Flow Cytometric Characterization of Antigen-Specific T Cells Based on RNA and Its Advantages in Detecting Infections and Immunological Disorders.

Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentation, fluorophores, and FISH methodologies will broaden and deepen the use of FISH-Flow, changing the immunological field by allowing determination of functional immune signatures at the mRNA level and the development of new diagnostic tools.

Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry.

Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from pre-existing in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.

Cutting edge: Vitamin D regulates lipid metabolism in Mycobacterium tuberculosis infection.

Vitamin D has long been linked to resistance to tuberculosis, an infectious respiratory disease that is increasingly hard to treat because of multidrug resistance. Previous work established that vitamin D induces macrophage antimicrobial functions against Mycobacterium tuberculosis. In this article, we report a novel, metabolic role for vitamin D in tuberculosis identified through integrated transcriptome and mechanistic studies. Transcriptome analysis revealed an association between vitamin D receptor (VDR) and lipid metabolism in human tuberculosis and infected macrophages. Vitamin D treatment of infected macrophages abrogated infection-induced accumulation of lipid droplets, which are required for intracellular M. tuberculosis growth. Additional transcriptomics results showed that vitamin D downregulates the proadipogenic peroxisome proliferator-activated receptor γ (PPARγ) in infected macrophages. PPARγ agonists reversed the antiadipogenic and the antimicrobial effects of VDR, indicating a link between VDR and PPARγ signaling in regulating both vitamin D functions. These findings suggest the potential for host-based, adjunct antituberculosis therapy targeting lipid metabolism.

Evidence for postinitiation regulation of mRNA biogenesis in tuberculosis.

Mycobacterium tuberculosis infection alters macrophage gene expression and macrophage response to IFN-γ, a critical host defense cytokine. However, regulation of these changes is poorly understood. We report discordance of changes in nascent transcript and total nuclear RNA abundance for the transcription factors STAT1 and IRF1, together with lack of effect on their RNA half-lives, in human THP-1 cells infected with M. tuberculosis and stimulated with IFN-γ. The results indicate that negative postinitiation regulation of mRNA biogenesis limits the expression of these factors, which mediate host defense against M. tuberculosis through the cellular response to IFN-γ. Consistent with the results for STAT1 and IRF1, transcriptome analysis reveals downregulation of postinitiation mRNA biogenesis processes and pathways by infection, with and without IFN-γ stimulation. Clinical relevance for regulation of postinitiation mRNA biogenesis is demonstrated by studies of donor samples showing that postinitiation mRNA biogenesis pathways are repressed in latent tuberculosis infection compared with cured disease and in active tuberculosis compared with ongoing treatment or with latent tuberculosis. For active disease and latent infection donors from two populations (London, U.K., and The Gambia), each analyzed using a different platform, pathway-related gene expression differences were highly correlated, demonstrating substantial specificity in the effect. Collectively, the molecular and bioinformatic analyses point toward downregulation of postinitiation mRNA biogenesis pathways as a means by which M. tuberculosis infection limits expression of immunologically essential transcription factors. Thus, negative regulation of postinitiation mRNA biogenesis can constrain the macrophage response to infection and overall host defense against tuberculosis.

Simvastatin increases the in vivo activity of the first-line tuberculosis regimen.

BACKGROUND: The need to develop new, improved treatments for tuberculosis (TB) remains urgent, and the repurposing of existing drugs represents a possible shortcut to market. Recently, there has been significant interest in host-directed adjuvant therapy to enhance bacillary killing. HMG-CoA reductase inhibitors (statins), which are among the most commonly prescribed drugs, have immunomodulatory properties and improve the clinical outcomes of bacterial infections. METHODS: We studied the tuberculocidal activity of simvastatin alone and in combination with first-line anti-TB drugs in J774 macrophages and during chronic TB infection. RESULTS: Exposure to 5 µM simvastatin significantly increased the tuberculocidal activity of isoniazid in J774 macrophages at Day 3 after infection versus isoniazid alone (P=0.02). Similarly, relative to the standard oral regimen of rifampicin (10 mg/kg), isoniazid (10 mg/kg) and pyrazinamide (150 mg/kg) given five times weekly, the addition of 25 mg/kg simvastatin enhanced bacillary killing, reducing the number of lung cfu by an additional 1 log10 at Day 28 (P<0.01) and by a further 1.25 log10 at Day 56 (P<0.01). CONCLUSIONS: The potential additive activity of simvastatin to first-line TB treatment holds promise. However, further studies to identify the optimal statin and dosing are required. In addition the ability of combination treatment with statins to accelerate the time required to achieve a stable cure remains to be explored.

