Abstract
Background: Inhibition of G-protein βγ (Gβγ) signaling was found previously to enhance T cell receptor (TCR)-stimulated increases in interleukin 2 (IL-2) mRNA in CD4+ T helper cells, suggesting that Gβγ might be a useful drug target for treating autoimmune diseases, as low dose IL-2 therapy can suppress autoimmune responses. Because IL-2 may counteract autoimmunity in part by shifting CD4+ T helper cells away from the Type 1 T helper cell (TH1) and TH17 subtypes towards the TH2 subtype, the purpose of this study was to determine if blocking Gβγ signaling affected the balance of TH1, TH17, and TH2 cytokine mRNAs produced by CD4+ T helper cells.Methods: Gallein, a small molecule inhibitor of Gβγ, and siRNA-mediated silencing of the G-protein β1 subunit (Gβ1) were used to test the effect of blocking Gβγ on mRNA levels of cytokines in primary human TCR-stimulated CD4+ T helper cells.Results: Gallein and Gβ1 siRNA decreased interferon-γ (IFN-γ) and IL-17A mRNA levels in TCR-stimulated CD4+ T cells grown under TH1-promoting conditions. Inhibiting Gβγ also decreased mRNA levels of STAT4, which plays a positive role in TH1 differentiation and IL-17A production. Moreover, mRNA levels of the STAT4-regulated TH1-associated proteins, IL-18 receptor β chain (IL-18Rβ), mitogen-activated protein kinase kinase kinase 8 (MAP3K8), lymphocyte activation gene 3 (LAG-3), natural killer cell group 7 sequence (NKG7), and oncostatin M (OSM) were also decreased upon Gβγ inhibition. Gallein also increased IL-4, IL-5, IL-9, and IL-13 mRNA levels in TCR-stimulated memory CD4+ T cells grown in TH2-promoting conditions.Conclusions: Inhibiting Gβγ to produce these shifts in cytokine mRNA production might be beneficial for patients with autoimmune diseases such as rheumatoid arthritis (RA), Crohn’s disease (CD), psoriasis, multiple sclerosis (MS), and Hashimoto’s thyroiditis (HT), in which both IFN-γ and IL-17A are elevated.
Highlights
We demonstrated previously that blocking G-protein βγ (Gβγ) signaling resulted in potentiation of T cell receptor (TCR)-stimulated interleukin 2 (IL-2) mRNA increases in human CD4+ T helper cells [1], suggesting this approach might be useful for treating autoimmune diseases, as low dose IL-2 therapy effectively suppressed immune responses in chronic graft-versus-host disease [2] and hepatitis C virus-induced vasculitis [3]
Gallein, a small molecule inhibitor of Gβγ signaling, and siRNA-mediated silencing of G-protein β1 subunit (Gβ1) decrease IFN-γ mRNA levels in human TCR-stimulated CD4+ T cells grown under TH1-promoting conditions We determined previously that blocking Gβγ signaling in primary human CD4+ T helper cells grown in TH1- or TH2-promoting conditions enhanced TCR-stimulated IL-2 mRNA levels by ~2-fold [1]
We focused on cytokine mRNAs because transcriptional regulation is the primary means of controlling expression of inducible cytokine genes [33,34,35,36,37,38] and because our own measurements of Quantitative PCR (qPCR)-determined IL-2 mRNA levels and secreted IL-2 [1] and those of others [39] demonstrated a strong correlation, and similar relationships have been determined for IFN-γ [40], IL-17 [41], and TH2 cytokines [42, 43]
Summary
We demonstrated previously that blocking Gβγ signaling resulted in potentiation of TCR-stimulated IL-2 mRNA increases in human CD4+ T helper cells [1], suggesting this approach might be useful for treating autoimmune diseases, as low dose IL-2 therapy effectively suppressed immune responses in chronic graft-versus-host disease [2] and hepatitis C virus-induced vasculitis [3]. Inhibition of G-protein βγ (Gβγ) signaling was found previously to enhance T cell receptor (TCR)-stimulated increases in interleukin 2 (IL-2) mRNA in CD4+ T helper cells, suggesting that Gβγ might be a useful drug target for treating autoimmune diseases, as low dose IL-2 therapy can suppress autoimmune responses. Because IL-2 may counteract autoimmunity in part by shifting CD4+ T helper cells away from the Type 1 T helper cell (TH1) and TH17 subtypes towards the TH2 subtype, the purpose of this study was to determine if blocking Gβγ signaling affected the balance of TH1, TH17, and TH2 cytokine mRNAs produced by CD4+ T helper cells. Conclusions: Inhibiting Gβγ to produce these shifts in cytokine mRNA production might be beneficial for patients with autoimmune diseases such as rheumatoid arthritis (RA), Crohn’s disease (CD), psoriasis, multiple sclerosis (MS), and Hashimoto’s thyroiditis (HT), in which both IFN-γ and IL-17A are elevated
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