Macrophage phagocytosis of SARS-CoV-2-infected cells mediates potent plasmacytoid dendritic cell activation.

Early and strong interferon type I (IFN-I) responses are usually associated with mild COVID-19 disease, whereas persistent or unregulated proinflammatory cytokine responses are associated with severe disease outcomes. Previous work suggested that monocyte-derived macrophages (MDMs) are resistant and unresponsive to SARS-CoV-2 infection. Here, we demonstrate that upon phagocytosis of SARS-CoV-2-infected cells, MDMs are activated and secrete IL-6 and TNF. Importantly, activated MDMs in turn mediate strong activation of plasmacytoid dendritic cells (pDCs), leading to the secretion of high levels of IFN-α and TNF. Furthermore, pDC activation promoted IL-6 production by MDMs. This kind of pDC activation was dependent on direct integrin-mediated cell‒cell contacts and involved stimulation of the TLR7 and STING signaling pathways. Overall, the present study describes a novel and potent pathway of pDC activation that is linked to the macrophage-mediated clearance of infected cells. These findings suggest that a high infection rate by SARS-CoV-2 may lead to exaggerated cytokine responses, which may contribute to tissue damage and severe disease.

Can the Prognosis of COVID-19 Disease Be Determined by Fecal Markers and Cytokines?

The coronavirus disease 2019 (COVID-19) pandemic has affected the entire world, and has a variety of clinical presentations. The aim of this study is to determine the relationships of fecal cytokines and markers with the symptoms and prognosis of children with COVID-19 infection, and to identify noninvasive markers during follow-up. In a cohort of 40 COVID-19-positive children and 40 healthy controls, fecal cytokines and markers were examined in stool samples. A binary logistic model was used to assess the potential of cytokines as risk factors for hospitalization. Odds ratios (ORs) with 95% confidence intervals (CIs) were reported. A P-value <0.05 was accepted as statistically significant. Levels of fecal lysozyme, myeloperoxidase, hemoglobin, and interleukin-5 (IL-5) (P < 0.05) were significantly higher among the patients than controls. In a logistic regression analysis, fecal IL-2 (OR = 3.83; 95% CI: 1.44-15.92), IL-4 (OR = 2.96; 95% CI: 1.09-12.93), IL-5 (OR = 4.56; 95% CI: 1.18-27.88), IL-10 (OR = 2.71 95% CI: 1.19-7.94), interferon-gamma (IFN-γ) (OR = 4.03; 95% CI: 1.44-15.73), IFN-α (OR = 3.02; 95% CI: 1.08-11.65), calcium-binding protein B S100 (S100 B) (OR = 4.78; 95% CI: 1.31-27.82), neutrophil elastase (NE) 2 (OR = 4.07; 95% CI: 1.17-19.69), and matrix metalloproteinase 1 (MMP-1) (OR = 3.67; 95% CI: 1.1-18.82) levels were significantly higher in hospitalized patients with COVID-19 infection than outpatients. We demonstrated that various fecal cytokines and markers were increased in patients who had COVID-19. Fecal IL-2, IL-4, IL-5, IL-10, IFN-γ, IFN-α, S100 B, NE, and MMP-1 levels were significantly elevated in hospitalized patients. We suggest that the fecal and serum levels of cytokines could be used to predict the prognosis of COVID-19 disease, although more studies are needed to confirm this.

Is IFN expression by NK cells a hallmark of severe COVID-19?

