Non-transcriptional IRF7 interacts with NF-κB to inhibit viral inflammation.

Interferon (IFN) regulatory factors (IRF) are key transcription factors in cellular antiviral responses. IRF7, a virus-inducible IRF, expressed primarily in myeloid cells, is required for transcriptional induction of interferon α and antiviral genes. IRF7 is activated by virus-induced phosphorylation in the cytoplasm, leading to its translocation to the nucleus for transcriptional activity. Here, we revealed a nontranscriptional activity of IRF7 contributing to its antiviral functions. IRF7 interacted with the pro-inflammatory transcription factor NF-κB-p65 and inhibited the induction of inflammatory target genes. Using knockdown, knockout, and overexpression strategies, we demonstrated that IRF7 inhibited NF-κB-dependent inflammatory target genes, induced by virus infection or toll-like receptor stimulation. A mutant IRF7, defective in transcriptional activity, interacted with NF-κB-p65 and suppressed NF-κB-induced gene expression. A single-action IRF7 mutant, active in anti-inflammatory function, but defective in transcriptional activity, efficiently suppressed Sendai virus and murine hepatitis virus replication. We, therefore, uncovered an anti-inflammatory function for IRF7, independent of transcriptional activity, contributing to the antiviral response of IRF7.

Interferons in Viral Infections.

Interferons (IFNs) are cytokines that inhibit viral replication in host cells by triggering innate immune responses through the transcriptional induction of various IFN-stimulated genes (ISGs) [...].

Differences in Responses of Immunosuppressed Kidney Transplant Patients to Moderna mRNA-1273 versus Pfizer-BioNTech.

Immunosuppressed kidney transplant (KT) recipients produce a weaker response to COVID-19 vaccination than immunocompetent individuals. We tested antiviral IgG response in 99 KT recipients and 66 healthy volunteers who were vaccinated with mRNA-1273 Moderna or BNT162b2 Pfizer-BioNTech vaccines. A subgroup of participants had their peripheral blood leukocytes (PBLs) evaluated for the frequency of T helper 1 (Th1) cells producing IL-2, IFN-γ and/or TNF-α, and IL-10-producing T-regulatory 1 (Tr) cells. Among KT recipients, 45.8% had anti-SARS-CoV-2 IgG compared to 74.1% of healthy volunteers (p = 0.009); also, anti-viral IgG levels were lower in recipients than in volunteers (p = 0.001). In terms of non-responders (≤2000 U/mL IgG), Moderna's group had 10.8% and Pfizer-BioNTech's group had 34.3% of non-responders at 6 months (p = 0.023); similarly, 15.7% and 31.3% were non-responders in Moderna and Pfizer-BioNTech groups at 12 months, respectively (p = 0.067). There were no non-responders among controls. Healthy volunteers had higher Th1 levels than KT recipients, while Moderna produced a higher Th1 response than Pfizer-BioNTech. In contrast, the Pfizer-BioNTech vaccine induced a higher Tr1 response than the Moderna vaccine (p < 0.05); overall, IgG levels correlated with Th1(fTTNF-α)/Tr1(fTIL-10) ratios. We propose that the higher number of non-responders in the Pfizer-BioNTech group than the Moderna group was caused by a more potent activity of regulatory Tr1 cells in KT recipients vaccinated with the Pfizer-BioNTech vaccine.

Retinoic Acid-Mediated Inhibition of Mouse Coronavirus Replication Is Dependent on IRF3 and CaMKK.

The ongoing COVID-19 pandemic has revealed the shortfalls in our understanding of how to treat coronavirus infections. With almost 7 million case fatalities of COVID-19 globally, the catalog of FDA-approved antiviral therapeutics is limited compared to other medications, such as antibiotics. All-trans retinoic acid (RA), or activated vitamin A, has been studied as a potential therapeutic against coronavirus infection because of its antiviral properties. Due to its impact on different signaling pathways, RA's mechanism of action during coronavirus infection has not been thoroughly described. To determine RA's mechanism of action, we examined its effect against a mouse coronavirus, mouse hepatitis virus strain A59 (MHV). We demonstrated that RA significantly decreased viral titers in infected mouse L929 fibroblasts and RAW 264.7 macrophages. The reduced viral titers were associated with a corresponding decrease in MHV nucleocapsid protein expression. Using interferon regulatory factor 3 (IRF3) knockout RAW 264.7 cells, we demonstrated that RA-induced suppression of MHV required IRF3 activity. RNA-seq analysis of wildtype and IRF3 knockout RAW cells showed that RA upregulated calcium/calmodulin (CaM) signaling proteins, such as CaM kinase kinase 1 (CaMKK1). When treated with a CaMKK inhibitor, RA was unable to upregulate IRF activation during MHV infection. In conclusion, our results demonstrate that RA-induced protection against coronavirus infection depends on IRF3 and CaMKK.

