FcγR-mediated SARS-CoV-2 infection of monocytes activates inflammation.

SARS-CoV-2 can cause acute respiratory distress and death in some patients1. Although severe COVID-19 is linked to substantial inflammation, how SARS-CoV-2 triggers inflammation is not clear2. Monocytes and macrophages are sentinel cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D, leading to inflammatory death (pyroptosis) and the release of potent inflammatory mediators3. Here we show that about 6% of blood monocytes of patients with COVID-19 are infected with SARS-CoV-2. Monocyte infection depends on the uptake of antibody-opsonized virus by Fcγ receptors. The plasma of vaccine recipients does not promote antibody-dependent monocyte infection. SARS-CoV-2 begins to replicate in monocytes, but infection is aborted, and infectious virus is not detected in the supernatants of cultures of infected monocytes. Instead, infected cells undergo pyroptosis mediated by activation of NLRP3 and AIM2 inflammasomes, caspase-1 and gasdermin D. Moreover, tissue-resident macrophages, but not infected epithelial and endothelial cells, from lung autopsies from patients with COVID-19 have activated inflammasomes. Taken together, these findings suggest that antibody-mediated SARS-CoV-2 uptake by monocytes and macrophages triggers inflammatory cell death that aborts the production of infectious virus but causes systemic inflammation that contributes to COVID-19 pathogenesis.

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in a minority of patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes and macrophages are sentinel immune cells in the blood and tissue, respectively, that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). We find that about 10% of blood monocytes in COVID-19 patients are infected with SARS-CoV-2. Monocyte infection depends on viral antibody opsonization and uptake of opsonized virus by the Fc receptor CD16. After uptake, SARS-CoV-2 begins to replicate in monocytes, as evidenced by detection of double-stranded RNA and subgenomic RNA and expression of a fluorescent reporter gene. However, infection is aborted, and infectious virus is not detected in infected monocyte supernatants or patient plasma. Instead, infected cells undergo inflammatory cell death (pyroptosis) mediated by activation of the NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD. Moreover, tissue-resident macrophages, but not infected epithelial cells, from COVID-19 lung autopsy specimens showed evidence of inflammasome activation. These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes/macrophages triggers inflammatory cell death that aborts production of infectious virus but causes systemic inflammation that contributes to severe COVID-19 disease pathogenesis.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in some patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes are sentinel blood cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that ~10% of blood monocytes in COVID-19 patients are dying and infected with SARS-CoV-2. Monocyte infection, which depends on antiviral antibodies, activates NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD cleavage and relocalization. Signs of pyroptosis (IL-1 family cytokines, LDH) in the plasma correlate with development of severe disease. Moreover, expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes triggers inflammation that contributes to severe COVID-19 disease pathogenesis. ONE SENTENCE SUMMARY: Antibody-mediated SARS-CoV-2 infection of monocytes activates inflammation and cytokine release.

Identification of a Distal Locus Enhancer Element That Controls Cell Type-Specific TNF and LTA Gene Expression in Human T Cells.

The human TNF/LT locus genes TNF, LTA, and LTB are expressed in a cell type-specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4+ T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF, LTA, and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type-specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9.

Cytoplasmic RNA Sensor Pathways and Nitazoxanide Broadly Inhibit Intracellular Mycobacterium tuberculosis Growth.

To establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.

The FDA-Approved Oral Drug Nitazoxanide Amplifies Host Antiviral Responses and Inhibits Ebola Virus.

Here, we show that the US Food and Drug Administration-approved oral drug nitazoxanide (NTZ) broadly amplifies the host innate immune response to viruses and inhibits Ebola virus (EBOV) replication. We find that NTZ enhances retinoic-acid-inducible protein I (RIG-I)-like-receptor, mitochondrial antiviral signaling protein, interferon regulatory factor 3, and interferon activities and induces transcription of the antiviral phosphatase GADD34. NTZ significantly inhibits EBOV replication in human cells through its effects on RIG-I and protein kinase R (PKR), suggesting that it counteracts EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV. NTZ also inhibits a second negative-strand RNA virus, vesicular stomatitis virus (VSV), through RIG-I and GADD34, but not PKR, consistent with VSV's distinct host innate immune evasion mechanisms. Thus, NTZ counteracts varied virus-specific immune evasion strategies by generally enhancing the RNA sensing and interferon axis that is triggered by foreign cytoplasmic RNA exposure, and holds promise as an oral therapy against EBOV.

