Immune interferon (IFN), also known as IFN-γ, promotes not only immunomodulation but also antimicrobial and anticancer activity. After IFN-γ binds to the complex of IFN-γ receptor (IFNGR) 1-IFNGR2 and subsequently activates its downstream signaling pathways, IFN-γ immediately causes transcriptional stimulation of a variety of genes that are principally involved in its biological activities. Regarding IFN-γ-dependent immunosurveillance, IFN-γ can directly suppress tumorigenesis and infection and/or can modulate the immunological status in both cancer cells and infected cells. Regarding the anticancer effects of IFN-γ, cancer cells develop strategies to escape from IFN-γ-dependent cancer immunosurveillance. Immune evasion, including the recruitment of immunosuppressive cells, secretion of immunosuppressive factors, and suppression of cytotoxic T lymphocyte responses, is speculated to be elicited by the oncogenic microenvironment. All of these events effectively downregulate IFN-γ-expressing cells and IFN-γ production. In addition to these extrinsic pathways, cancer cells may develop cellular tolerance that manifests as hyporesponsiveness to IFN-γ stimulation. This review discusses the potential escape mechanisms from IFN-γ-dependent immunosurveillance in tumorigenesis.

The antiparasitic agent niclosamide has been demonstrated to inhibit the arthropod-borne Zika virus. Here, we investigated the antiviral capacity of niclosamide against dengue virus (DENV) serotype 2 infection in vitro and in vivo. Niclosamide effectively retarded DENV-induced infection in vitro in human adenocarcinoma cells (A549), mouse neuroblastoma cells (Neuro-2a), and baby hamster kidney fibroblasts (BHK-21). Treatment with niclosamide did not retard the endocytosis of DENV while niclosamide was unable to enhance the antiviral type I interferon response. Furthermore, niclosamide did not cause a direct effect on viral replicon-based expression. Niclosamide has been reported to competitively inhibit the mTOR (mammalian target of rapamycin), STAT3 (signal transducer and activator of transcription 3), and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathways; however, selective inhibitors of those pathways did not reduce DENV infection. Similar to the vacuolar-type H⁺-ATPase inhibitor bafilomycin A1, both niclosamide and other protonophores, such as CCCP (carbonyl cyanide m-chlorophenyl hydrazone), and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), effectively reduced endosomal acidification and viral dsRNA replication. Co-administration of a single dose of niclosamide partially decreased viral replication, viral encephalitis, and mortality in DENV-infected ICR suckling mice. These results demonstrate that niclosamide diminishes viral infection by hindering endosomal acidification. Dengue and severe dengue cause global health concerns annually. Without antiviral drugs, supportive care is the only treatment option for patients with DENV infection. A current vaccine has been approved for protection against DENV infection; however, the potential risks and challenges associated with the immunopathogenesis of DENV remain unresolved. For anti-dengue therapy, the repurposing of drugs with antimicrobial and anticancer properties is a possible pharmacological strategy. In this study, we evaluated the potential antiviral effects of the antiparasitic drug niclosamide, considering its current pharmacological efficacy against arthropod-borne Zika virus infection. Using in vitro and in vivo models of DENV infection, we demonstrated that one of the therapeutic effects of niclosamide is to significantly target endosomal acidification. Following safety screening, repurposing niclosamide treatment may facilitate the development of anti-dengue drugs in the near future.

Patients with dengue virus (DENV) infection may also present acute viral encephalitis through an unknown mechanism. Here, we report that encephalitic DENV-infected mice exhibited progressive hunchback posture, limbic seizures, limbic weakness, paralysis and lethality 7 days post-infection. These symptoms were accompanied by CNS inflammation, neurotoxicity and blood-brain barrier destruction. Microglial cells surrounding the blood vessels and injured hippocampus regions were activated by DENV infection. Pharmacologically depleting microglia unexpectedly increased viral replication, neuropathy and mortality in DENV-infected mice. In microglia-depleted mice, the DENV infection-mediated expression of antiviral cytokines and the infiltration of CD8-positive cytotoxic T lymphocytes (CTLs) was abolished. DENV infection prompted the antigen-presenting cell-like differentiation of microglia, which in turn stimulated CTL proliferation and activation. These results suggest that microglial cells play a key role in facilitating antiviral immune responses against DENV infection and acute viral encephalitis.