Interferon-stimulated gene TDRD7 interacts with AMPK and inhibits its activation to suppress viral replication and pathogenesis.

IMPORTANCE: Virus infection triggers induction of interferon (IFN)-stimulated genes (ISGs), which ironically inhibit viruses themselves. We identified Tudor domain-containing 7 (TDRD7) as a novel antiviral ISG, which inhibits viral replication by interfering with autophagy pathway. Here, we present a molecular basis for autophagy inhibitory function of TDRD7. TDRD7 interacted with adenosine monophosphate (AMP)-activated protein kinase (AMPK), the kinase that initiates autophagy, to inhibit its activation. We identified domains required for the interaction; deleting AMPK-interacting domain blocked antiAMPK and antiviral activities of TDRD7. We used primary cells and mice to evaluate the TDRD7-AMPK antiviral pathway. TDRD7-deficient primary mouse cells exhibited enhanced AMPK activation and viral replication. Finally, TDRD7 knockout mice showed increased susceptibility to respiratory virus infection. Therefore, our study revealed a new antiviral pathway of IFN and its contribution to host response. Our results have therapeutic potential; a TDRD7-derived peptide may be an effective AMPK inhibitor with application as antiviral agent.

Evaluation of intra- and inter-lab variability in quantifying SARS-CoV-2 in a state-wide wastewater monitoring network.

In December 2019, SARS-CoV-2, the virus that causes coronavirus disease 2019, was first reported and subsequently triggered a global pandemic. Wastewater monitoring, a strategy for quantifying viral gene concentrations from wastewater influents within a community, has served as an early warning and management tool for the spread of SARS-CoV-2 in a community. Ohio built a collaborative statewide wastewater monitoring network that is supported by eight labs (university, government, and commercial laboratories) with unique sample processing workflows. Consequently, we sought to characterize the variability in wastewater monitoring results for network labs. Across seven trials between October 2020 and November 2021, eight participating labs successfully quantified two SARS-CoV-2 RNA targets and human fecal indicator virus targets in wastewater sample aliquots with reproducible results, although recovery efficiencies of spiked surrogates ranged from 3 to 75%. When SARS-CoV-2 gene fragment concentrations were adjusted for recovery efficiency and flow, the proportion of variance between laboratories was minimized, serving as the best model to account for between-lab variance. Another adjustment factor (alone and in different combinations with the above factors) considered to account for sample and measurement variability includes fecal marker normalization. Genetic quantification variability can be attributed to many factors, including the methods, individual samples, and water quality parameters. In addition, statistically significant correlations were observed between SARS-CoV-2 RNA and COVID-19 case numbers, supporting the notion that wastewater surveillance continues to serve as an effective monitoring tool. This study serves as a real-time example of multi-laboratory collaboration for public health preparedness for infectious diseases.

Ohio Coronavirus Wastewater Monitoring Network: Implementation of Statewide Monitoring for Protecting Public Health.

CONTEXT: Prior to the COVID-19 pandemic, wastewater influent monitoring for tracking disease burden in sewered communities was not performed in Ohio, and this field was only on the periphery of the state academic research community. PROGRAM: Because of the urgency of the pandemic and extensive state-level support for this new technology to detect levels of community infection to aid in public health response, the Ohio Water Resources Center established relationships and support of various stakeholders. This enabled Ohio to develop a statewide wastewater SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) monitoring network in 2 months starting in July 2020. IMPLEMENTATION: The current Ohio Coronavirus Wastewater Monitoring Network (OCWMN) monitors more than 70 unique locations twice per week, and publicly available data are updated weekly on the public dashboard. EVALUATION: This article describes the process and decisions that were made during network initiation, the network progression, and data applications, which can inform ongoing and future pandemic response and wastewater monitoring. DISCUSSION: Overall, the OCWMN established wastewater monitoring infrastructure and provided a useful tool for public health professionals responding to the pandemic.

