Involvement of tyrosine kinase-2 in both the IL-12/Th1 and IL-23/Th17 axes in vivo.

Tyrosine kinase-2 (Tyk2), a member of the Jak family of kinases, mediates the signals triggered by various cytokines, including type I IFNs, IL-12, and IL-23. In the current study, we investigated the in vivo involvement of Tyk2 in several IL-12/Th1- and IL-23/Th17-mediated models of experimental diseases, including methylated BSA injection-induced footpad thickness, imiquimod-induced psoriasis-like skin inflammation, and dextran sulfate sodium- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. In these disease models, Tyk2 deficiency influenced the phenotypes in immunity and/or inflammation. Our findings demonstrate a somewhat broader contribution of Tyk2 to immune systems than previously expected and suggest that Tyk2 may represent an important candidate for drug development by targeting both the IL-12/Th1 and IL-23/Th17 axes.

Xanthine oxidoreductase promotes the inflammatory state of mononuclear phagocytes through effects on chemokine expression, peroxisome proliferator-activated receptor-{gamma} sumoylation, and HIF-1{alpha}.

The protective effects of pharmacological inhibitors of xanthine oxidoreductase (XOR) have implicated XOR in many inflammatory diseases. Nonetheless, the role played by XOR during inflammation is poorly understood. We previously observed that inhibition of XOR within the inflammatory mononuclear phagocytes (MNP) prevented neutrophil recruitment during adoptive transfer demonstrating the role of XOR in MNP-mediated neutrophil recruitment. To further explore the role of XOR in the inflammatory state of MNP, we studied MNP isolated from inflammatory lungs combined with analyses of MNP cell lines. We demonstrated that XOR activity was increased in inflammatory MNP following insufflation of Th-1 cytokines in vivo and that activity was specifically increased by MNP differentiation. Inhibition of XOR reduced levels of CINC-1 secreted by MNP. Expression of peroxisome proliferator-activated receptor γ (PPARγ) in purified rat lung MNP and MNP cell lines reflected both the presence of PPARγ isoforms and PPARγ SUMOylation, and XOR inhibitors increased levels of SUMO-PPARγ in MNP cell lines. Both ectopic overexpression of XOR cDNA and uric acid supplementation reduced SUMO-PPARγ in MNP cells. Levels of the M2 markers CD36, CD206, and arginase-1 were modulated by uric acid and oxonic acid, whereas siRNA to SUMO-1 or PIAS-1 also reduced arginase-1 in RAW264.7 cells. We also observed that HIF-1α was increased by XOR inhibitors in inflammatory MNP and in MNP cell lines. These data demonstrate that XOR promotes the inflammatory state of MNP through effects on chemokine expression, PPARγ SUMOylation, and HIF-1α and suggest that strategies for inhibiting XOR may be valuable in modulating lung inflammatory disorders.

The effects of electroacupuncture on TH1/TH2 cytokine mRNA expression and mitogen-activated protein kinase signaling pathways in the splenic T cells of traumatized rats.

BACKGROUND: Surgical trauma contributes to postoperative immune suppression, which is associated with an increased susceptibility to subsequent infections. Electroacupuncture (EA) can alleviate pain and exert immunoregulatory effects. However, the mechanism underlying the immnuomodulation effects of EA is not fully elucidated. Therefore, we investigated the effects of EA on T helper (Th)1/Th2 cytokine production and mRNA expression and evaluated the signaling regulatory mechanism of EA effects. METHODS: Rats were divided into four groups (n = 24 each): control, trauma, trauma (T) + sham EA, and T + EA. EA was applied to Zusanli (ST36) and Lanwei (Extra37) acupoints at 20 min after surgery for 30 min, and then performed once a day on postoperative days 1-5. Splenic T cells were isolated and the production and mRNA expression of interleukin (IL)-2, interferon-gamma, IL-4, and IL-10 were assayed. The activation of mitogen-activated protein kinase and the DNA binding activity of nuclear factor (NF)-kappaB and activator protein (AP)-1 were examined. RESULTS: Paw withdrawal threshold and paw withdrawal latency were significantly increased in the T + EA group compared with the trauma group from postoperative day 1 (paw withdrawal threshold: 5.8 +/- 0.7 vs 3.0 +/- 0.7 g; paw withdrawal latency: 7.0 +/- 0.8 vs 4.5 +/- 0.5 s; P < 0.001) to day 5 (9.0 +/- 0.6 vs 5.5 +/- 0.6 g; 12.0 +/- 1.3 vs 7.0 +/- 0.8 s; P < 0.001). Th1 cytokine (IL-2 and interferon-gamma) production and mRNA expression in splenic T cells of traumatized rats were significantly decreased on postoperative day 3 (P < 0.001, trauma group versus control group), whereas Th2 cytokine (IL-4 and IL-10) production and mRNA expression were increased (P < 0.001). This was accompanied with a significant depression in the activity of extracellular-regulated protein kinase (ERK)1/2, p38, NF-kappaB, and AP-1 (P < 0.001, trauma group versus control group). EA administration increased Th1 cytokine protein and mRNA expression, suppressed Th2 cytokine protein and mRNA expression (P < 0.05, T + EA group versus trauma group), and increased the activity of ERK1/2, p38, NF-kappaB, and AP-1 (P < 0.001, T + EA group versus trauma group). CONCLUSIONS: EA regulates a balance between Th1 and Th2 cytokines at protein and mRNA levels in splenic T cells, and, at least in part, involves the signaling pathways of ERK1/2, p38, NF-kappaB, and AP-1. The findings suggest that EA may improve immune suppression after surgical trauma.

