Fig 1.
Cytokine secretion by LPS-stimulated WT and TNFRp55-/- splenocytes on ReA onset.
Splenic cells were obtained from WT and TNFRp55-/-mice on day 14 after Ye infection (ReA onset), and stimulated in vitro with LPS. Splenocytes from uninfected mice (PBS) were used as controls. After 3, 12 and 24h of stimulation, supernatants were collected and assayed by ELISA for TNF (A), IL-12/23p40 (B), IFN-γ (C), IL-17A (D) and IL-10 (E). Values represent the mean ± SEM of quadruplets at each time point of one out of two independent experiments, with five mice per group. *P<0.05; ** P<0.01; *** P<0.001.
Fig 2.
IL-12/23p40+ dendritic cells in spleen of WT and TNFRp55-/- mice on ReA onset.
Splenic cells were obtained from WT and TNFRp55-/- mice on day 14 after Ye infection (ReA onset). Dendritic cells (DCs) were stained for cell surface CD11c and MHC-II, and analyzed by flow cytometry. (A) Absolute DC number in the spleen of Ye-infected WT and TNFRp55-/- mice are presented. Splenic DCs of uninfected (PBS) mice were used as controls. (B) Representative dot plot showing analysis of IL-12/23p40+ DCs (CD11c+MHC-II+ gate) in splenocytes from control (PBS), and Ye-infected WT and TNFRp55-/- mice. The cells were stimulated with PMA/Ionomycin and brefeldin for 5 h, and then stained for cell surface CD11c and MHC-II, and intracellular IL-12/23p40, and then analyzed by flow cytometry. The numbers in the plots indicate the percentages of labeled cells in representative mice. (C) Percentage of IL-12/23p40+ DCs of the sum of three independent experiments. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SEM. ***P<0.001. ns: not significant.
Fig 3.
Activation profile of and IL-12/23p40 secretion by isolated dendritic cells.
Dendritic cells (DCs) were expanded in vivo in WT and TNFRp55-/- mice. The cells were isolated and infected in vitro with Ye. Representative overlaid flow cytometry histogram analysis showing CD80 (A) and CD86 (C) expressions of uninfected and Ye-infected splenic DCs. Percentages of DCs CD80+(B) and CD86+ (D), and IL-12/23p40 secretion in the supernatants (E) expressed as the mean ± SEM of quadruplets of one out of two independent experiments with four mice per group. *P<0.05; ** P<0.01.
Fig 4.
Th1 differentiation of CD4+ lymphocytes by WT or TNFRp55-/-DCs.
Ye-infected splenic DCs of WT or TNFRp55-/-were co-cultured with purified CD4+ lymphocytes of Ye-infected WT (A), TNFRp55-/-mice (D). The cells were stained with cell surface CD4 and intracellular IFN-γ, and analyzed by flow cytometry. Dot plots from CD3+ CD4+gate, represent a sample of the statistical analysis shown in B and E (A and D). Percentages of WT CD4+ IFN-γ+ (B) and TNFRp55-/- CD4+ IFN-γ+ (E) T cells. Levels of IFN-γ measured in culture supernatants by ELISA(C and F). Results are expressed as the mean ± SEM of quadruplets of one out of two independent experiments with four mice per group. *P<0.05; ** P<0.01; *** P<0.001.
Fig 5.
Th17 differentiation of CD4+ lymphocytes by WT or TNFRp55-/- DCs.
Ye-infected splenic DCs of WT or TNFRp55-/-were co-cultured with purified CD4+ lymphocytes of Ye-infected WT (A) or TNFRp55-/-mice (D) as described in methods. The cells were stained with cell surface CD4 and intracellular IL-17, and analyzed by flow cytometry. Dot plots from CD3+ CD4+ gate, represent a sample of the statistical analysis shown in B and E (A and D). Percentages of WT CD4+ IL-17A+ (B) and TNFRp55-/- CD4+ IL-17A+ (E) T cells. Levels of IL-17A measured in culture supernatants by ELISA (C and F). Results are expressed as the mean ± SEM of quadruplets of one out of two independent experiments with four mice per group. *P<0.05; *** P<0.001. ns: not significant.
Fig 6.
Participation of MAPKs on IL-12/23p40 overproduction by dendritic cells.
Splenic cells were obtained from uninfected (A and B) and Ye-infected WT and TNFRp55-/- mice (on ReA onset) (C and D), and stimulated in vitro with LPS in the presence of the indicated MAPK inhibitors. After 12 and 24h, supernatants were collected from the cell culture, and assayed by ELISA for IL-12/23p40. The same experimental scheme was performed with in vivo expanded and isolated WT and TNFRp55-/- DCs. After 24h, IL-12/23p40 was assessed in the 24h-supernatants of WT (E) and TNFRp55-/- (F) DCs. A comparison of IL-12/23p40 levels in the supernatants of WT versus TNFRp55-/- DCs was performed (G). Results are expressed as the mean ± SEM of quadruplets of one out of two independent experiments with four mice per group. ** P<0.01; *** P<0.001. ns: not significant.
Fig 7.
Phenotypical and functional characterization of JAWS II cell line.
Representative overlaid flow cytometry histogram (A) and percentage (B) analysis, showing dendritic cell surface markers and TNF receptors in JAWS II cell line after 24h of stimulation with LPS. Unstimulated cells (basal) were used as controls. The levels of TNF (C) were assessed in the supernatants by ELISA. JAWS II cells were stimulated with LPS alone or in combination with human TNF (D), or in the presence of MAPK inhibitors (E). IL-12/23p40 secretion was analyzed in these supernatants. The results represent the mean ± SEM of quadruplets of one out of two independent experiments. *P<0.05; ** P<0.01; *** P<0.001.
Fig 8.
Contribution of TNFRp75 and TNFRp55 to IL-12/23p40 production by WT and TNFRp55-/- dendritic cells.
Isolated WT (A, C, E and G) and TNFRp55-/- (B, D, F and H) DCs were stimulated with LPS alone or in the presence of Etanercept (A, B, E, F) that inhibits both TNFRp75 and TNFRp55; or CAY10500 (C, D, G and H) that blocks specifically TNFRp55. After 24h, the supernatants were collected and assayed by ELISA for IL-12/23p40 (A-D), and IL-10 (E-H). Results are expressed as the mean ± SEM of quadruplets of one out of two independent experiments. *P<0.05; ** P<0.01. ns: not significant.
Fig 9.
Proposal model of the participation of DCs controlling IL-12/23p40 through TNFRp55 and TNFRp75 under bacterial antigen stimulation.
TNF through TNFRp75 triggers IL-12/23p40 secretion by DCs. This IL-12/23p40 production is controlled by TNF/TNFRp55 pathway involving IL-10 and other unknown mechanisms. In the absence of the regulatory TNF/TNFRp55 circuit, DCs increase IL-12/23p40 secretion, driving the adaptive response to chronic Th1 and Th17 programs. Black spots represent inflammatory cytokines, and grey spots represent regulatory cytokines.