Natural Killer cells (NK) are crucial in host defense against viruses. There are many unanswered questions about the immune system in COVID-19, especially the mechanisms that contribute to the development of mild or severe forms of the disease. Although NK cells may have an essential role in the pathogenesis of COVID-19, the mechanisms involved in this process are not yet fully elucidated. Here, we demonstrate that CD3-CD56+ NK cells frequency in the volunteers who recovered from mild COVID-19 (Mild CoV) presented a significant increase compared to the healthy control (HC) and individuals recovering from severe COVID-19 (Severe CoV) groups. Furthermore, distinct IFN profiles in recovered COVID-19 patients with mild or severe clinical forms of the disease were observed in the total NK cells (CD3-CD56+). In the first group, NK cells express increased levels of IFN-α compared to the severe CoV, while higher production of IFN-γ in severe CoV was found. Moreover, NK cells in mild CoV express more cytolytic granules depicted by granzyme B and perforin. Compared to HC, PBMCs from mild CoV presented higher Ki-67 and TIM-3 production after Pool CoV-2 and Pool Spike CoV-2 peptides stimulus. In addition, non-stimulated PBMCs in the mild CoV group had higher NK TIM-3+ frequency than severe CoV. In the mild CoV group, Pool Spike CoV-2 and Pool CoV-2 peptides stimuli elicited higher granzyme B and perforin coexpression and IFN-α production by PBMCs. However, in severe CoV, Pool Spike CoV-2 reduced the coexpression of granzyme B, perforin, and CD107a suggesting a decrease in the cytotoxic activity of NK cells. Therefore, our study shows that NK cells may have a crucial role in COVID-19 with the involvement of IFN-α and cytotoxic properties that aid in developing qualified immune responses. Furthermore, the data suggest that higher amounts of IFN-γ may be linked to the severity of this disease.

Activation of the AIM2 Receptor in Circulating Cells of Post-COVID-19 Patients With Signs of Lung Fibrosis Is Associated With the Release of IL-1α, IFN-α and TGF-ß.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for COVID-19, has caused a global pandemic. Observational studies revealed a condition, herein called as Long-COVID syndrome (PC), that affects both moderately and severely infected patients, reducing quality-of-life. The mechanism/s underlying the onset of fibrotic-like changes in PC are still not well defined. The goal of this study was to understand the involvement of the Absent in melanoma-2 (AIM2) inflammasome in PC-associated lung fibrosis-like changes revealed by chest CT scans. Peripheral blood mononuclear cells (PBMCs) obtained from PC patients who did not develop signs of lung fibrosis were not responsive to AIM2 activation by Poly dA:dT. In sharp contrast, PBMCs from PC patients with signs of lung fibrosis were highly responsive to AIM2 activation, which induced the release of IL-1α, IFN-α and TGF-ß. The recognition of Poly dA:dT was not due to the activation of cyclic GMP-AMP (cGAMP) synthase, a stimulator of interferon response (cGAS-STING) pathways, implying a role for AIM2 in PC conditions. The release of IFN-α was caspase-1- and caspase-4-dependent when AIM2 was triggered. Instead, the release of pro-inflammatory IL-1α and pro-fibrogenic TGF-ß were inflammasome independent because the inhibition of caspase-1 and caspase-4 did not alter the levels of the two cytokines. Moreover, the responsiveness of AIM2 correlated with higher expression of the receptor in circulating CD14+ cells in PBMCs from patients with signs of lung fibrosis.

Impact of BNT162b2 mRNA anti-SARS-CoV-2 vaccine on interferon-alpha production by plasmacytoid dendritic cells and autoreactive T cells in patients with systemic lupus erythematosus: The COVALUS project.