Proteome-scale antibody responses and outcome of Mycobacterium tuberculosis infection in nonhuman primates and in tuberculosis patients.

BACKGROUND: Biomarkers of progression from latent Mycobacterium tuberculosis infection to active tuberculosis are needed. We assessed correlations between infection outcome and antibody responses in macaques and humans by high-throughput, proteome-scale serological studies. METHODS: Mycobacterium tuberculosis proteome microarrays were probed with serial sera from macaques representing various infection outcomes and with single-point human sera from tuberculosis suspects. Fluorescence intensity data were analyzed by calculating Z scores and associated P values. Temporal changes in macaque antibody responses were analyzed by polynomial regression. Correlations between human responses and sputum bacillary burden were assessed by quantile and hurdle regression. RESULTS: Macaque outcome groups exhibited distinct antibody profiles: early, transient responses in latent infection and stable antibody increase in active and reactivation disease. In humans, antibody levels and reactive protein numbers increased with bacillary burden. Responses to a subset of 10 proteins were more tightly associated with disease state than reactivity to the broader reactive proteome. CONCLUSIONS: Integration of macaque and human data reveals dynamic properties of antibody responses in relation to outcome and leads to actionable findings for translational research. These include the potential of antibody responses to detect acute infection and preclinical tuberculosis and to identify serodiagnostic proteins for the spectrum of bacillary burden in tuberculosis.

Bystander inhibition of dendritic cell differentiation by Mycobacterium tuberculosis-induced IL-10.

Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen-presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon-α and granulocyte-macrophage colony-stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat-inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb-stimulated cells (SN Mtb) remained CD14(+) and poorly differentiated into CD1a(+) cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin-1ß, interleukin-6, interleukin-10 and tumor necrosis factor-α upon Mtb stimulation. However, only interleukin-10 neutralization through the addition of soluble interleukin-10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin-10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen-activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin-10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non-infected monocytes into DCs through the release of interleukin-10. These results shed light on new aspects of the host-pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence.

Physical therapy for nocturnal lower limb cramping: A case report.

BACKGROUND AND PURPOSE: Nocturnal lower limb cramps are sudden, intensely painful, and can decrease sleep, increase anxiety, and reduce quality of life. The purpose of this case report is to describe the effectiveness of an evidence-based physical therapy intervention for a person with lower limb cramps. CASE DESCRIPTION: The patient was a 34-year-old female who presented with idiopathic bilateral lower limb foot pain and cramps. INTERVENTION: Rehabilitation addressed muscle strength, joint mobility, soft tissue extensibility, and biomechanical influences during functional movement, for a total of seven sessions over seven weeks. OUTCOMES: The frequency and severity of cramps decreased from three to four times/night, lasting several minutes down to one episode/week, lasting less than a minute. Her Numeric Pain Rating Scale (NPRS) score at worst decreased from 6/10 at the initial examination to 0/10 upon discharge. Lower extremity strength and functional mobility outcomes also exhibited improvement over the course of care. The patient's Lower Extremity Functional Scale (LEFS) score decreased from 87.5% to 80.0%, indicating greater disability, though this did not reach minimal detectable change levels. DISCUSSION: While her cramping and resulting pain improved, perceived participation restrictions persisted. Lack of improvement in the participation measure may have been related to the lack of a structured biopsychosocial approach to the rehabilitation process.

Gene expression profiles of bronchoalveolar cells in pulmonary TB.

