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.

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.

T cells and adaptive immunity to Mycobacterium tuberculosis in humans.

The adaptive immune response mediated by T cells is critical for control of Mycobacterium tuberculosis (M. tuberculosis) infection in humans. However, the M. tuberculosis antigens and host T-cell responses that are required for an effective adaptive immune response to M. tuberculosis infection are yet to be defined. Here, we review recent findings on CD4(+) and CD8(+) T-cell responses to M. tuberculosis infection and examine the roles of distinct M. tuberculosis-specific T-cell subsets in control of de novo and latent M. tuberculosis infection, and in the evolution of T-cell immunity to M. tuberculosis in response to tuberculosis treatment. In addition, we discuss recent studies that elucidate aspects of M. tuberculosis-specific adaptive immunity during human immunodeficiency virus co-infection and summarize recent findings from vaccine trials that provide insight into effective adaptive immune responses to M. tuberculosis infection.

TB-IRIS, T-cell activation, and remodeling of the T-cell compartment in highly immunosuppressed HIV-infected patients with TB.

OBJECTIVE: To investigate the impact of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) upon immunological recovery and the T-cell compartment after initiation of TB and antiretroviral therapy (ART). DESIGN AND METHODS: We prospectively evaluated T-cell immunophenotypes by flow cytometry and cytokines by Luminex assays in a subset (n = 154) of highly immunosuppressed HIV-infected patients with TB from the Cambodian Early versus Late Introduction of Antiretrovirals randomized clinical trial. We compared findings from patients who developed TB-IRIS with findings from patients who did not develop TB-IRIS. Data were evaluated with mixed-effect linear regression, Kaplan-Meier estimates, and Wilcoxon rank-sum tests, and q-values were calculated to control for multiple comparisons. RESULTS: Development of TB-IRIS was associated with significantly greater pre-ART frequencies of HLA-DRCD45ROCD4, CCR5CD4, OX40CD4, and Fas effector memory CD8 T cells, and significantly elevated levels of plasma interleukin (IL)-6, IL-1ß, IL-8, and IL-10, and viral load. Post-ART initiation, effector memory CD4 and Fas effector memory CD4 T-cell frequencies significantly expanded, and central memory CD4 T-cell frequencies significantly contracted in patients who experienced TB-IRIS. By week 34 post-TB treatment initiation, effector memory/central memory CD4 T-cell ratios were markedly higher in TB-IRIS versus non-TB-IRIS patients. CONCLUSIONS: A distinct pattern of pre-ART T-cell and cytokine markers appear to poise the immune response of certain patients to develop TB-IRIS. Experience of TB-IRIS is then associated with long-term remodeling of the CD4 T-cell memory compartment towards an effector memory-dominated phenotype. We speculate that these pre and post-ART TB-IRIS-associated immune parameters may contribute to superior immune control of TB/HIV co-infection and better clinical outcome.

A distal locus element mediates IFN-γ priming of lipopolysaccharide-stimulated TNF gene expression.

Interferon γ (IFN-γ) priming sensitizes monocytes and macrophages to lipopolysaccharide (LPS) stimulation, resulting in augmented expression of a set of genes including TNF. Here, we demonstrate that IFN-γ priming of LPS-stimulated TNF transcription requires a distal TNF/LT locus element 8 kb upstream of the TNF transcription start site (hHS-8). IFN-γ stimulation leads to increased DNase I accessibility of hHS-8 and its recruitment of interferon regulatory factor 1 (IRF1), and subsequent LPS stimulation enhances H3K27 acetylation and induces enhancer RNA synthesis at hHS-8. Ablation of IRF1 or targeting the hHS-8 IRF1 binding site in vivo with Cas9 linked to the KRAB repressive domain abolishes IFN-γ priming, but does not affect LPS induction of the gene. Thus, IFN-γ poises a distal enhancer in the TNF/LT locus by chromatin remodeling and IRF1 recruitment, which then drives enhanced TNF gene expression in response to a secondary toll-like receptor (TLR) stimulus.