OBJECTIVE: To evaluate the specific response of SLE patients to BNT162b2 vaccination and its impact on autoimmunity defined as in vivo production of interferon-alpha (IFNα) by plasmacytoid dendritic cells (pDCs) and autoreactive immune responses. METHODS: Our prospective study included SLE patients and healthy volunteers (HV) who received 2 doses of BNT162b2 vaccine 4 weeks apart. Subjects under immunosuppressive drugs or with evidence of prior COVID-19 were excluded. IgG anti-Spike SARS-CoV-2 (anti-S) antibodies, anti-S specific-B cells, anti-S specific T cells, in vivo INF-α production by pDCs, activation marker expression by pDCs and autoreactive anti-nuclear T cells were quantified before first injection, before second injection, and 3 and 6 months after first injection. RESULTS: Vaccinated SLE patients produced significantly lower IgG antibodies and specific B cells against SARS-CoV-2 as compared to HV. In contrast, anti-S T cell response did not significantly differ between SLE patients and HV. Following vaccination, the surface expression of HLA-DR and CD86 and the in vivo production of IFNα by pDCs significantly increased in SLE patients. The boosted expression of HLA-DR on pDCs induced by BNT162b2 vaccine correlated with the overall immune responses against SARS-CoV-2 (anti-S antibodies: r = 0.27 [0.05-0.46], p = 0.02; anti-S B cells: r = 0.19 [-0.03-0.39], p = 0.09); anti-S T cells: r = 0.28 [0.05-0.47], p = 0.016). Eventually, anti-SARS-CoV-2 vaccination was associated with an overall decrease of autoreactive T cells (slope = - 0.00067, p = 0.015). CONCLUSION: BNT162b2 vaccine induces a transient in vivo activation of pDCs in SLE that contributes to the immune responses against SARS-CoV-2. Unexpectedly BNT162b2 vaccine also dampens the pool of circulating autoreactive T cells, suggesting that vaccination may have a beneficial impact on SLE disease.

Rapid Reversible Osmoregulation of Cytoplasmic Biomolecular Condensates of Human Interferon-α-Induced Antiviral MxA GTPase.

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1-2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40-50 mOsm). Both reassembled into new structures within 1-2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90-175 mOsm) first rapidly disassembled within 1-3 min, and then, in most cells, spontaneously reassembled 7-15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.

Propofol directly binds to and inhibits TLR7.

Sedatives/anesthetics are important medical tools to facilitate medical care and increase patients' comfort. Increasingly, there is recognition that sedatives/anesthetics can modulate immune functions. Toll-like receptors (TLRs) are major pattern recognition receptors involved in the recognition of microbial components. TLR7 recognizes single-strand RNA virus such as influenza and SARS-CoV2 viruses and initiates interferon (IFN) responses. IFN production triggered by TLR7 stimulation is a critical anti-viral response. For example, patients with TLR7 variants including loss-of- function variants were associated with severe COVID-19. Taken together, it is important to determine if sedatives/anesthetics mitigate TLR7 function. We have previously showed that TLR7-mediated activation was not affected by volatile anesthetics. However, we found that propofol attenuated TLR7 activation among intravenous sedatives in the reporter assay. TLR7 agonist R837 stimulation increased TNF-α, IL-1ß, IL-6, IL-10, and IFN-ß mRNA levels in bone marrow-derived dendritic cells, while these levels were attenuated by propofol. Our murine lung slice experiments showed that propofol attenuated IFN production. R837 increased IFN-ß expression in the lungs, and propofol attenuated IFN-ß expression in an in vivo model of R837 intranasal instillation. We also found that propofol directly bound to and hindered its association of TLR7 with MyD88. Our analysis using fropofol, propofol derivative showed that the hydroxyl group in propofol was important for propofol-TLR7 interaction.

Middle East respiratory syndrome coronavirus ORF4b protein inhibits TLR7- and TLR9-dependent alpha interferon induction.

The Toll-like receptor (TLR)7- and TLR9-dependent signalling cascade is responsible for production of a large amount of alpha interferon by plasmacytoid dendritic cells upon viral infection. Here, we show that Middle East respiratory syndrome coronavirus (MERS-CoV) accessory protein ORF4b has the most potential among the MERS-CoV accessory proteins to inhibit the TLR7/9-signaling-dependent alpha interferon production. ORF4b protein, which has a bipartite nuclear localization signal, was found to bind to IKKα, a kinase responsible for phosphorylation of interferon regulatory factor (IRF)7. This interaction caused relocation of a large proportion of IKKα from the cytoplasm to the nucleus. Studies using ORF4b and IKKα mutants demonstrated that ORF4b protein inhibited IKKα-mediated IRF7 phosphorylation by sequestering IKKα in the nucleus and by impeding the phosphorylation process of cytoplasmic IKKα.