The host response to Mycobacterium tuberculosis includes macrophage activation, inflammation with increased immune effector cells, tissue necrosis, and cavity formation, and fibrosis, distortion, and bronchiectasis. To evaluate the molecular basis of the immune response in the lungs of patients with active pulmonary tuberculosis (TB), we used bronchoalveolar lavage to obtain cells at the site of infection. Affymetrix GeneChip microarrays and cDNA nylon filter microarrays interrogated gene expression in bronchoalveolar lavage (BAL) cells from 11 healthy controls and 17 patients with active pulmonary TB. We found altered gene expression for 69 genes in TB versus normal controls that included cell surface markers, cytokines, chemokines, receptors, transcription factors, and complement components. In addition, TB BAL cell gene expression patterns segregated into 2 groups: one suggestive of a T helper type 1 (Th1) cellular immune response with increased signal transducer and activator of transcription-4 (STAT-4), interferon-gamma (IFN-gamma receptor), and monokine induced by IFN-gamma (MIG) expression with increased IFN-gamma protein levels in BAL fluid; the other group displayed characteristics of Th2 immunity with increased STAT-6, CD81, and IL-10 receptor expression. We were able to demonstrate that a Th2 presentation could change to a Th1 pattern after anti-tuberculous treatment in 1 TB patient studied serially. These gene expression data support the conclusion that pulmonary TB produces a global change in the BAL cell transcriptome with manifestations of either Th1 or Th2 immunity.

Tuberculosis State Is Associated with Expression of Toll-Like Receptor 2 in Sputum Macrophages.

During tuberculosis, macrophages are critical for both pathogen survival and host immune activation. Since expression of particular cell surface markers reflects cell function, we used flow cytometry to measure the abundance of surface markers associated with polarity, lipid uptake, or pattern recognition on macrophages found in induced sputum. Nine macrophage surface markers were examined from three groups of donors: infection-free, latent tuberculosis infection, and active pulmonary tuberculosis. Using a trend test, we found that expression of Toll-like receptor 2 was greater from absence of infection to latent infection and from latent infection to active tuberculosis. The results point to the possibility that innate immune cell phenotypes be used to distinguish among tuberculosis infection stages. Moreover, this study shows that readily accessible sputum macrophages have potential for tuberculosis diagnosis and prognosis. IMPORTANCEMycobacterium tuberculosis is an intracellular pathogen that parasitizes the host macrophage. While approximately two billion people are infected worldwide, only 5 to 10% become diseased with pulmonary tuberculosis, at least in the absence of comorbidities. Tuberculosis control requires development of noninvasive methods probing the host immune status to help distinguish latent infection from active tuberculosis. With such methods, high-risk individuals could be targeted for treatment before disease manifestation. Previous investigations have been based on examination of peripheral blood cells or, more rarely, lung macrophages obtained with invasive procedures, such as bronchoalveolar lavages. Here we show that differences exist in the expression of a surface protein (Toll-like receptor 2) between macrophages recovered from the sputum of individuals in different diagnostic groups: i.e., infection free, latent tuberculosis infection, and active pulmonary tuberculosis. Thus, phenotypic analysis of local macrophages obtained with noninvasive procedures can help distinguish among tuberculosis infection stages.

FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry.

We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ∼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.

Protein kinase I of Mycobacterium tuberculosis: cellular localization and expression during infection of macrophage-like cells.

Protein kinase I of Mycobacterium tuberculosis, which has an unusual amino acid composition in its catalytic loop, displayed autophosphorylation and transphosphorylation activity. Immunoblot analysis of sub-cellular fractions of M. tuberculosis, using anti-PknI antibodies raised in rabbits, showed that PknI localizes to the bacterial cytosol. In contrast, PknA was membrane-bound. Relative expression of pknI, when measured by combining molecular beacons and RT-PCR, decreased during infection of THP-1 human macrophages. Expression of pknA and pknB was upregulated during infection. Thus PknI represents a group of protein kinases that is distinct from the more extensively studied enzymes PknA and PknB.

Arsenic enhances the activation of Stat1 by interferon gamma leading to synergistic expression of IRF-1.

Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia (APL), including those who have relapsed after treatment with all-trans-retinoic acid (RA). In vitro studies with the APL-derived NB4 cell line showed that As2O3 exerts a dose-dependent dual effect, which induces apoptosis at 1 microM, whereas at a lower concentration of 0.1 microM, a partial differentiation of APL is observed. In non-APL cells, interferon (IFN) alpha and 1 microM As2O3 act synergistically to induce apoptosis. In this report, we show that in NB4 cells and in two RA-resistant NB4-derived cell lines, NB4-R1 and NB4-R2, IFNalpha or IFNgamma combined with 0.1 microM As2O3 lead to an increased maturation effect. Moreover, IFNgamma alone is able to differentiate RA-sensitive and -resistant cells with a higher maturation effect on NB4-R2 cells. In contrast, all these cells underwent apoptosis in the presence of the cytokine and a higher concentration of As2O3. IFNgamma boosted As2O3-induced apoptosis in APL cells as tested by TUNEL, Annexin V staining and activation of caspase 3. As2O3 differently altered IFN-induced gene products; it downregulated PML/RARalpha and PML, did not alter PKR and Stat1, and upregulated interferon regulatory family (IRF)-1. Synergism by IFNgamma and arsenic on IRF-1 expression is mediated by a composite element in the IRF-1 promoter that includes an IFNgamma-activation site (GAS) overlapped by a nonconsensus site for nuclear factor kappa B (NFkappaB). Arsenic has no effect on NFkappaB, whereas it enhances the activation of Stat1 by IFNgamma in NB4 cells leading to an increase in IRF-1 expression.

Infection with Mycobacterium tuberculosis induces the Warburg effect in mouse lungs.

To elucidate the little-known bioenergetic pathways of host immune cells in tuberculosis, a granulomatous disease caused by the intracellular pathogen Mycobacterium tuberculosis, we characterized infected murine lung tissue by transcriptomic profiling and confocal imaging. Transcriptomic analysis revealed changes of host energy metabolism during the course of infection that are characterized by upregulation of key glycolytic enzymes and transporters for glucose uptake, and downregulation of enzymes participating in the tricarboxylic acid cycle and oxidative phosphorylation. Consistent with elevated glycolysis, we also observed upregulation of a transporter for lactate secretion and a V type H(+) -ATPase involved in cytosolic pH homeostasis. Transcription profiling results were corroborated by immunofluorescence microscopy showing increased expression of key glycolytic enzymes in macrophages and T cells in granulomatous lesions. Moreover, we found increased mRNA and protein levels in macrophages and T cells of hypoxia inducible factor 1 alpha (HIF-1α), the regulatory subunit of HIF-1, a master transcriptional regulator. Thus, our findings suggest that immune cells predominantly utilize aerobic glycolysis in response to M. tuberculosis infection. This bioenergetic shift is similar to the Warburg effect, the metabolic signature of cancer cells. Finding immunometabolic changes during M. tuberculosis infection opens the way to new strategies for immunotherapy against tuberculosis.

Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status.

RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. We tested whether antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI), a condition affecting one-third of the world population. Peripheral-blood mononuclear cells from donors, identified as positive or negative for LTBI by current medical practice, were stimulated ex vivo with mycobacterial antigen. IFNG and IL2 mRNA production was assayed by FISH-Flow. Concurrently, immunophenotypes of the cytokine mRNA-positive cells were characterized by conventional, antibody-based staining of cell-surface markers. An association was found between donor LTBI status and antigen-specific induction of IFNG and IL2 transcripts. Induction of these cytokine genes, which was detected by FISH-Flow in a quarter the time required to see release of the corresponding proteins by ELISA, occurred primarily in activated CD4+ T cells via T-cell receptor engagement. Moreover, NK cells contributed to IFNG gene induction. These results show that antigen-driven induction of T-cell cytokine mRNA is a measurable single-cell parameter of the host responses associated with latent tuberculosis. FISH-Flow read-outs contribute a multi-scale dimension to the immunophenotyping afforded by antibody-based flow cytometry. Multi-scale, single-cell analyses may satisfy the need to determine disease stage and therapy response for tuberculosis and other infectious pathologies.

