HIV-1 protein Vpr suppresses IL-12 production from human monocytes by enhancing glucocorticoid action: potential implications of Vpr coactivator activity for the innate and cellular immunity deficits observed in HIV-1 infection

The HIV-1 protein Vpr has glucocorticoid receptor coactivator activity, potently increasing the sensitivity of glucocorticoid target tissues to cortisol. Patients with AIDS and normal cortisol secretion have manifestations compatible with glucocorticoid hypersensitivity of the immune system, such as suppression of innate and cellular immunities. The latter can be explained by glucocorticoid-induced inhibition of cytokine networks regulating innate and Th1-driven cellular immunity. We demonstrated that extracellularly administered Vpr protein dose-dependently potentiated glucocorticoid-induced suppression of both mRNA expression and secretion of IL-12 subunit p35 and IL-12 holo-protein, but not IL-12 subunit p40 or IL-10, by human monocytes/macrophages stimulated with LPS or heat-killed, formalin-fixed Staphylococcus aureus (Cowan strain 1). This effect was inhibited by the glucocorticoid receptor antagonist RU 486. Also, Vpr changed the expression of an additional five glucocorticoid-responsive genes in the same direction as dexamethasone and was active in potentiating the trans-activation, but not the trans-repression, properties of the glucocorticoid receptor on nuclear factor kappaB- or activating protein 1-regulated simple promoters. Thus, extracellular Vpr enhances the suppressive actions of the ligand-activated glucocorticoid receptor on IL-12 secretion by human monocytes/macrophages. Through this effect, Vpr may contribute to the suppression of innate and cellular immunities of HIV-1-infected individuals and AIDS patients.     

Tyrphostin B42 inhibits IL-12-induced tyrosine phosphorylation and activation of Janus kinase-2 and prevents experimental allergic encephalomyelitis

IL-12 is a macrophage-derived cytokine that induces proliferation, cytokine production, and cytotoxic activity of T and NK cells. Signaling through its receptor, IL-12 induces these cellular responses by tyrosine phosphorylation and activation of Janus kinase-2 (Jak-2), Tyk-2, Stat3, and Stat4. We have used tyrphostin B42 (AG490), a Jak-2 inhibitor, to determine the role of Jak-2 kinase in IL-12 signaling and IL-12-induced T cell functions. Treatment of activated T cells with tyrphostin B42 inhibited the IL-12-induced tyrosine phosphorylation and activation of Jak-2 without affecting Tyk-2 kinase. In contrast, treatment with tyrphostin A1 inhibited the tyrosine phosphorylation of Tyk-2 but not that of Jak-2 kinase. Inhibition of either Jak-2 or Tyk-2 leads to a decrease in the IL-12-induced tyrosine phosphorylation of Stat3, but not of Stat4, protein. While inhibition of Jak-2 lead to programmed cell death, the inhibition of Jak-2 or Tyk-2 resulted a decrease in IFN-gamma production. We have further tested the in vivo effects of tyrphostin B42 in experimental allergic encephalomyelitis, a Th1 cell-mediated autoimmune disease. In vivo treatment with tyrphostin B42 decreased the proliferation and IFN-gamma production of neural Ag-specific T cells. Treatment of mice with tyrphostin B42 also reduced the incidence and severity of active and passive EAE. These results suggest that tyrphostin B42 prevents EAE by inhibiting IL-12 signaling and IL-12-mediated Th1 differentiation in vivo.     

Inhibition of IFN-gamma-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion

Mycobacterium tuberculosis (MTB) persists inside macrophages despite vigorous immune responses. MTB and MTB 19-kDa lipoprotein inhibit class II MHC (MHC-II) expression and Ag processing by a Toll-like receptor 2-dependent mechanism that is shown in this study to involve a defect in IFN-gamma induction of class II transactivator (CIITA). Exposure of macrophages to MTB or MTB 19-kDa lipoprotein inhibited IFN-gamma-induced MHC-II expression, but not IL-4-induced MHC-II expression, by preventing induction of mRNA for CIITA (total, type I, and type IV), IFN regulatory factor-1, and MHC-II. MTB 19-kDa lipoprotein induced mRNA for suppressor of cytokine signaling (SOCS)1 but did not inhibit IFN-gamma-induced Stat1 phosphorylation. Furthermore, the lipoprotein inhibited MHC-II Ag processing in SOCS1(-/-) macrophages. MTB 19-kDa lipoprotein did not inhibit translocation of phosphorylated Stat1 to the nucleus or Stat1 binding to and transactivation of IFN-gamma-sensitive promoter constructs. Thus, MTB 19-kDa lipoprotein inhibited IFN-gamma signaling independent of SOCS1 and without interfering with the activation of Stat1. Inhibition of IFN-gamma-induced CIITA by MTB 19-kDa lipoprotein may allow MTB to evade detection by CD4(+) T cells.     

IL-4 and IL-2 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis.