A Novel Antiviral Protein Derived from Oenanthe javanica: Type I Interferon-Dependent Antiviral Signaling and Its Pharmacological Potential.

Pathogenesis-related (PR) proteins produced in plants play a crucial role in self-defense against microbial attacks. Previously, we have identified a novel PR-1-like protein (OPRP) from Oenanthe javanica and examined its pharmacologic relevance and cell signaling in mammalian cells. Purified full-length OPRP protein significantly increased toll-like receptor 4 (TLR4)-dependent expression levels of genes such as inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and CD80. We also found that small peptides (OPRP2 and OPRP3) designed from OPRP remarkably upregulated myxovirus resistance (Mx1), 2'-5' oligoadenylate sythetase (OAS), and interferon (IFN) α/ß genes in mouse splenocytes as well as human epithelial cells. Notably, OPRP protein distinctively activated STAT1 phosphorylation and ISGF-3γ. Interestingly, OPRP2 and OPRP3 were internalized to the cytoplasm and triggered dimerization of STAT1/STAT2, followed by upregulation of type I IFN-dependent antiviral cytokines. Moreover, OPRP1 successfully inhibited viral (Pseudo SARS-CoV-2) entry into host cells. Taken together, we conclude that OPRP and its small peptides (OPRP1 to 3) present a new therapeutic intervention for modulating innate immune activity through type I IFN-dependent antiviral signaling and a new therapeutic approach that drives an antiviral state in non-immune cells by producing antiviral cytokines.

Inhibition of the IFN-α JAK/STAT Pathway by MERS-CoV and SARS-CoV-1 Proteins in Human Epithelial Cells.

Coronaviruses (CoVs) have caused several global outbreaks with relatively high mortality rates, including Middle East Respiratory Syndrome coronavirus (MERS)-CoV, which emerged in 2012, and Severe Acute Respiratory Syndrome (SARS)-CoV-1, which appeared in 2002. The recent emergence of SARS-CoV-2 highlights the need for immediate and greater understanding of the immune evasion mechanisms used by CoVs. Interferon (IFN)-α is the body's natural antiviral agent, but its Janus kinase/signal transducer and activators of transcription (JAK/STAT) signalling pathway is often antagonized by viruses, thereby preventing the upregulation of essential IFN stimulated genes (ISGs). Therapeutic IFN-α has disappointingly weak clinical responses in MERS-CoV and SARS-CoV-1 infected patients, indicating that these CoVs inhibit the IFN-α JAK/STAT pathway. Here we show that in lung alveolar A549 epithelial cells expression of MERS-CoV-nsp2 and SARS-CoV-1-nsp14, but not MERS-CoV-nsp5, increased basal levels of total and phosphorylated STAT1 & STAT2 protein, but reduced IFN-α-mediated phosphorylation of STAT1-3 and induction of MxA. While MERS-CoV-nsp2 and SARS-CoV-1-nsp14 similarly increased basal levels of STAT1 and STAT2 in bronchial BEAS-2B epithelial cells, unlike in A549 cells, they did not enhance basal pSTAT1 nor pSTAT2. However, both viral proteins reduced IFN-α-mediated induction of pSTAT1-3 and ISGs (MxA, ISG15 and PKR) in BEAS-2B cells. Furthermore, even though IFN-α-mediated induction of pSTAT1-3 was not affected by MERS-CoV-nsp5 expression in BEAS-2B cells, downstream ISG induction was reduced, revealing that MERS-CoV-nsp5 may use an alternative mechanism to reduce antiviral ISG induction in this cell line. Indeed, we subsequently discovered that all three viral proteins inhibited STAT1 nuclear translocation in BEAS-2B cells, unveiling another layer of inhibition by which these viral proteins suppress responses to Type 1 IFNs. While these observations highlight cell line-specific differences in the immune evasion effects of MERS-CoV and SARS-CoV-1 proteins, they also demonstrate the broad spectrum of immune evasion strategies these deadly coronaviruses use to stunt antiviral responses to Type IFN.