Pertussis toxin enhances Th1 responses by stimulation of dendritic cells.

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-gamma and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-gamma. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-alpha production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-gamma production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.

A minimal IFN-gamma promoter confers Th1 selective expression.

Th1 and Th2 cells differentiate from naive precursors to effector cells that produce either IFN-gamma or IL-4, respectively. To identify transcriptional paths leading to activation and silencing of the IFN-gamma gene, we analyzed transgenic mice that express a reporter gene under the control of the 5' IFN-gamma promoter. We found that as the length of the promoter is increased, -110 to -225 to -565 bp, the activity of the promoter undergoes a transition from Th1 nonselective to Th1 selective. This is due, at least in part, to a T box expressed in T cells-responsive unit within the -565 to -410 region of the IFN-gamma promoter. The -225 promoter is silent when compared with the -110 promoter and silencing correlates with Yin Yang 1 binding to the promoter. The p38 mitogen-activated protein kinase signaling pathway, which also regulates IFN-gamma gene transcription, regulates the -70- to -44-bp promoter element. Together, the results demonstrate that a minimal IFN-gamma promoter contains a T box expressed in T cells responsive unit and is sufficient to confer Th1 selective expression upon a reporter.

Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production.

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE(2) production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE(2) synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-gamma present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE(2) synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-gamma. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-gamma in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.

Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice.

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-gamma production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-gamma production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-gamma production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-gamma production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-gamma and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.

Co-regulation of antigen-specific T lymphocyte responses by type I and type II cyclic AMP-dependent protein kinases (cAK).

While a differential sensitivity to cyclic AMP (cAMP)-mediated signaling between Th1 and Th2 cells has been hypothesized, differential activity of downstream signaling through cAMP-dependent protein kinase (cAK) isoforms remains unexplored. We herein report the effects of type 1- and type 2-specific cAK agonists and antagonists on proliferative responses and cytokine generation from ragweed-driven peripheral blood mononuclear cells (PBMCs) and Amb a 1-specific Th1 and Th2 clones. Rp-8-Cl- and Rp-8-CPT-cAMP were utilized as single agent antagonists of cAKI and cAKII, respectively; 8-AHA-cAMP, with and without 8-PIP-cAMP, and 8-CPT-cAMP, with and without 6-Bnz-cAMP, were used as synergistic agonist pairs specific for the cAKI and cAKII, respectively. Activation of either cAKI or cAKII individually was ineffective in down-regulating proliferative responses of PBMCs or T cell clones; concentration-response curves for the Th1 and Th2 clones were identical. Moreover, inhibition of either cAKI or cAKII individually was ineffective in overcoming the down-regulatory effects of phosphodiesterase inhibition. Activation of either cAKI or cAKII individually was ineffective in down-regulating proinflammatory cytokine generation from T cell clones (interleukin-4 from Th2; interferon-gamma from Th1). However, concurrent activation of both cAKI and cAKII produced down-regulatory effects equivalent to those of the phosphodiesterase inhibitor on both proliferation and cytokine generation. These data suggest a critical role for concurrent activation of cAKI and cAKII in the functional efficacy of antigen-driven downstream signaling due to elevations of intracellular cAMP and argue against differential regulation of Th1 and Th2 responses by cAK subtypes.