IRF and tuberculosis.

Tuberculosis is the most prevalent infectious disease and causes more deaths than any other, yet only 5%-10% of people infected by the causative agent, Mycobacterium tuberculosis, will develop the disease. Thus, natural resistance among humans is the norm. Fundamental immune responses to M. tuberculosis are being elucidated, including induction of interferon regulatory factor-1 (IRF-1). Moreover, IRF-1 has been found necessary for normal resistance to infection by mycobacteria in mice. Roles for IRF-1 in a plethora of immune system functions have been described. This review considers molecular responses to infection by M. tuberculosis that might account for induction of IRF-1 and highlights putative connections between immunomodulatory functions of IRF-1 and immune responses relevant to infection by M. tuberculosis. However, the complexity inherent in pleiotropy and redundancy limits the ability to draw firm conclusions. In many cases, it remains to be demonstrated that a particular function of IRF-1 is the basis for a known response to infection. For example, although IRF-1 is required for a Th1 cell-mediated, adaptive immune response in some circumstances, it is not known if the Th1 response to infection by M. tuberculosis requires IRF-1. Conversely, some known contributions by IRF-1 to fundamental aspects of the immune system are not yet proven relevant in the host response to infection. For example, it is not known if control of T cell subset development by IRF-1 is significant for host defense against M. tuberculosis. Functions of other IRF that overlap with or are distinct from the functions of IRF-1 also could be important for the immune response to M. tuberculosis.

Mycobacterium tuberculosis sigma factor E regulon modulates the host inflammatory response.

Mycobacterium tuberculosis survives in macrophages and usually subverts the bactericidal mechanisms of these phagocytes. The understanding of this host-pathogen interaction is relevant for the development of new treatments for tuberculosis. The adaptation of M. tuberculosis to intracellular life depends on its ability to regulate the expression of its genes. Sigma factors are important bacterial transcription activators that bind to the RNA polymerase and give it promoter specificity. Sigma factor E (SigE) controls the expression of genes that are essential for virulence. We have identified the SigE regulon during infection of macrophages, and we analyzed the impact of this regulon on the transcriptional response of phagocytes. Our results indicate that SigE regulates the expression of genes involved in the maintenance of M. tuberculosis cell envelope integrity and function during macrophage infection. Analysis of the phagocytes' transcriptional response indicates that the SigE regulon is involved in the modulation of the inflammatory response.

Mechanisms of polymorphonuclear neutrophil-mediated induction of HIV-1 replication in macrophages during pulmonary tuberculosis.

BACKGROUND: Pulmonary tuberculosis (TB) can present with polymorphonuclear neutrophil (PMN)-predominant alveolitis. TB accelerates acquired immunodeficiency syndrome by increasing human immunodeficiency virus type 1 (HIV-1) replication and mutation in alveolar macrophages. A 16-kDa CCAAAT/enhancer-binding protein beta (C/EBP beta ) isoform is a strong transcriptional repressor of the HIV long terminal repeat (LTR) in resting alveolar macrophages, leading to latent viral infection; its expression is lost during TB, derepressing the HIV LTR. METHODS: Lung segments were sampled from HIV/Mycobacterium tuberculosis-coinfected patients by means of bronchoalveolar lavage. In vitro coculture experiments defined the mechanism of induction of HIV-1 infection in macrophages by PMNs. RESULTS: Lung segments from patients with PMN-predominant TB had a markedly elevated viral load. Direct contact between activated PMNs and macrophages stimulated HIV-1 replication and LTR transcription and down-regulated inhibitory C/EBP beta . Isolated PMN membranes substituted for PMN contact, derepressing the HIV-1 LTR. The lipid raft fraction of PMN membranes expressed CD40 ligand (CD40L), CD28, and leukocyte function-associated antigen 1 (LFA-1 [i.e., CD11a and CD18]), and PMN activation increased lipid raft expression of CD40L and CD28. Blocking antibodies to CD40L, CD28, and LFA-1 inhibited PMN membrane-mediated HIV-1 LTR derepression. Alternately, cross-linking of macrophage receptors for CD40L, CD28, and LFA-1 (CD40, CD80/86, and intercellular adhesion molecule 1) abolished inhibitory C/EBP beta expression. CONCLUSION: PMN-macrophage contact derepresses the HIV-1 LTR and enhances HIV-1 replication in alveolar macrophages during pulmonary TB. Derepression is mediated through costimulatory molecule signaling.