Critically ill patients: Histopathological evidence of thyroid dysfunction.

PURPOSE: Critical illness is characterized by severe biphasic physical and metabolic stress as result of systemic inflammatory response syndrome and/or multiple organ dysfunction syndrome, and is frequently associated with non-thyroidal illness. Purpose of this study is to better understand the cytomorphological basis of NTI by performing histopathological examinations of thyroid gland on autopsies of patients who died from critical illness. METHODS: Histopathological examination of thyroid gland of 58 critically ill patients was performed in our hospital. The cases included 24 cases of burn injury, 24 cases of traumatic brain injury, and 10 cases of cerebral stroke. Thyroid samples obtained during autopsy were preserved in formol saline and stained with hematoxylin and eosin. The sections were visualized under light microscopy. RESULTS: Out of 58 cases examined, 21 patients showed normal thyroid findings, and rest of the cases had unusual thyroid findings in the histopathological study. The principal finding was distortion of thyroid follicular architecture. Other findings include mononuclear cell infiltration, clumping of thyroglobulin, and exhaustion of thyroid follicles. CONCLUSION: Critical illness produces metabolically damaging effects on thyroid gland, which functionally corresponds to a state of low T3 syndrome. These changes are more pronounced in BI and cerebral stroke than in TBI.

Inflammatory Control of Viral Infection.

Inflammatory responses during virus infection differentially impact the host. Managing inflammatory responses is essential in controlling viral infection and related diseases. Recently, we identified a cellular anti-inflammatory mechanism, RIKA (Repression of IRF3-mediated inhibition of NF-κB activity), which controls viral inflammation and pathogenesis. The RIKA function of IRF3 may be explored further in other inflammatory diseases beyond viral infection.

How Different Pathologies Are Affected by IFIT Expression.

The type-I interferon (IFN) system represents the first line of defense against viral pathogens. Recognition of the virus initiates complex signaling pathways that result in the transcriptional induction of IFNs, which are then secreted. Secreted IFNs stimulate nearby cells and result in the production of numerous proinflammatory cytokines and antiviral factors. Of particular note, IFN-induced tetratricopeptide repeat (IFIT) proteins have been thoroughly studied because of their antiviral activity against different viral pathogens. Although classically studied as an antiviral protein, IFIT expression has recently been investigated in the context of nonviral pathologies, such as cancer and sepsis. In oral squamous cell carcinoma (OSCC), IFIT1 and IFIT3 promote metastasis, while IFIT2 exhibits the opposite effect. The role of IFIT proteins during bacterial/fungal sepsis is still under investigation, with studies showing conflicting roles for IFIT2 in disease severity. In the setting of viral sepsis, IFIT proteins play a key role in clearing viral infection. As a result, many viral pathogens, such as SARS-CoV-2, employ mechanisms to inhibit the type-I IFN system and promote viral replication. In cancers that are characterized by upregulated IFIT proteins, medications that decrease IFIT expression may reduce metastasis and improve survival rates. Likewise, in cases of viral sepsis, therapeutics that increase IFIT expression may improve viral clearance and reduce the risk of septic shock. By understanding the effect of IFIT proteins in different pathologies, novel therapeutics can be developed to halt disease progression.

Autophagic checks and balances of cellular immune responses.

IRF3 (interferon regulatory factor 3) is a critical component of the antiviral innate immune response. IRF3 deficiency causes detrimental effects to the host during virus infection. Dysregulation of IRF3 functions is associated with viral, inflammatory, and hepatic diseases. Both transcriptional and pro-apoptotic activities of IRF3 are involved in the exacerbated inflammation and apoptosis in liver injury induced by ethanol and high-fat diets. Therefore, regulation of IRF3 activities has consequences, and it is a potential therapeutic target for infectious and inflammatory diseases. We recently revealed that IRF3 is degraded by a small molecule, auranofin, by activating the cellular macroautophagy/autophagy pathway. Autophagy is a catabolic pathway that contributes to cellular homeostasis and antiviral host defense. Degradation of IRF3 by autophagy may be a novel strategy used by the viruses to their benefit. In addition, IRF3 functions are harmful in other diseases, including liver injury and bacterial infection. A better understanding of the role of autophagy in regulating IRF3 functions has significant implications in developing therapeutic strategies. Therefore, autophagy provides checks and balances in the innate immune response.

