kits according to the manufacturer?s instructions. Differences in the amounts of cytokines in the groups were compared using one or two-way analysis of variance (ANOVA), followed by Tukey?s test or Bonferroni multiple comparison test, respectively. A p value less than 0.05 was considered as statistically significant. All analyses were performed using GraphPad Prism 5 software. We found that at 24h after LPS stimulation, splenocytes from WT mice activated during Ye infection were more susceptible to inhibition of IL-12p40 production and to induction of IL-10 than cells from naïve animals (p<0.001, compared naïve and Ye- infected mice for both cytokines). In contrast, this inhibition was not exhibited by splenocytes of infected TNFRp55 -/- mice suggesting a regulatory role for TNFRp55 at day 14 of infection. Accordingly, LPS-stimulated splenocytes derived from Ye-infected TNFRp55 -/- mice secreted significant more IL-12p40 than cells from Ye-infected WT mice at 12 and 24 h after LPS-stimulation (p<0.001 compared with Ye-infected WT mice). These results were matched with the studies in LPS/IFN-g-stimulated RAW 264.7 line in which preincubation with TNF regulated IL-12p40 production (p<0.05). Consequently, we analyzed the kinetic of TNF production in splenocytes and found that the secretion of this cytokine in cells from naïve mice occurred earlier than IL-12/23p40 and IL-10 after LPS stimulation peaking at 12 h. By comparison with WT cells, TNF secretion by cells from infected TNFRp55 -/- mice resulted higher than cells of WT cells at 24 h after LPS stimulation indicating TNF induction as an attempt to compensate its function in absent of TNFRp55 signaling (p<0.001, compared with Ye-infected WT mice). Both IL-12p40 and IL-10 production significantly decreased in stimulated TNFRp55-/- naïve cells when JNK or p38 inhibitions were used (p<0.0001, compared with DMSO/LPS control). Moreover, at 12 h after LPS stimulation, both JNK and p38 inhibitions reduced IL-12p40 secretion in naïve TNFRp55-/- cells and consequently resulted similar to those WT cells suggesting that these pathways may be involved in the overproduction of IL-12p40 in TNFRp55 deficient cells. In addition, IL-17 production was increased in both Ye-infected WT and TNFRp55 mice but not in naïve cells demonstrating association of this cytokine with in vivo infection. Moreover, IL-12p40 differences correlated with IFN-g levels since splenocytes from Ye-infected TNFRp55-/- secreted significant higher levels of IFN-g than the cells from Ye-infected WT mice at 12 and 24 h after LPS-stimulation (p<0.001, compared with infected WT mice). We concluded that TNFRp55-pathway controls IL-12p40, and consequently impact on IFN-g production in Ye-induced ReA. Moreover, JNK and p38 MAPK activation mediate IL-12p40 and IL-10 secretion. The higher inhibition of IL-12p40 production associated with higher IL-10 induction in infected WT cells suggest that IL-10 may play a role in IL-12p40 regulation by TNFRp55 signaling under Ye infection. These findings support the dual role of TNF in this ReA murine model and contribute to elucidate the critical role of TNFRp55 pathway in controlling arthritis development with impact on the ReA treatment.