It is well established that T cell maturation and activation are negatively regulated by a mechanism termed apoptosis. We now present evidence that glucocorticoids, known to possess immunosuppressive properties, cause apoptosis in mature Th cells, similarly to what has been reported for thymocytes. Th cells treated with the synthetic glucocorticoid dexamethasone show genome fragmentation into oligonucleosomal fragments, and proliferation of growth factor stimulated Th cells is inhibited by glucocorticoids. We show that IL-4 specifically rescues Th2 cells from dexamethasone-mediated apoptosis, whereas IL-2 and IL-1 are ineffective in these cells. However, IL-2 is the relevant rescue-factor of glucocorticoid-treated Th1 cells. The rescue induced by IL-4 and IL-2 is thought to be mediated by protein kinases (possibly protein kinase C), as evidenced by the fact that the protein kinase inhibitor H7 blocks the action of IL-4 and IL-2 in glucocorticoid-treated cells. Our in vitro data show that mature T cells can be protected by their own growth factors from the deleterious effects of the synthetic glucocorticoid dexamethasone, and suggest that specific interactions occur between lymphokines and naturally produced glucocorticoids in vivo, which may play a role in the regulation of the immune response.     

Stat5a inhibits IL-12-induced Th1 cell differentiation through the induction of suppressor of cytokine signaling 3 expression

In previous studies, we have shown that Th2 cell differentiation is diminished but Th1 cell differentiation is increased in Stat5a-deficient (Stat5a(-/-)) CD4(+) T cells. In the present study, we clarified the molecular mechanisms of Stat5a-mediated Th cell differentiation. We found that enhanced Th1 cell differentiation and the resultant IFN-gamma production played a dominant inhibitory role in the down-regulation of IL-4-induced Th2 cell differentiation of Stat5a(-/-) CD4(+) T cells. We also found that IL-12-induced Stat4 phosphorylation and Th1 cell differentiation were augmented in Stat5a(-/-) CD4(+) T cells. Importantly, the expression of suppressor of cytokine signaling (SOCS)3, a potent inhibitor of IL-12-induced Stat4 activation, was decreased in Stat5a(-/-) CD4(+) T cells. Moreover, a reporter assay showed that a constitutively active form of Stat5a but not Stat6 activated the SOCS3 promoter. Furthermore, chromatin immunoprecipitation assays revealed that Stat5a binds to the SOCS3 promoter in CD4(+) T cells. Finally, the retrovirus-mediated expression of SOCS3 restored the impaired Th cell differentiation of Stat5a(-/-) CD4(+) T cells. These results suggest that Stat5a forces the Th1/Th2 balance toward a Th2-type by preventing IL-12-induced Th1 cell differentiation through the induction of SOCS3.     

Setaria digitata secreted filarial lipids modulate IL-12 signaling through JAK-STAT pathway leading to the development of Th1 response

Filariasis is a debilitating parasitic disease in many tropical countries. Despite the highly evolved immune system, the filarial parasites successfully evade host immunity to persist for a sustained period of time. Earlier studies have shown that the filarial parasites achieve this long-term survival through release of immunosuppressive materials in the host. In this study, we show that the secreted filarial lipids (SFL) isolated from Setaria digitata suppress Th1 immune response. While immunization with myelin antigen induces Th1 response in mice, in vitro treatment with SFL resulted in a dose-dependent decrease in myelin antigen-induced proliferation and secretion of IL-12 and IFNgamma. The SFL also inhibited IL-12-induced T cell proliferation and Th1 differentiation in vitro. The inhibition of T cell responses by SFL associates with the blockade of IL-12-induced activation of JAK-STAT signaling pathway in T cells. These findings suggest that the SFL modulates Th1 immune response by blocking IL-12 signaling in T cells and thus play a role in host immune evasion of filarial parasites.     

Dendritic cells transduced with SOCS-3 exhibit a tolerogenic/DC2 phenotype that directs type 2 Th cell differentiation in vitro and in vivo

Dendritic cells (DCs) have been suggested to direct a type of Th differentiation through their cytokine profile, e.g., high IL-12/IL-23 for Th1 (named DC1/immunogenic DCs) and IL-10 for Th2 (DC2/tolerogenic DCs). Suppressor of cytokine signaling (SOCS)-3 is a potent inhibitor of Stat3 and Stat4 transduction pathways for IL-23 and IL-12, respectively. We thus hypothesize that an enhanced SOCS-3 expression in DCs may block the autocrine response of IL-12/IL-23 in these cells, causing them to become a DC2-type phenotype that will subsequently promote Th2 polarization of naive T cells. Indeed, in the present study we found that bone marrow-derived DCs transduced with SOCS-3 significantly inhibited IL-12-induced activation of Stat4 and IL-23-induced activation of Stat3. These SOCS-3-transduced DCs expressed a low level of MHC class II and CD86 on their surface, produced a high level of IL-10 but low levels of IL-12 and IFN-gamma, and expressed a low level of IL-23 p19 mRNA. Functionally, SOCS-3-transduced DCs drove naive myelin oligodendrocyte glycoprotein-specific T cells to a strong Th2 differentiation in vitro and in vivo. Injection of SOCS-3-transduced DCs significantly suppressed experimental autoimmune encephalomyelitis, a Th1 cell-mediated autoimmune disorder of the CNS and an animal model of multiple sclerosis. These results indicate that transduction of SOCS-3 in DCs is an effective approach to generating tolerogenic/DC2 cells that then skew immune response toward Th2, thus possessing therapeutic potential in Th1-dominant autoimmune disorders such as multiple sclerosis.     