IRF3 inhibits nuclear translocation of NF-κB to prevent viral inflammation.

Interferon (IFN) regulatory factor 3 (IRF3) is a transcription factor activated by phosphorylation in the cytoplasm of a virus-infected cell; by translocating to the nucleus, it induces transcription of IFN-ß and other antiviral genes. We have previously reported IRF3 can also be activated, as a proapoptotic factor, by its linear polyubiquitination mediated by the RIG-I pathway. Both transcriptional and apoptotic functions of IRF3 contribute to its antiviral effect. Here, we report a nontranscriptional function of IRF3, namely, the repression of IRF3-mediated NF-κB activity (RIKA), which attenuated viral activation of NF-κB and the resultant inflammatory gene induction. In Irf3-/- mice, consequently, Sendai virus infection caused enhanced inflammation in the lungs. Mechanistically, RIKA was mediated by the direct binding of IRF3 to the p65 subunit of NF-κB in the cytoplasm, which prevented its nuclear import. A mutant IRF3 defective in both the transcriptional and the apoptotic activities was active in RIKA and inhibited virus replication. Our results demonstrated IRF3 deployed a three-pronged attack on virus replication and the accompanying inflammation.

SARS-CoV-2 Monitoring in Wastewater Reveals Novel Variants and Biomarkers of Infection.

Wastewater-based epidemiology (WBE) is a popular tool for the early indication of community spread of infectious diseases. WBE emerged as an effective tool during the COVID-19 pandemic and has provided meaningful information to minimize the spread of infection. Here, we present a combination of analyses using the correlation of viral gene copies with clinical cases, sequencing of wastewater-derived RNA for the viral mutants, and correlative analyses of the viral gene copies with the bacterial biomarkers. Our study provides a unique platform for potentially using the WBE-derived results to predict the spread of COVID-19 and the emergence of new variants of concern. Further, we observed a strong correlation between the presence of SARS-CoV-2 and changes in the microbial community of wastewater, particularly the significant changes in bacterial genera belonging to the families of Lachnospiraceae and Actinomycetaceae. Our study shows that microbial biomarkers could be utilized as prediction tools for future infectious disease surveillance and outbreak responses. Overall, our comprehensive analyses of viral spread, variants, and novel bacterial biomarkers will add significantly to the growing body of literature on WBE and COVID-19.

Autophagy in Virus Infection: A Race between Host Immune Response and Viral Antagonism.

Virus-infected cells trigger a robust innate immune response and facilitate virus replication. Here, we review the role of autophagy in virus infection, focusing on both pro-viral and anti-viral host responses using a select group of viruses. Autophagy is a cellular degradation pathway operated at the basal level to maintain homeostasis and is induced by external stimuli for specific functions. The degradative function of autophagy is considered a cellular anti-viral immune response. However, autophagy is a double-edged sword in viral infection; viruses often benefit from it, and the infected cells can also use it to inhibit viral replication. In addition to viral regulation, autophagy pathway proteins also function in autophagy-independent manners to regulate immune responses. Since viruses have co-evolved with hosts, they have developed ways to evade the anti-viral autophagic responses of the cells. Some of these mechanisms are also covered in our review. Lastly, we conclude with the thought that autophagy can be targeted for therapeutic interventions against viral diseases.

Autophagic degradation of IRF3 induced by the small-molecule auranofin inhibits its transcriptional and proapoptotic activities.

