Dexamethasone for Severe COVID-19: How Does It Work at Cellular and Molecular Levels?

The coronavirus disease 2019 (COVID-19) caused by infection of the severe respiratory syndrome coronavirus-2 (SARS-CoV-2) significantly impacted human society. Recently, the synthetic pure glucocorticoid dexamethasone was identified as an effective compound for treatment of severe COVID-19. However, glucocorticoids are generally harmful for infectious diseases, such as bacterial sepsis and severe influenza pneumonia, which can develop respiratory failure and systemic inflammation similar to COVID-19. This apparent inconsistency suggests the presence of pathologic mechanism(s) unique to COVID-19 that renders this steroid effective. We review plausible mechanisms and advance the hypothesis that SARS-CoV-2 infection is accompanied by infected cell-specific glucocorticoid insensitivity as reported for some other viruses. This alteration in local glucocorticoid actions interferes with undesired glucocorticoid to facilitate viral replication but does not affect desired anti-inflammatory properties in non-infected organs/tissues. We postulate that the virus coincidentally causes glucocorticoid insensitivity in the process of modulating host cell activities for promoting its replication in infected cells. We explore this tenet focusing on SARS-CoV-2-encoding proteins and potential molecular mechanisms supporting this hypothetical glucocorticoid insensitivity unique to COVID-19 but not characteristic of other life-threatening viral diseases, probably due to a difference in specific virally-encoded molecules and host cell activities modulated by them.

MxA is a positive regulator of type I IFN signaling in HCV infection.

Type I interferons (IFNs) are a family of primordial cytokines that respond to various pathogen infections including Hepatitis C virus (HCV). Type I IFNs signal through Jak/STAT pathway leading to the production of a few hundred interferon stimulated genes (ISGs). The aim of this study was to explore the role of one of these ISGs, MxA in HCV infection and type I IFN production. Plasmid encoding MxA was cloned into PcDNA3.1-3×tag vector and MxA expression was confirmed both at mRNA (RT-PCR) and protein (Western blot, WB) levels. IFNα and IFNß productions were quantified by RT-PCR from cell lysate and by ELISA kit from culture medium following MxA over-expression in Huh7.5.1 cells. The activation status of Jak/STAT signaling pathway was examined at three levels: p-STAT1 (WB), interferon sensitive response element (ISRE) activity (dual luciferase reporter gene assay), and levels of ISG expression (RT-qPCR). J6/JFH1 HCV culture system was used to study the role of MxA in HCV replication. Our findings indicated that MxA over-expression inhibited HCV replication and potentiated the IFNα-mediated anti-HCV activity; MxA stimulated the production of IFNα, IFNß, and enhanced IFNα-induced activation of Jak-STAT signaling pathway. We concluded that MxA is a positive regulator of type I IFN signaling in HCV infection.

Poly(I:C)-Encapsulating Nanoparticles Enhance Innate Immune Responses to the Tuberculosis Vaccine Bacille Calmette-Guérin (BCG) via Synergistic Activation of Innate Immune Receptors.

The attenuated live vaccine strain bacille Calmette-Guérin (BCG) is currently the only available vaccine against tuberculosis (TB), but is largely ineffective against adult pulmonary TB, the most common disease form. This is in part due to BCG's ability to interfere with the host innate immune response, a feature that might be targeted to enhance the potency of this vaccine. Here, we investigated the ability of chitosan-based nanoparticles (pIC-NPs) containing polyinosinic-polycytidylic acid (poly(I:C)), an inducer of innate immunity via Toll-like receptor 3 (TLR3), to enhance the immunogenicity of BCG in mouse bone marrow derived macrophages (BMDM) in vitro. Incorporation of poly(I:C) into NPs protected it against degradation by ribonucleases and increased its uptake by mouse BMDM. Whereas soluble poly(I:C) was ineffective, pIC-NPs strongly enhanced the proinflammatory immune response of BCG-infected macrophages in a synergistic fashion, as evident by increased production of cytokines and induction of nitric oxide synthesis. Using macrophages from mice deficient in key signaling molecules involved in the pathogen recognition response, we identified combined activation of MyD88- and TRIF-dependent TLR signaling pathways to be essential for the synergistic effect between BCG and NP. Moreover, synergy was strongly dependent on the order of the two stimuli, with TLR activation by BCG functioning as the priming event for the subsequent pIC-NP stimulus, which acted through an auto-/paracrine type I interferon (IFN) feedback loop. Our results provide a foundation for a promising new approach to enhance BCG-vaccine immunogenicity by costimulation with NPs. They also contribute to a molecular understanding of the observed synergistic interaction between the pIC-NPs and BCG vaccine.

Type I interferons contribute to experimental cerebral malaria development in response to sporozoite or blood-stage Plasmodium berghei ANKA.