Helicobacter pylori infection interferes with epithelial Stat6-mediated interleukin-4 signal transduction independent of cagA,cagE, or VacA

Helicobacter pylori is a bacterial pathogen evolved to chronically colonize the gastric epithelium, evade immune clearance by the host, and cause gastritis, peptic ulcers, and even gastric malignancies in some infected humans. In view of the known ability of this bacterium to manipulate gastric epithelial cell signal transduction cascades, we determined the effects of H. pylori infection on epithelial IL-4-Stat6 signal transduction. HEp-2 and MKN45 epithelial cells were infected with H. pylori strains LC11 or 8823 (type 1; cagA(+)/cagE(+)/VacA(+)), LC20 (type 2; cagA(-), cagE(-), VacA(-)), and cagA, cagE, and vacA isogenic mutants of strain 8823, with some cells receiving subsequent treatment with the Th2 cytokine IL-4, a known Stat6 activator. Immunofluorescence showed a disruption of Stat6-induced nuclear translocation by IL-4 in LC11-infected HEp-2 cells. IL-4-inducible Stat6 DNA binding in HEp-2 and MKN45 cells was abrogated by infection, but MKN45 cell viability was unaffected. A decrease in IL-4-mediated Stat6 tyrosine phosphorylation in nuclear and whole cell lysates was also observed following infection with strains LC11 and LC20, while neither strain altered IL-4 receptor chain alpha or Janus kinase 1 protein expression. Furthermore, parental strain 8823 and its isogenic cagA, cagE, and vacA mutants also suppressed IL-4-induced Stat6 tyrosine phosphorylation to comparable degrees. Thus, H. pylori did not directly activate Stat6, but blocked the IL-4-induced activation of epithelial Stat6. This may represent an evolutionarily conserved strategy to disrupt a Th2 response and evade the host immune system, allowing for successful chronic infection.     

gC1q receptor ligation selectively down-regulates human IL-12 production through activation of the phosphoinositide 3-kinase pathway

gC1qR, a complement receptor for C1q, plays a pivotal role in the regulation of inflammatory and antiviral T cell responses. Several pathogens, including hepatitis C virus, exploit gC1qR-dependent regulatory pathways to manipulate host immunity. However, the molecular mechanism(s) of gC1qR signaling involved in regulating inflammatory responses remains unknown. We report the selective inhibition of TLR4-induced IL-12 production after cross-linking of gC1qR on the surface of macrophages and dendritic cells. Suppression of IL-12 did not result from increased IL-10 or TGF-beta, but was dependent on PI3K activation. Activation of PI3K and subsequent phosphorylation of Akt define an intracellular pathway mediating gC1qR signaling and cross-talk with TLR4 signaling. This is the first report to identify signaling pathways used by gC1qR-mediated immune suppression, and it establishes a means of complement-mediated immune suppression to inhibit Th1 immunity crucial for clearing pathogenic infection.     

Modulation of mast cell proteinase-activated receptor expression and IL-4 release by IL-12

It has been recognized that protease-activated receptors (PARs), interleukin (IL)-4 and IL-6 are involved in the pathogenesis of allergic diseases, and that IL-12 plays a role in adaptive immune response. However, little is known of the effect of IL-12 on protease-induced cytokine release from mast cells. In the present study, we examined potential influence of IL-12 on mast cell PAR expression and IL-4 and IL-6 release. The results showed that IL-12 downregulated the expression of PAR-2 and upregulated expression of PAR-4 on P815 cells. It also downregulated expression of PAR-2 mRNA, and upregulated expression of PAR-1, PAR-3 and PAR-4 mRNAs. However, IL-12 enhanced trypsin- and tryptase-induced PAR-2 and PAR-2 mRNA expression. It was observed that IL-12 induced release of IL-4, but reduced trypsin- and tryptase-stimulated IL-4 secretion from P815 cells. PD98059, U0126 and LY294002 not only abolished IL-12-induced IL-4 release but also inhibited IL-12-induced phosphorylation of extracellular signal-regulated kinase and Akt. In conclusion, IL-12 may serve as a regulator in keeping the balance of Th1 and Th2 cytokine production in allergic inflammation.