The ubiquitously expressed transcription factor interferon (IFN) regulatory factor 3 (IRF3) is critical for the induction of antiviral genes, e.g., type-I IFN. In addition to its transcriptional function, IRF3 also activates a nontranscriptional, proapoptotic signaling pathway. While the proapoptotic function of IRF3 protects against viral infections, it is also involved in harmful immune responses that trigger hepatocyte cell death and promote liver disease. Thus, we hypothesized that a small-molecule inhibitor of the proapoptotic activity of IRF3 could alleviate fatty-acid-induced hepatocyte cell death. We conducted a high-throughput screen, which identified auranofin as a small-molecule inhibitor of the proapoptotic activity of IRF3. In addition to the nontranscriptional apoptotic pathway, auranofin also inhibited the transcriptional activity of IRF3. Using biochemical and genetic tools in human and mouse cells, we uncovered a novel mechanism of action for auranofin, in which it induces cellular autophagy to degrade IRF3 protein, thereby suppressing IRF3 functions. Autophagy-deficient cells were unable to degrade IRF3 upon auranofin treatment, suggesting that the autophagic degradation of IRF3 is a novel approach to regulate IRF3 activities. Using a physiologically relevant in vitro model, we demonstrated that auranofin inhibited fatty-acid-induced apoptotic cell death of hepatocytes. In summary, auranofin is a novel inhibitor of IRF3 functions and may represent a potential therapeutic option in diseases where IRF3 is deleterious.

14-3-3ζ: A suppressor of inflammatory arthritis.

Inflammatory arthritis (IA) is a common disease that affects millions of individuals worldwide. Proinflammatory events during IA pathogenesis are well studied; however, loss of protective immunity remains underexplored. Earlier, we reported that 14-3-3zeta (ζ) has a role in T-cell polarization and interleukin (IL)-17A signal transduction. Here, we demonstrate that 14-3-3ζ knockout (KO) rats develop early-onset severe arthritis in two independent models of IA, pristane-induced arthritis and collagen-induced arthritis. Arthritic 14-3-3ζ KO animals showed an increase in bone loss and immune cell infiltration in synovial joints. Induction of arthritis coincided with the loss of anti-14-3-3ζ antibodies; however, rescue experiments to supplement the 14-3-3ζ antibody by passive immunization did not suppress arthritis. Instead, 14-3-3ζ immunization during the presymptomatic phase resulted in significant suppression of arthritis in both wild-type and 14-3-3ζ KO animals. Mechanistically, 14-3-3ζ KO rats exhibited elevated inflammatory gene signatures at the messenger RNA and protein levels, particularly for IL-1ß. Furthermore, the immunization with recombinant 14-3-3ζ protein suppressed IL-1ß levels, significantly increased anti-14-3-3ζ antibody levels and collagen production, and preserved bone quality. The 14-3-3ζ protein increased collagen expression in primary rat mesenchymal cells. Together, our findings indicate that 14-3-3ζ causes immune suppression and extracellular remodeling, which lead to a previously unrecognized IA-suppressive function.

Transcriptional and Non-Transcriptional Activation, Posttranslational Modifications, and Antiviral Functions of Interferon Regulatory Factor 3 and Viral Antagonism by the SARS-Coronavirus.

The immune system defends against invading pathogens through the rapid activation of innate immune signaling pathways. Interferon regulatory factor 3 (IRF3) is a key transcription factor activated in response to virus infection and is largely responsible for establishing an antiviral state in the infected host. Studies in Irf3-/- mice have demonstrated the absence of IRF3 imparts a high degree of susceptibility to a wide range of viral infections. Virus infection causes the activation of IRF3 to transcribe type-I interferon (e.g., IFNß), which is responsible for inducing the interferon-stimulated genes (ISGs), which act at specific stages to limit virus replication. In addition to its transcriptional function, IRF3 is also activated to trigger apoptosis of virus-infected cells, as a mechanism to restrict virus spread within the host, in a pathway called RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA). These dual functions of IRF3 work in concert to mediate protective immunity against virus infection. These two pathways are activated differentially by the posttranslational modifications (PTMs) of IRF3. Moreover, PTMs regulate not only IRF3 activation and function, but also protein stability. Consequently, many viruses utilize viral proteins or hijack cellular enzymes to inhibit IRF3 functions. This review will describe the PTMs that regulate IRF3's RIPA and transcriptional activities and use coronavirus as a model virus capable of antagonizing IRF3-mediated innate immune responses. A thorough understanding of the cellular control of IRF3 and the mechanisms that viruses use to subvert this system is critical for developing novel therapies for virus-induced pathologies.

EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions.

STING (STimulator of INterferon Genes) mediates protective cellular response to microbial infection and tissue damage, but its aberrant activation can lead to autoinflammatory diseases. Upon ligand stimulation, the endoplasmic reticulum (ER) protein STING translocates to endosomes for induction of interferon production, while an alternate trafficking route delivers it directly to the autophagosomes. Here, we report that phosphorylation of a specific tyrosine residue in STING by the epidermal growth factor receptor (EGFR) is required for directing STING to endosomes, where it interacts with its downstream effector IRF3. In the absence of EGFR-mediated phosphorylation, STING rapidly transits into autophagosomes, and IRF3 activation, interferon production, and antiviral activity are compromised in cell cultures and mice, while autophagic activity is enhanced. Our observations illuminate a new connection between the tyrosine kinase activity of EGFR and innate immune functions of STING and suggest new experimental and therapeutic approaches for selective regulation of STING functions.

14-3-3ζ-TRAF5 axis governs interleukin-17A signaling.

IL-17A is a therapeutic target in many autoimmune diseases. Most nonhematopoietic cells express IL-17A receptors and respond to extracellular IL-17A by inducing proinflammatory cytokines. The IL-17A signal transduction triggers two broad, TRAF6- and TRAF5-dependent, intracellular signaling pathways to produce representative cytokines (IL-6) and chemokines (CXCL-1), respectively. Our limited understanding of the cross-talk between these two branches has generated a crucial gap of knowledge, leading to therapeutics indiscriminately blocking IL-17A and global inhibition of its target genes. In previous work, we discovered an elevated expression of 14-3-3 proteins in inflammatory aortic disease, a rare human autoimmune disorder with increased levels of IL-17A. Here we report that 14-3-3ζ is essential for IL-17 signaling by differentially regulating the signal-induced IL-6 and CXCL-1. Using genetically manipulated human and mouse cells, and ex vivo and in vivo rat models, we uncovered a function of 14-3-3ζ. As a part of the molecular mechanism, we show that 14-3-3ζ interacts with several TRAF proteins; in particular, its interaction with TRAF5 and TRAF6 is increased in the presence of IL-17A. In contrast to TRAF6, we found TRAF5 to be an endogenous suppressor of IL-17A-induced IL-6 production, an effect countered by 14-3-3ζ. Furthermore, we observed that 14-3-3ζ interaction with TRAF proteins is required for the IL-17A-induced IL-6 levels. Together, our results show that 14-3-3ζ is an essential component of IL-17A signaling and IL-6 production, an effect that is suppressed by TRAF5. To the best of our knowledge, this report of the 14-3-3ζ-TRAF5 axis, which differentially regulates IL-17A-induced IL-6 and CXCL-1 production, is unique.

The interferon-inducible protein TDRD7 inhibits AMP-activated protein kinase and thereby restricts autophagy-independent virus replication.

The interferon system is the first line of defense against virus infection. Recently, using a high-throughput genetic screen of a human interferon-stimulated gene short-hairpin RNA library, we identified a viral restriction factor, TDRD7 (Tudor domain-containing 7). TDRD7 inhibits the paramyxo-/pneumoviruses (e.g. Sendai virus and respiratory syncytial virus) by interfering with the virus-induced cellular autophagy pathway, which these viruses use for their replication. Here, we report that TDRD7 is a viral restriction factor against herpes simplex virus (HSV-1). Using knockdown, knockout, and ectopic expression systems, we demonstrate the anti-HSV-1 activity of TDRD7 in multiple human and mouse cell types. TDRD7 inhibited the virus-activated AMP-activated protein kinase (AMPK), which was essential for HSV-1 replication. Genetic ablation or chemical inhibition of AMPK activity suppressed HSV-1 replication in multiple human and mouse cells. Mechanistically, HSV-1 replication after viral entry depended on AMPK but not on its function in autophagy. The antiviral activity of TDRD7 depended on its ability to inhibit virus-activated AMPK. In summary, our results indicate that the newly identified viral restriction factor TDRD7 inhibits AMPK and thereby blocks HSV-1 replication independently of the autophagy pathway. These findings suggest that AMPK inhibition represents a potential strategy to manage HSV-1 infections.