Klotho deficiency intensifies hypoxia-induced expression of IFN-α/ß through upregulation of RIG-I in kidneys.

Hypoxia is a common pathway to the progression of end-stage kidney disease. Retinoic acid-inducible gene I (RIG-I) encodes an RNA helicase that recognizes viruses including SARS-CoV2, which is responsible for the production of interferon (IFN)-α/ß to prevent the spread of viral infection. Recently, RIG-I activation was found under hypoxic conditions, and klotho deficiency was shown to intensify the activation of RIG-I in mouse brains. However, the roles of these functions in renal inflammation remain elusive. Here, for in vitro study, the expression of RIG-I and IFN-α/ß was examined in normal rat kidney (NRK)-52E cells incubated under hypoxic conditions (1% O2). Next, siRNA targeting RIG-I or scramble siRNA was transfected into NRK52E cells to examine the expression of RIG-I and IFN-α/ß under hypoxic conditions. We also investigated the expression levels of RIG-I and IFN-α/ß in 33 human kidney biopsy samples diagnosed with IgA nephropathy. For in vivo study, we induced renal hypoxia by clamping the renal artery for 10 min in wild-type mice (WT mice) and Klotho-knockout mice (Kl-/- mice). Incubation under hypoxic conditions increased the expression of RIG-I and IFN-α/ß in NRK52E cells. Their upregulation was inhibited in NRK52E cells transfected with siRNA targeting RIG-I. In patients with IgA nephropathy, immunohistochemical staining of renal biopsy samples revealed that the expression of RIG-I was correlated with that of IFN-α/ß (r = 0.57, P<0.001, and r = 0.81, P<0.001, respectively). The expression levels of RIG-I and IFN-α/ß were upregulated in kidneys of hypoxic WT mice and further upregulation was observed in hypoxic Kl-/- mice. These findings suggest that hypoxia induces the expression of IFN-α/ß through the upregulation of RIG-I, and that klotho deficiency intensifies this hypoxia-induced expression in kidneys.

Ferret Interferon (IFN)-Inducible Transmembrane Proteins Are Upregulated by both IFN-α and Influenza Virus Infection.

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.

Dysregulation of Pulmonary Responses in Severe COVID-19.

Patients with coronavirus disease 2019 (COVID-19) predominantly have a respiratory tract infection with various symptoms and high mortality is associated with respiratory failure second to severe disease. The risk factors leading to severe disease remain unclear. Here, we reanalyzed a published single-cell RNA-Seq (scRNA-Seq) dataset and found that bronchoalveolar lavage fluid (BALF) of patients with severe disease compared to those with mild disease contained decreased TH17-type cells, decreased IFNA1-expressing cells with lower expression of toll-like receptor 7 (TLR7) and TLR8, increased IgA-expressing B cells, and increased hyperactive epithelial cells (and/or macrophages) expressing matrix metalloproteinases (MMPs), hyaluronan synthase 2 (HAS2), and plasminogen activator inhibitor-1 (PAI-1), which may together contribute to the pulmonary pathology in severe COVID-19. We propose IFN-I (and TLR7/TLR8) and PAI-1 as potential biomarkers to predict the susceptibility to severe COVID-19.

SARS-CoV-2 Infection Induces Psoriatic Arthritis Flares and Enthesis Resident Plasmacytoid Dendritic Cell Type-1 Interferon Inhibition by JAK Antagonism Offer Novel Spondyloarthritis Pathogenesis Insights.

