MEK/ERK MAP kinase limits poly I:C-induced antiviral gene expression in RAW264.7 macrophages by reducing interferon-beta expression.

Toll-like receptor 3 (TLR3) recognizes viral double-stranded RNA (or the synthetic dsRNA analog poly I:C) and induces a signal transduction pathway that results in activation of transcription factors that induce expression of antiviral genes including type I interferon (IFN-I). Secreted IFN-I positively feeds back to amplify antiviral gene expression. In this report, we study the role of MEK/ERK MAP kinase in modulating antiviral gene expression downstream of TLR3. We find MEK/ERK is a negative regulator of antiviral gene expression by limiting expression of IFN-ß. However, MEK/ERK does not limit antiviral responses downstream of the type I interferon receptor. These findings provide insights into regulatory mechanisms of antiviral gene expression and reveal potential targets for modulating antiviral immunity.

Interferon regulatory factor 3 plays a role in macrophage responses to interferon-γ.

IFN-γ produced during viral infections activates the IFN-γ receptor (IFNGR) complex for STAT1 transcriptional activity leading to expression of Interferon Regulatory Factors (IRF). Simultaneous activation of TBK/IKKε via TLR3 during viral infections activates the transcription factor IRF3. Together these transcription factors contributes to expression of intracellular proteins (e.g. ISG49, ISG54) and secreted proteins (e.g. IFN-ß, IP-10, IL-15) that are essential to innate antiviral immunity. Here we examined the role of IRF3 in expression of innate anti-viral proteins produced in response to IFN-γ plus TLR3 agonist. Wild-type (WT) and IRF3KO RAW264.7 cells, each with ISG54-promoter-luciferase reporter vectors, were stimulated with IFN-γ, poly I:C, or both together. ISG54 promoter activity was significantly reduced in IRF3KO RAW264.7 cells responding to IFN-γ, poly I:C, or IFN-γ plus poly I:C, compared with WT RAW264.7 cells. These data were confirmed with western blot and qRT-PCR. Primary macrophages and dendritic cells (DCs) from IRF3KO mice also showed decreased ISG54 in response to IFN-γ, poly I:C, or IFN-γ plus poly I:C compared with those from WT mice. Moreover, pharmacological inhibition of TBK/IKKε significantly reduced ISG54 promoter activity in response to IFN-γ, poly I:C, or IFN-γ plus poly I:C. Similarly, expression of ISG49 and IL-15, but not IP-10, was impaired in IRF3KO RAW264.7 cells responding to IFN-γ or poly I:C, which also had impaired STAT1 phosphorylation and IRF1 expression. These data show that IRF3 contributes to IFN-γ/IFNGR signaling for expression of innate anti-viral proteins in macrophages.

IFN-γ synergism with poly I:C reduces growth of murine and human cancer cells with simultaneous changes in cell cycle and immune checkpoint proteins.

Previously, we reported that IFN-γ and poly I:C, a TLR3 Pattern Recognition Receptor (PRR) agonist, reduces growth of and induces Cleaved-Caspase-3, ISG54 and p27Kip in B16 melanoma cells. Here, analysis of IFN-γ/PRR synergism was expanded with UM-SCC1 and UM-SCC38 human squamous carcinoma cells and other PRR agonists. As in B16 cells, poly I:C plus IFN-γ synergism reduced UM-SCC1 and UM-SCC38 growth, and no more than 24 h was needed for significant growth reduction. IFN-γ synergism to stem B16 growth also occurred with TLR7, TLR9, TLR4, and STING agonists, but not TLR2 agonist. IFN-γ synergized with TLR3 and TLR4 agonists reducing UM-SCC1 growth, and with TLR7 and TLR3 agonists reducing UM-SCC38 growth. IFN-γ plus poly I:C, which had the most pronounced effect, decreased cyclin-D1, increased G1 cell cycle arrest, and increased Cleaved caspase-3 in B16 cells, as well as RAW264.7, a virus-transformed murine macrophage cell line. Finally, IFN-γ plus poly I:C modulated total but not cell surface expression of immune checkpoint protein PD-L1, as well as cell cycle checkpoint proteins in B16 cells. Thus IFN-γ plus poly I:C, and other PRR agonists, may well be effective adjuvants to cancer immunotherapy against several tumor cell types.