Cerebral malaria is a severe complication of Plasmodium falciparum infection. Although T-cell activation and type II IFN-γ are required for Plasmodium berghei ANKA (PbA)-induced murine experimental cerebral malaria (ECM), the role of type I IFN-α/ß in ECM development remains unclear. Here, we address the role of the IFN-α/ß pathway in ECM devel-opment in response to hepatic or blood-stage PbA infection, using mice deficient for types I or II IFN receptors. While IFN-γR1⁻/⁻ mice were fully resistant, IFNAR1⁻/⁻ mice showed delayed and partial protection to ECM after PbA infection. ECM resistance in IFN-γR1⁻/⁻ mice correlated with unaltered cerebral microcirculation and absence of ischemia, while WT and IFNAR1⁻/⁻ mice developed distinct microvascular pathologies. ECM resistance appeared to be independent of parasitemia. Instead, key mediators of ECM were attenuated in the absence of IFNAR1, including PbA-induced brain sequestration of CXCR3⁺-activated CD8⁺ T cells. This was associated with reduced expression of Granzyme B, IFN-γ, IL-12Rß2, and T-cell-attracting chemokines CXCL9 and CXCL10 in IFNAR1⁻/⁻ mice, more so in the absence of IFN-γR1. Therefore, the type I IFN-α/ß receptor pathway contributes to brain T-cell responses and microvascular pathology, although it is not as essential as IFN-γ for the development of cerebral malaria upon hepatic or blood-stage PbA infection.

Pteropus vampyrus TRIM40 Is an Interferon-Stimulated Gene That Antagonizes RIG-I-like Receptors.

Nipah virus (NiV; genus: Henipavirus; family: Paramyxoviridae) naturally infects Old World fruit bats (family Pteropodidae) without causing overt disease. Conversely, NiV infection in humans and other mammals can be lethal. Comparing bat antiviral responses with those of humans may illuminate the mechanisms that facilitate bats' tolerance. Tripartite motif proteins (TRIMs), a large family of E3-ubiquitin ligases, fine-tune innate antiviral immune responses, and two human TRIMs interact with Henipavirus proteins. We hypothesize that NiV infection induces the expression of an immunosuppressive TRIM in bat, but not human cells, to promote tolerance. Here, we show that TRIM40 is an interferon-stimulated gene (ISG) in pteropodid but not human cells. Knockdown of bat TRIM40 increases gene expression of IFNß, ISGs, and pro-inflammatory cytokines following poly(I:C) transfection. In Pteropus vampyrus, but not human cells, NiV induces TRIM40 expression within 16 h after infection, and knockdown of TRIM40 correlates with reduced NiV titers as compared to control cells. Bats may have evolved to express TRIM40 in response to viral infections to control immunopathogenesis.

Reprogramming viral immune evasion for a rational design of next-generation vaccines for RNA viruses.

Type I interferons (IFNs-α/ß) are antiviral cytokines that constitute the innate immunity of hosts to fight against viral infections. Recent studies, however, have revealed the pleiotropic functions of IFNs, in addition to their antiviral activities, for the priming of activation and maturation of adaptive immunity. In turn, many viruses have developed various strategies to counteract the IFN response and to evade the host immune system for their benefits. The inefficient innate immunity and delayed adaptive response fail to clear of invading viruses and negatively affect the efficacy of vaccines. A better understanding of evasion strategies will provide opportunities to revert the viral IFN antagonism. Furthermore, IFN antagonism-deficient viruses can be generated by reverse genetics technology. Such viruses can potentially serve as next-generation vaccines that can induce effective and broad-spectrum responses for both innate and adaptive immunities for various pathogens. This review describes the recent advances in developing IFN antagonism-deficient viruses, their immune evasion and attenuated phenotypes in natural host animal species, and future potential as veterinary vaccines.

The African swine fever virus protease pS273R inhibits DNA sensing cGAS-STING pathway by targeting IKKε.

African swine fever virus (ASFV), a large and complex cytoplasmic double-stranded DNA virus, has developed multiple strategies to evade the antiviral innate immune responses. Cytosolic DNA arising from invading ASFV is mainly detected by the cyclic GMP-AMP synthase (cGAS) and then triggers a series of innate immune responses to prevent virus invasion. However, the immune escape mechanism of ASFV remains to be fully clarified. The pS273R of ASFV is a member of the SUMO-1-specific protease family and is crucial for valid virus replication. In this study, we identified pS273R as a suppressor of cGAS-STING pathway mediated type I interferon (IFN) production by ASFV genomic open reading frame screening. The pS273R was further confirmed as an inhibitor of IFN production as well as its downstream antiviral genes in cGAS-STING pathway. Mechanistically, pS273R greatly decreased the cGAS-STING signaling by targeting IKKε but not TBK1, and pS273R was found to disturb the interaction between IKKε and STING through its interaction with IKKε. Further, mutational analyses revealed that pS273R antagonized the cGAS-STING pathway by enzyme catalytic activity, which might affect the IKKε sumoylation state required for the interaction with STING. In summary, our results revealed for the first time that pS273R acts as an obvious negative regulator of cGAS-STING pathway by targeting IKKε via its enzymatic activity, which shows a new immune evasion mechanism of ASFV.