Objective: Bacterial and viral infectious triggers are linked to spondyloarthritis (SpA) including psoriatic arthritis (PsA) development, likely via dendritic cell activation. We investigated spinal entheseal plasmacytoid dendritic cells (pDCs) toll-like receptor (TLR)-7 and 9 activation and therapeutic modulation, including JAK inhibition. We also investigated if COVID-19 infection, a potent TLR-7 stimulator triggered PsA flares. Methods: Normal entheseal pDCs were characterized and stimulated with imiquimod and CpG oligodeoxynucleotides (ODN) to evaluate TNF and IFNα production. NanoString gene expression assay of total pDCs RNA was performed pre- and post- ODN stimulation. Pharmacological inhibition of induced IFNα protein was performed with Tofacitinib and PDE4 inhibition. The impact of SARS-CoV2 viral infection on PsA flares was evaluated. Results: CD45+HLA-DR+CD123+CD303+CD11c- entheseal pDCs were more numerous than blood pDCs (1.9 ± 0.8% vs 0.2 ± 0.07% of CD45+ cells, p=0.008) and showed inducible IFNα and TNF protein following ODN/imiquimod stimulation and were the sole entheseal IFNα producers. NanoString data identified 11 significantly upregulated differentially expressed genes (DEGs) including TNF in stimulated pDCs. Canonical pathway analysis revealed activation of dendritic cell maturation, NF-κB signaling, toll-like receptor signaling and JAK/STAT signaling pathways following ODN stimulation. Both tofacitinib and PDE4i strongly attenuated ODN induced IFNα. DAPSA scores elevations occurred in 18 PsA cases with SARS-CoV2 infection (9.7 ± 4 pre-infection and 35.3 ± 7.5 during infection). Conclusion: Entheseal pDCs link microbes to TNF/IFNα production. SARS-CoV-2 infection is associated with PsA Flares and JAK inhibition suppressed activated entheseal plasmacytoid dendritic Type-1 interferon responses as pointers towards a novel mechanism of PsA and SpA-related arthropathy.

MAIT cell activation augments adenovirus vector vaccine immunogenicity.

Mucosal-associated invariant T (MAIT) cells are innate sensors of viruses and can augment early immune responses and contribute to protection. We hypothesized that MAIT cells may have inherent adjuvant activity in vaccine platforms that use replication-incompetent adenovirus vectors. In mice and humans, ChAdOx1 (chimpanzee adenovirus Ox1) immunization robustly activated MAIT cells. Activation required plasmacytoid dendritic cell (pDC)-derived interferon (IFN)-α and monocyte-derived interleukin-18. IFN-α-induced, monocyte-derived tumor necrosis factor was also identified as a key secondary signal. All three cytokines were required in vitro and in vivo. Activation of MAIT cells positively correlated with vaccine-induced T cell responses in human volunteers and MAIT cell-deficient mice displayed impaired CD8+ T cell responses to multiple vaccine-encoded antigens. Thus, MAIT cells contribute to the immunogenicity of adenovirus vectors, with implications for vaccine design.

Potent and Selective Knockdown of Tyrosine Kinase 2 by Antisense Oligonucleotides.

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor tyrosine kinase, together with JAK1, JAK2, and JAK3. JAKs are important signaling mediators of many proinflammatory cytokines and represent compelling pharmacological targets for autoimmune and inflammatory diseases. Pan-acting small-molecule JAK inhibitors were approved for the treatment of rheumatoid arthritis and ulcerative colitis. However, their limited selectivity among JAK members have led to undesirable side effects, driving a search toward specific JAK inhibitors. Recently, TYK2 has emerged as a target of choice for the treatment of autoimmune diseases and severe COVID-19 with an optimum balance between efficacy and safety, based on observations from human genetics studies and clinical outcomes of several agents targeting cytokine pathways for which TYK2 plays an essential role. In this article, we address selective targeting of TYK2 from the genetic sequence space through development of antisense oligonucleotides (ASOs) against TYK2 mRNA. Potent ASO candidates were identified from the screening of over 200 ASOs using locked nucleic acid gapmer design. The lead ASOs exhibited potent and selective knockdown of TYK2 mRNA and protein across a panel of model human cell lines in a dose-dependent manner, showing no reduction in the mRNA and protein expression levels of other JAK paralogs. In agreement with the depletion of TYK2 proteins, several TYK2-mediated cytokine signaling pathways, including IFN-α and IL-12, were inhibited upon ASO treatment. Our results established the TYK2 ASOs as investigational tool compound and potential therapeutic agent for the treatment of autoimmune diseases and severe COVID-19.