Interleukin (IL)-10 levels are increased in dengue virus (DENV)-infected patients with severe disorders. A hypothetical intrinsic pathway has been proposed for the IL-10 response during antibody-dependent enhancement (ADE) of DENV infection; however, the mechanisms of IL-10 regulation remain unclear. We found that DENV infection and/or attachment was sufficient to induce increased expression of IL-10 and its downstream regulator suppressor of cytokine signaling 3 in human monocytic THP-1 cells and human peripheral blood monocytes. IL-10 production was controlled by activation of cyclic adenosine monophosphate response element-binding (CREB), primarily through protein kinase A (PKA)- and phosphoinositide 3-kinase (PI3K)/PKB-regulated pathways, with PKA activation acting upstream of PI3K/PKB. DENV infection also caused glycogen synthase kinase (GSK)-3β inactivation in a PKA/PI3K/PKB-regulated manner, and inhibition of GSK-3β significantly increased DENV-induced IL-10 production following CREB activation. Pharmacological inhibition of spleen tyrosine kinase (Syk) activity significantly decreased DENV-induced IL-10 production, whereas silencing Syk-associated C-type lectin domain family 5 member A caused a partial inhibition. ADE of DENV infection greatly increased IL-10 expression by enhancing Syk-regulated PI3K/PKB/GSK-3β/CREB signaling. We also found that viral load, but not serotype, affected the IL-10 response. Finally, modulation of IL-10 expression could affect DENV replication. These results demonstrate that, in monocytes, IL-10 production is regulated by ADE through both an extrinsic and an intrinsic pathway, all involving a Syk-regulated PI3K/PKB/GSK-3β/CREB pathway, and both of which impact viral replication. IL-10 has multiple cellular functions, including anti-inflammatory and immunomodulatory effects. Clinical studies have demonstrated that the serum levels of IL-10 are significantly increased in DENV-infected patients with severe disorders. However, the molecular mechanism underlying DENV-induced IL-10 production is still unresolved. In this study, we demonstrate a molecular mechanism for DENV-induced IL-10 production, which may be exacerbated by ADE through Fcγ receptor-mediated extrinsic and intrinsic pathways, leading to IL-10/SOCS3-mediated advantages for viral replication. With or without Fcγ receptor- or CLEC5A-mediated DENV infection, a common Syk/PKA-regulated PI3K/PKB activation results in a decrease in GSK-3β activity followed by an increase in CREB-mediated IL-10 expression not only in THP-1 monocytic cells but also in human monocytes. Taken together, we demonstrate a potential regulation and a pathological role for ADE-induced IL-10 overproduction during DENV replication. Therefore, inhibiting immunosuppression by targeting the IL-10 pathways identified in this study may help to prevent the progression of severe dengue diseases.

Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms, including cytokine storm, brain stem encephalitis, and fulminant pulmonary edema. Gamma interferon (IFN-γ) may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. This study, investigating the role of IFN-γ in EV71 infection using a murine model, showed that IFN-γ was elevated. Moreover, IFN-γ receptor-deficient mice showed higher mortality rates and more severe disease progression with slower viral clearance than wild-type mice.In vitroresults showed that IFN-γ pretreatment reduced EV71 yield, whereas EV71 infection caused IFN-γ resistance with attenuated IFN-γ signaling in IFN regulatory factor 1 (IRF1) gene transactivation. To study the immunoediting ability of EV71 proteins in IFN-γ signaling, 11 viral proteins were stably expressed in cells without cytotoxicity; however, viral proteins 2A and 3D blocked IFN-γ-inducedIRF1transactivation following a loss of signal transducer and activator of transcription 1 (STAT1) nuclear translocation. Viral 3D attenuated IFN-γ signaling accompanied by a STAT1 decrease without interfering with IFN-γ receptor expression. Restoration of STAT1 or blocking 3D activity was able to rescue IFN-γ signaling. Interestingly, viral 2A attenuated IFN-γ signaling using another mechanism by reducing the serine phosphorylation of STAT1 following the inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. These results demonstrate the anti-EV71 ability of IFN-γ and the immunoediting ability by EV71 2A and 3D, which attenuate IFN-γ signaling through different mechanisms.IMPORTANCEImmunosurveillance by gamma interferon (IFN-γ) may confer anti-enterovirus 71 (anti-EV71) activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. IFN-γ receptor-deficient mice showed higher mortality and more severe disease progression, indicating the anti-EV71 property of IFN-γ. However, EV71 infection caused cellular insusceptibility in response to IFN-γ stimulation. We used anin vitrosystem with viral protein expression to explore the novel IFN-γ inhibitory properties of the EV71 2A and 3D proteins through the different mechanisms. According to this study, targeting either 2A or 3D pharmacologically and/or genetically may sustain a cellular susceptibility in response to IFN-γ, particularly for IFN-γ-mediated anti-EV71 activity.

Infection by the dengue virus (DENV) threatens global public health due to its high prevalence and the lack of effective treatments. Host factors may contribute to the pathogenesis of DENV; herein, we investigated the role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is activated by DENV in mononuclear phagocytes. DENV infection selectively activates Nrf2 following nuclear translocation. Following endoplasmic reticular (ER) stress, protein kinase R-like ER kinase (PERK) facilitated Nrf2-mediated transcriptional activation of C-type lectin domain family 5, member A (CLEC5A) to increase CLEC5A expression. Signaling downstream of the Nrf2-CLEC5A interaction enhances Toll-like receptor 3 (TLR3)-independent tumor necrosis factor (TNF)-α production following DENV infection. Forced expression of the NS2B3 viral protein induces Nrf2 nuclear translocation/activation and CLEC5A expression which increases DENV-induced TNF-α production. Animal studies confirmed Nrf2-induced CLEC5A and TNF-α in brains of DENV-infected mice. These results demonstrate that DENV infection causes Nrf2-regulated TNF-α production by increasing levels of CLEC5A.