Cells was grown in 5% CO2/air flow at 37C in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin G, 100 g/ml streptomycin, and 1 g/ml fungizone. and TNF, which have been implicated PF-5006739 in Th17 reactions, but not IL-12 or IFN.28C32 At present, the mechanisms that govern sponsor reactions to including the neutrophil influx are not well understood. We propose that Th17 cells play a significant part in the immune response to through recruitment of neutrophils and additional innate defense factors. We have tested this hypothesis inside a mouse model of genital tract illness. Our results indicate that induces IL-17 production in vitro and in vivo, leading to IL-17-dependent secretion of IL-6, LIX, and MIP-2 from genital tract cells. Furthermore, obstructing of IL-17A with antibody, or deletion of IL-17RA in mice prolongs the course of illness with and delays the recruitment of neutrophils. RESULTS Production of cytokines in response to is definitely capable of inducing cytokines standard of a Th17 response, we incubated mouse splenic mononuclear cells with either or gonococcal OMV and teste d the supernatants for secreted cytokines. After three days, the cells produced IL-17, in response to either FA1090 or its outer membranes, as well as the mitogen ConA (Number 1A, B). IL-17 production improved with time of incubation and dose of OMV, through 5 days with 5 g/ml of OMV; no significant increase in IL-17 occurred at higher OMV concentrations. Related results were seen with strain MS11, and with an Opa-protein deletion mutant of strain FA1090 (ref. 33; data not demonstrated). Heat treatment of OMV preparations (100C for 10 min) did not abrogate the induction of IL-17 (data not demonstrated), suggesting the stimulatory components were heat-stable. Consequently to determine whether lipo-oligosaccharide (LOS) was responsible for inducing IL-17, LOS from strain PID2 and related gonococci were cultured with spleen cells from either C3H/HeJ (TLR4-deficient), C3H/FeJ (TLR4-normal), or TLR2-knockout mice. Gonococcal LOS induced IL-17 production in TLR4-normal (and TLR2-deficient) cells, but not in TLR4-deficient cells (Number 1C). Furthermore the IL-17 response to gonococci or OMV was diminished (but not completely abrogated) in TLR4-deficient cells, whereas TLR2-knockout cells were responsive to gonococci or OMV (Number 1C). Open in a separate window Number 1 ITGA9 induces Th17-connected cytokines, but not Th1-connected cytokines. (A) Production of IL-17 from mouse splenic mononuclear cells, incubated in medium only (control) or with 2 g/ml ConA or outer membrane vesicles (OMV) at numerous concentrations for either 1, 3, or 5 days. Supernatants were assayed for IL-17 by ELISA. (B) Production of IL-17 from mouse splenic mononuclear cells, incubated in medium only (control) or with 2 g/ml ConA or at numerous multiplicities of illness (MOI) for either 1, 3, or 5 days. Supernatants were assayed for IL-17 by ELISA. (C) Production of IL-17 from C3H/HeJ (TLR4-deficient), C3H/FeJ (TLR4-normal), or TLR2-knockout mouse splenic mononuclear cells incubated for 3 days in medium only (control), or with PID2 at MOI 10:1, or with OMV (5g/ml), or LOS (5g/ml). Supernatants were assayed for IL-17 by ELISA. (D) Production of IL-6, IL-12, IL-17, IL-22, and IFN- from mouse splenic mononuclear PF-5006739 cells incubated for 3 days in medium only (control) or with 2 g/ml or 5 g/ml of OMV, or at an MOI of 10:1. Supernatants were assayed for cytokines by ELISA. All experiments (A-D) were carried out in triplicate, and results are demonstrated as mean SD; * shows cytokine secretion significantly above control levels (P 0.01; College students t). (E) Circulation cytometry profiles of murine spleen cells cultured for 3 days with gonococcal OMV (ideal panel), compared to control unstimulated cells (remaining panel). Cells were stained for intracellular IL-17 (PE) and surface -T cell PF-5006739 receptor (FITC). In addition, supernatants from spleen cells cultured with gonococci or OMV showed production of IL-22 and IL-6, but a lack of IFN- (Number 1D), consistent with the development of a Th17 response. Production of IL-12, a hallmark of a Th1 response, was not observed at any time point in response to gonococcal activation. The ability of to induce secretion of IL-17, IL-22, and IL-6, but not IFN- in mouse spleen cell cultures, suggests that it is capable of eliciting Th17 reactions. As determined by flow cytometry, some of the cells that produced IL-17 were rather than T cells (Number 1E). However, IL-23, was not recognized in cultures of spleen mononuclear cells stimulated with gonococci or ConA. It is possible that IL-23 was produced but not detected because of quick uptake by T cells. Consequently, we identified whether gonococci could selectively induce IL-23 production by APC only. When mouse BMDC were incubated with or its OMV for 24 hours, IL-6 and IL-23 were recognized in the supernatants, whereas IL-12 production was produced only.
Cells was grown in 5% CO2/air flow at 37C in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin G, 100 g/ml streptomycin, and 1 g/ml fungizone