Imaging flow cytometry analysis of intracellular pathogens.

Imaging flow cytometry has been applied to address questions in infection biology, in particular, infections induced by intracellular pathogens. This methodology, which utilizes specialized analytic software makes it possible to analyze hundreds of quantified features for hundreds of thousands of individual cellular or subcellular events in a single experiment. Imaging flow cytometry analysis of host cell-pathogen interaction can thus quantitatively addresses a variety of biological questions related to intracellular infection, including cell counting, internalization score, and subcellular patterns of co-localization. Here, we provide an overview of recent achievements in the use of fluorescently labeled prokaryotic or eukaryotic pathogens in human cellular infections in analysis of host-pathogen interactions. Specifically, we give examples of Imagestream-based analysis of cell lines infected with Toxoplasma gondii or Mycobacterium tuberculosis. Furthermore, we illustrate the capabilities of imaging flow cytometry using a combination of standard IDEAS™ software and the more recently developed Feature Finder algorithm, which is capable of identifying statistically significant differences between researcher-defined image galleries. We argue that the combination of imaging flow cytometry with these software platforms provides a powerful new approach to understanding host control of intracellular pathogens.

TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice.

Despite their antiviral effect, the in vivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. In explants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3-4 weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

A Role for IFITM Proteins in Restriction of Mycobacterium tuberculosis Infection.

The interferon (IFN)-induced transmembrane (IFITM) proteins are critical mediators of the host antiviral response. Here, we expand the role of IFITM proteins to host defense against intracellular bacterial infection by demonstrating that they restrict Mycobacterium tuberculosis (MTb) intracellular growth. Simultaneous knockdown of IFITM1, IFITM2, and IFITM3 by RNAi significantly enhances MTb growth in human monocytic and alveolar/epithelial cells, whereas individual overexpression of each IFITM impairs MTb growth in these cell types. Furthermore, MTb infection, Toll-like receptor 2 and 4 ligands, and several proinflammatory cytokines induce IFITM1-3 gene expression in human myeloid cells. We find that IFITM3 co-localizes with early and, in particular, late MTb phagosomes, and overexpression of IFITM3 enhances endosomal acidification in MTb-infected monocytic cells. These findings provide evidence that the antiviral IFITMs participate in the restriction of mycobacterial growth, and they implicate IFITM-mediated endosomal maturation in its antimycobacterial activity.

Regulation of Mycobacterium tuberculosis-dependent HIV-1 transcription reveals a new role for NFAT5 in the toll-like receptor pathway.

Tuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load. Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages. We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb. Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression. Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter. These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression.

The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells.

BACKGROUND: Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells. FINDINGS: Here, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface ß1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1Lai-Halo), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers. CONCLUSIONS: Use of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.

Arc of a vicious circle: pathways activated by Mycobacterium tuberculosis that target the HIV-1 long terminal repeat.

In this review, we examine how a subset of signal transduction cascades initiated by Mycobacterium tuberculosis (Mtb) infection modulates transcription mediated by the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). We describe two distinct phases of signaling that target transcription factors known to bind the HIV-1 LTR, and thus drive viral transcription and replication, in cells of the Mtb-infected host. First, Mtb-derived molecules, including cell wall components and DNA, interact with a number of host pattern recognition receptors. Second, cytokines and chemokines secreted in response to Mtb infection initiate signal transduction cascades through their cognate receptors. Given the variation in cell wall components among distinct clinical Mtb strains, the initial pattern recognition receptor interaction leading to direct LTR activation and differential cytokine and chemokine production is likely to be an important aspect of Mtb strain-specific regulation of HIV-1 transcription and replication. Improved understanding of these molecular mechanisms in the context of bacterial and host genetics should provide key insights into the accelerated viral replication and disease progression characteristic of HIV/TB coinfection.