IFN-alpha beta secreted during infection is necessary but not sufficient for negative feedback regulation of IFN-alpha beta signaling by Mycobacterium tuberculosis.

IFN-alphabeta functions in the transition from innate to adaptive immunity and may impinge on the interaction of Mycobacterium tuberculosis with its host. Infection by M. tuberculosis causes IFN-alphabeta secretion and down-regulation of IFN-alphabeta signaling in human APC and the human monocytic cell line THP-1, which provides a model for these studies. Neutralization of secreted IFN-alphabeta prevents inhibition of IFN-alpha signaling during infection, but several lines of evidence distinguish inhibition due to infection from a negative feedback response to only IFN-alphabeta. First, greater inhibition of IFN-alpha-stimulated STAT-1 tyrosine phosphorylation occurs 3 days postinfection than 1 or 3 days after IFN-alphabeta pretreatment. Second, LPS also induces IFN-alphabeta secretion and causes IFN-alphabeta-dependent down-regulation of IFN-alpha signaling, yet the inhibition differs from that caused by infection. Third, IFN-alpha signaling is inhibited when cells are grown in conditioned medium collected from infected cells 1 day postinfection, but not if it is collected 3 days postinfection. Because IFN-alphabeta is stable, the results with conditioned medium suggest the involvement of an additional, labile substance during infection. Further characterizing signaling for effects of infection, we found that cell surface IFN-alphabeta receptor is not reduced by infection, but that infection increases association of protein tyrosine phosphatase 1c with the receptor and with tyrosine kinase 2. Concomitantly, IFN-alpha stimulation of tyrosine kinase 2 tyrosine phosphorylation and kinase activity decreases in infected cells. Moreover, infection reduces the abundance of JAK-1 and tyrosine-phosphorylated JAK-1. Thus, the distinctive down-regulation of IFN-alpha signaling by M. tuberculosis occurs together with a previously undescribed combination of inhibitory intracellular events.

Interleukin-10 induces inhibitory C/EBPbeta through STAT-3 and represses HIV-1 transcription in macrophages.

Pulmonary tuberculosis (TB) has been characterized by inflammation with increased pro- or anti-inflammatory cytokines produced by macrophages. We have reported that IFN produces inhibitory C/EBPbeta and represses transcription of the HIV-1 LTR in macrophages. STAT-1 and type I IFN receptor knockout mice have macrophages that are defective in IFN signaling, yet LPS stimulation induces inhibitory C/EBPbeta, demonstrating that other cytokines can induce this repressor. LPS or Mycobacterium tuberculosis-derived lipoarabinomannan induce the anti-inflammatory cytokine interleukin (IL)-10, which represses the HIV-1 LTR in differentiated THP-1 macrophages by inducing inhibitory C/EBPbeta. In contrast, in undifferentiated THP-1 monocytes, IL-10 did not inhibit HIV-1 replication or induce C/EBPbeta. IL-10 signal transduction uses STAT-3, and macrophages from STAT-3-/- mice fail to produce inhibitory C/EBPbeta after LPS or IL-10 stimulation. Transfection of STAT-3 into THP-1 cells enhances C/EBPbeta promoter activity. THP-1 differentiation also increases STAT-3 protein, but not STAT-3 gene transcription, and induces a translational regulator, CUG-binding protein, that was essential for production of C/EBPbeta. Differentiation induced post-transcriptional regulation is required to produce inhibitory C/EBPbeta in response to IL-10. Only macrophages are able to repress HIV-1 LTR promoter activity and inhibit viral replication in response to IL-10 or type I IFN.