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.     

Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral 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.     

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.     

Parthenolide inhibits activation of signal transducers and activators of transcription (STATs) induced by cytokines of the IL-6 family

Progression of inflammatory processes correlates with the release of cell-derived mediators from the local site of inflammation. These mediators, including cytokines of the IL-1 and IL-6 families, act on host cells and exert their action by activating their signal transduction pathways leading to specific target gene activation. Parthenolide, a sesquiterpene lactone found in many medical plants, is an inhibitor of IL-1-type cytokine signaling that blocks the activation of NF-kappaB. Here we show that parthenolide is also an effective inhibitor of IL-6-type cytokines. It inhibits IL-6-type cytokine-induced gene expression by blocking STAT3 phosphorylation on Tyr705. This prevents STAT3 dimerization necessary for its nuclear translocation and consequently STAT3-dependent gene expression. This is a new molecular mechanism of parthenolide action that additionally explains its anti-inflammatory activities.     

Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis

Stat6 and IRS-2 are two important signaling proteins that associate with the cytoplasmic tail of the interleukin 4 (IL-4) receptor. Data from numerous in vitro experiments have led to a model for IL-4 signal transduction in which the Stat6 signaling pathway is responsible for the IL-4 induced changes in gene expression and differentiation events, while the IRS-2 signaling pathway provides mitogenic and antiapoptotic signals. In order to determine the relative contributions of these signaling molecules in primary lymphocytes, we have examined IL-4 responses in T cells from mice deficient for either Stat6 or IRS-2 as well as from mice doubly deficient for both genes. Both IRS-2 and, especially, Stat6 are shown to be critically involved in IL-4-induced proliferation of T cells, presumably through the cooperative regulation of the Cdk inhibitor p27kip1. Like Stat6-deficient Th cells, IRS-2-deficient cells are also compromised in their ability to secrete Th2 cytokines, revealing a previously unrecognized role for IRS-2 in Th2 cell development. Although Stat6 and/or IRS-2 expression is required for IL-4-induced proliferative and differentiative responses, both signaling proteins are dispensable for the antiapoptotic effect of IL-4. However, treatment of lymphocytes with a protein tyrosine phosphatase inhibitor is able to block the antiapoptotic effect of IL-4 specifically in Stat6- or IRS-2-deficient cells and not in wild-type cells. Our results suggest that Stat6 and IRS-2 cooperate in promoting both IL-4-induced proliferative and differentiating responses, while an additional signaling mediator that depends on protein tyrosine phosphatase activity contributes to the antiapoptotic activities of IL-4 in primary T cells.     

Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability,during RANKL-mediated osteoclastogenesis

Parthenolide, a natural product derived from Feverfew, prevents septic shock and inflammation. We aimed to identify the effects of parthenolide on the RANKL (receptor activator of NF-κB ligand)-induced differentiation and bone resorbing activity of osteoclasts. In this study, parthenolide dose-dependently inhibited RANKL-mediated osteoclast differentiation in BMMs, without any evidence of cytotoxicity and the phosphorylation of p38, ERK, and IκB, as well as IκB degradation by RANKL treatment. Parthenolide suppressed the expression of NFATc1, OSCAR, TRAP, DC-STAMP, and cathepsin K in RANKL-treated BMMs. Furthermore, parthenolide down-regulated the stability of c-Fos protein, but could not suppress the expression of c-Fos. Overexpression of NFATc1 and c-Fos in BMMs reversed the inhibitory effect of parthenolide on RANKL-mediated osteoclast differentiation. Parthenolide also inhibited the bone resorbing activity of mature osteoclasts. Parthenolide inhibits the differentiation and bone-resolving activity of osteoclast by RANKL, suggesting its potential therapeutic value for bone destructive disorders associated with osteoclast-mediated bone resorption. [BMB Reports 2014; 47(8): 451-456]     

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.     

Parthenolide inhibits TRIF-dependent signaling pathway of toll-like receptors in RAW264.7 macrophages

Toll-like receptors (TLRs) play an important role in induction of innate immune responses for host defense against invading microbial pathogens. Microbial component engagement of TLRs can trigger the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent downstream signaling pathways. Parthenolide, an active ingredient of feverfew (Tanacetum parthenium), has been used for centuries to treat many chronic diseases. Parthenolide inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-κB kinase. However, it is not known whether parthenolide inhibits the TRIF-dependent pathway. To evaluate the therapeutic potential of parthenolide, its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly [I:C]) was examined. Parthenolide inhibited nuclear factor-κB and interferon regulatory factor 3 activation induced by LPS or poly[I:C], and the LPS-induced phosphorylation of interferon regulatory factor 3 as well as interferon-inducible genes such as interferon inducible protein-10. These results suggest that parthenolide can modulate TRIF-dependent signaling pathways of TLRs, and may be the basis of effective therapeutics for chronic inflammatory diseases.     

Crosstalk between PKCzeta and the IL4/Stat6 pathway during T-cell-mediated hepatitis

PKCzeta is required for nuclear factor kappa-B (NF-kappaB) activation in several cell systems. NF-kappaB is a suppressor of liver apoptosis during development and in concanavalin A (ConA)-induced T-cell-mediated hepatitis. Here we show that PKCzeta-/- mice display inhibited ConA-induced NF-kappaB activation and reduced damage in liver. As the IL-4/Stat6 pathway is necessary for ConA-induced hepatitis, we addressed here the potential role of PKCzeta in this cascade. Interestingly, the loss of PKCzeta severely attenuated serum IL-5 and liver eotaxin-1 levels, two critical mediators of liver damage. Stat6 tyrosine phosphorylation and Jak1 activation were ablated in the liver of ConA-injected PKCzeta-/- mice and in IL-4-stimulated PKCzeta-/- fibroblasts. PKCzeta interacts with and phosphorylates Jak1 and PKCzeta activity is required for Jak1 function. In contrast, Par-4-/- mice have increased sensitivity to ConA-induced liver damage and IL-4 signaling. This unveils a novel and critical involvement of PKCzeta in the IL-4/Stat6 signaling pathway in vitro and in vivo.