JAK1 Inhibition Blocks Lethal Immune Hypersensitivity in a Mouse Model of Down Syndrome.

Individuals with Down syndrome (DS; trisomy 21) display hyperactivation of interferon (IFN) signaling and chronic inflammation, which could potentially be explained by the extra copy of four IFN receptor (IFNR) genes encoded on chromosome 21. However, the clinical effects of IFN hyperactivity in DS remain undefined. Here, we report that a commonly used mouse model of DS overexpresses IFNR genes and shows hypersensitivity to IFN ligands in diverse immune cell types. When treated repeatedly with a TLR3 agonist to induce chronic inflammation, these animals overexpress key IFN-stimulated genes, induce cytokine production, exhibit liver pathology, and undergo rapid weight loss. Importantly, the lethal immune hypersensitivity and cytokine production and the ensuing pathology are ameliorated by JAK1 inhibition. These results indicate that individuals with DS may experience harmful hyperinflammation upon IFN-inducing immune stimuli, as observed during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, pointing to JAK1 inhibition as a strategy to restore immune homeostasis in DS.

Type I Interferon Susceptibility Distinguishes SARS-CoV-2 from SARS-CoV.

SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero E6 and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, whereas SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures, we observe the absence of IFN-I stimulation by SARS-CoV-2 alone but detect the failure to counteract STAT1 phosphorylation upon IFN-I pretreatment, resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment postinfection and found that SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame 3b (ORF3b) and genetic differences versus ORF6 suggest that the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development.IMPORTANCE With the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection.

Impaired HVJ-stimulated Interferon producing capacity in MPO-ANCA-associated vasculitis with rapidly progressive glomerulonephritis lead to susceptibility to infection.

ANCA-associated RPGN leads to renal failure through systemic vasculitis and diffuse crescentic glomerulonephritis. MPO-ANCA-RPGN patients are highly susceptible to infections. Our aim in this study was to uncover reasons why these patients were susceptible to infections. We analyzed various aspects of type I interferon system including HVJ-stimulated IFN-α producing capacity and plasmacytoid dendritic cell (pDC) number in whole blood in MPO-ANCA-RPGN patients. Compared with healthy subjects, MPO-ANCA-RPGN patients showed impaired HVJ-stimulated IFN-α producing capacity and lower pDC number with or without glucocorticoid treatment. Immuno-histological staining of MPO-ANCA-RPGN kidney samples revealed a few but apparent pDC in T cell infiltrating regions even in patients with low pDC number in their peripheral blood. Patients' low HVJ-stimulated IFN-α producing capacity and pDC numbers persisted even after patients underwent several years of treatment. Former infection was determined using patients' serum BPI, Lamp-2 and Calprotectin, since they are reflective of a history of infection. These markers were higher in MPO-ANCA-RPGN patients than in healthy subjects. These results indicate that impaired HVJ-stimulated IFN-α production as well as dysfunction of the IFN system might have resulted from a previous bout of infection and can be partially implicated in patients' long-term susceptibility and vulnerability to infection.

Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients.

Coronavirus disease 2019 (COVID-19) is characterized by distinct patterns of disease progression that suggest diverse host immune responses. We performed an integrated immune analysis on a cohort of 50 COVID-19 patients with various disease severity. A distinct phenotype was observed in severe and critical patients, consisting of a highly impaired interferon (IFN) type I response (characterized by no IFN-ß and low IFN-α production and activity), which was associated with a persistent blood viral load and an exacerbated inflammatory response. Inflammation was partially driven by the transcriptional factor nuclear factor-κB and characterized by increased tumor necrosis factor-α and interleukin-6 production and signaling. These data suggest that type I IFN deficiency in the blood could be a hallmark of severe COVID-19 and provide a rationale for combined therapeutic approaches.