Epigenetic control of cytokine gene expression: regulation of the TNF/LT locus and T helper cell differentiation.

Epigenetics encompasses transient and heritable modifications to DNA and nucleosomes in the native chromatin context. For example, enzymatic addition of chemical moieties to the N-terminal "tails" of histones, particularly acetylation and methylation of lysine residues in the histone tails of H3 and H4, plays a key role in regulation of gene transcription. The modified histones, which are physically associated with gene regulatory regions that typically occur within conserved noncoding sequences, play a functional role in active, poised, or repressed gene transcription. The "histone code" defined by these modifications, along with the chromatin-binding acetylases, deacetylases, methylases, demethylases, and other enzymes that direct modifications resulting in specific patterns of histone modification, shows considerable evolutionary conservation from yeast to humans. Direct modifications at the DNA level, such as cytosine methylation at CpG motifs that represses promoter activity, are another highly conserved epigenetic mechanism of gene regulation. Furthermore, epigenetic modifications at the nucleosome or DNA level can also be coupled with higher-order intra- or interchromosomal interactions that influence the location of regulatory elements and that can place them in an environment of specific nucleoprotein complexes associated with transcription. In the mammalian immune system, epigenetic gene regulation is a crucial mechanism for a range of physiological processes, including the innate host immune response to pathogens and T cell differentiation driven by specific patterns of cytokine gene expression. Here, we will review current findings regarding epigenetic regulation of cytokine genes important in innate and/or adaptive immune responses, with a special focus upon the tumor necrosis factor/lymphotoxin locus and cytokine-driven CD4+ T cell differentiation into the Th1, Th2, and Th17 lineages.

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.

ALIX/AIP1 is required for NP incorporation into Mopeia virus Z-induced virus-like particles.

During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions.

Minigenome-based reporter system suitable for high-throughput screening of compounds able to inhibit Ebolavirus replication and/or transcription.

We describe an Ebolavirus minigenome-based system that is suitable for high-throughput screening of compounds able to impair Ebolavirus virus replication and/or transcription. The assay is robust (Z' factor, >0.6) and can be carried out in low-biosafety containment. Results from a pilot screen of 960 compounds are presented.

Ebola virus VP40 late domains are not essential for viral replication in cell culture.

Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.

Filovirus budding.

Family Filoviridae, which includes Ebola virus (EBOV) and Marburg virus (MARV), is a growing threat to human and non-human primate populations in central Africa. Although many facets of the filovirus life cycle remain to be deciphered, a great deal has been learned in recent years. In particular, a clearer understanding of the roles played by viral, as well as cellular, proteins in the assembly and budding processes has been achieved. This review will discuss the current state of filovirus budding research, with especial emphasis placed on the viral matrix protein VP40 and its relationship with the cellular vesicular sorting pathway. Possible budding functions of the viral glycoprotein (GP), as well as the membrane-associated viral protein 24 (VP24), will also be described, and a model for filovirus budding will be proposed.

Production of novel ebola virus-like particles from cDNAs: an alternative to ebola virus generation by reverse genetics.

We established a plasmid-based system for generating infectious Ebola virus-like particles (VLPs), which contain an Ebola virus-like minigenome consisting of a negative-sense copy of the green fluorescent protein gene. This system produced nearly 10(3) infectious particles per ml of supernatant, equivalent to the titer of Ebola virus generated by a reverse genetics system. Interestingly, infectious Ebola VLPs were generated, even without expression of VP24. Transmission and scanning electron microscopic analyses showed that the morphology of the Ebola VLPs was indistinguishable from that of authentic Ebola virus. Thus, this system allows us to study Ebola virus entry, replication, and assembly without biosafety level 4 containment. Furthermore, it may be useful in vaccine production against this highly pathogenic agent.