SRP54 Negatively Regulates IFN-Beta Production and Antiviral Response by Targeting RIG-I and MDA5.

During virus infection, RIG-I-like receptors (RLRs) recognize viral RNAs and recruit the adaptor protein VISA to activate downstream signaling, leading to activation of transcription factors NF-κB and IRF3, which collaborate to induce type I interferons (IFNs). IFNs further induce expression of hundreds of IFN-stimulated genes (ISGs) that suppress viral replication and facilitate the adaptive immune response. Dysregulated production of IFNs is implicated in various immune diseases. Here we identified Signal Recognition Particle 54 (SRP54) as a negative regulator of RLRs-induced antiviral signaling. Overexpression of SRP54 inhibited RNA virus-triggered induction of IFN-ß and increased viral replication, whereas knockdown of SRP54 had opposite effects. Mechanistically, SRP54 interacted with both RIG-I and MDA5 and impaired their association with VISA. Our findings demonstrate that SRP54 acts as a negative regulator of RLRs-mediated innate immune response by disrupting the recruitment of VISA to RIG-I/MDA5.

Interferon Regulatory Factor 9 Promotes Lung Cancer Progression via Regulation of Versican.

Transcription factors can serve as links between tumor microenvironment signaling and oncogenesis. Interferon regulatory factor 9 (IRF9) is recruited and expressed upon interferon stimulation and is dependent on cofactors that exert in tumor-suppressing or oncogenic functions via the JAK-STAT pathway. IRF9 is frequently overexpressed in human lung cancer and is associated with decreased patient survival; however, the underlying mechanisms remain to be elucidated. Here, we used stably transduced lung adenocarcinoma cell lines (A549 and A427) to overexpress or knockdown IRF9. Overexpression led to increased oncogenic behavior in vitro, including enhanced proliferation and migration, whereas knockdown reduced these effects. These findings were confirmed in vivo using lung tumor xenografts in nude mice, and effects on both tumor growth and tumor mass were observed. Using RNA sequencing, we identified versican (VCAN) as a novel downstream target of IRF9. Indeed, IRF9 and VCAN expression levels were found to be correlated. We showed for the first time that IRF9 binds at a newly identified response element in the promoter region of VCAN to regulate its transcription. Using an siRNA approach, VCAN was found to enable the oncogenic properties (proliferation and migration) of IRF9 transduced cells, perhaps with CDKN1A involvement. The targeted inhibition of IRF9 in lung cancer could therefore be used as a new treatment option without multimodal interference in microenvironment JAK-STAT signaling.

Mutation in Irf8 Gene (Irf8R294C ) Impairs Type I IFN-Mediated Antiviral Immune Response by Murine pDCs.

Plasmacytoid dendritic cells (pDCs) are the key producers of type I interferons (IFNs), thus playing a central role in initiating antiviral immune response. Besides robust type I IFN production, pDCs also act as antigen presenting cells post immunogenic stimulation. Transcription factor Irf8 is indispensable for the development of both pDC and cDC1 subset. However, the mechanism underlying the differential regulation by IRF8 in cDC1- and pDC-specific genomic architecture of developmental pathways still remains to be fully elucidated. Previous studies indicated that the Irf8R294C mutation specifically abrogates development of cDC1 without affecting that of pDC. In the present study using RNA-seq based approach, we have found that though the point mutation Irf8R294C did not affect pDC development, it led to defective type I IFN production, thus resulting in inefficient antiviral response. This observation unraveled the distinctive roles of IRF8 in these two subpopulations-regulating the development of cDC1 whereas modulating the functionality of pDCs without affecting development. We have reported here that Irf8R294C mutation also caused defect in production of ISGs as well as defective upregulation of costimulatory molecules in pDCs in response to NDV infection (or CpG stimulation). Through in vivo studies, we demonstrated that abrogation of type I IFN production was concomitant with reduced upregulation of costimulatory molecules in pDCs and increased NDV burden in IRF8R294C mice in comparison with wild type, indicating inefficient viral clearance. Further, we have also shown that Irf8R294C mutation abolished the activation of type I IFN promoter by IRF8, justifying the low level of type I IFN production. Taken together, our study signifies that the single point mutation in Irf8, Irf8R294C severely compromised type I IFN-mediated immune response by murine pDCs, thereby causing impairment in antiviral immunity.