HIV-1 replication is differentially regulated by distinct clinical strains of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is the largest cause of death in human immunodeficiency virus type 1 (HIV-1) infection, having claimed an estimated one third to one half of the 30 million AIDS deaths that have occurred worldwide. Different strains of Mycobacterium tuberculosis (MTb), the causative agent of TB, are known to modify the host immune response in a strain-specific manner. However, a MTb strain-specific impact upon the regulation of HIV-1 replication has not previously been established. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] We isolated normal human peripheral blood mononuclear cells (PBMC) and co-infected them with HIV-1 and with either the well characterized CDC1551 or HN878 MTb clinical isolate. We show that HIV-1 co-infection with the CDC1551 MTb strain results in higher levels of virus replication relative to co-infection with the HN878 MTb strain ex vivo. Furthermore, we show that the distinct pattern of CDC1551 or HN878 induced HIV-1 replication is associated with significantly increased levels of TNF and IL-6, and of the transcription and nuclear translocation of the p65 subunit of the transcription factor NF-kappaB, by CDC1551 relative to HN878. CONCLUSIONS/SIGNIFICANCE: These results provide a precedent for TB strain-specific effects upon HIV-1 replication and thus for TB strain-specific pathogenesis in the outcome of HIV-1/TB co-infection. MTb strain-specific factors and mechanisms involved in the regulation of HIV-1 during co-infection will be of importance in understanding the basic pathogenesis of HIV-1/TB co-infection.

NFAT5 regulates HIV-1 in primary monocytes via a highly conserved long terminal repeat site.

To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor-viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5-LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.

Transactivator of transcription from HIV type 1 subtype E selectively inhibits TNF gene expression via interference with chromatin remodeling of the TNF locus.

The transactivator of transcription (Tat) protein is essential for efficient HIV type 1 (HIV-1) replication and is involved in the transcriptional regulation of the host immune response gene, TNF. In this study, we demonstrate that Tat proteins from representative HIV-1 subtype E isolates, but not from subtypes B or C, selectively inhibit TNF gene transcription and protein production in CD4(+) Jurkat T cells. Strikingly, we show that this repression is due to a tryptophan at residue 32 of Tat E and is secondary to interference with recruitment of the histone acetyltransferase P/CAF to the TNF promoter and with chromatin remodeling of the TNF locus. This study presents a novel mechanism by which HIV-1 manipulates a host immune response gene that is important in its own replication. Moreover, these results demonstrate a new mechanism by which the TNF gene is regulated via chromatin remodeling secondary to viral infection.

Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV.

Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIV(IIIB) strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.

Mycobacterium tuberculosis recall antigens suppress HIV-1 replication in anergic donor cells via CD8+ T cell expansion and increased IL-10 levels.

Mycobacterium tuberculosis (MTb) is the leading cause of death in the setting of AIDS. MTb enhances the pathogenicity and accelerates the course of HIV disease and, furthermore, infection with HIV-1 increases the risk of reactivation or reinfection with MTb. In this study, we show that host-specific recall responses to one pathogen, MTb, has a direct effect upon the regulation of a second pathogen, HIV-1. Using cells from immunocompetent former tuberculosis (TB) patients who displayed either a persistently positive (responsive) or negative (anergic), delayed-type hypersensitivity (DTH) reaction to intradermal injection of purified protein derivative (PPD), we investigated the effect of recall Ags to MTb upon the replication of HIV-1 primary isolates in vitro. We show that HIV-1 replication of a T cell-tropic isolate was significantly impaired in MTb-stimulated PBMC from PPD-anergic donors. Furthermore, these donors displayed a significant increase in CD8(+) T cells and IL-10 levels and lower levels of IL-2 and TNF-alpha relative to PPD-responsive donors in response to PPD stimulation. Strikingly, CD8(+) T cell depletion and blocking of IL-10 significantly increased HIV-1 replication in these PPD-anergic donors, indicating that an immunosuppressive response to MTb recall Ags inhibits HIV-1 replication in PPD-anergic individuals. Therefore, immunotherapeutic approaches aimed at recapitulating Ag-specific MTb anergy in vivo could result in novel and effective approaches to inhibit HIV-1 disease progression in MTb/HIV-1 coinfection.