Richard Pine

Regulation of LMP2 and TAP1 Genes by IRF-1 Explains the Paucity of CD8+ T Cells in IRF-1−/− Mice

The TAP1 and LMP2 genes are central for class I MHC function and share a common promoter. Here, we analyze the molecular mechanism of IFN gamma up-regulation of TAP1 and LMP2. In vivo footprinting indicates IFN gamma up-regulates protein-DNA contacts at an IRF-E that is essential for the up-regulation of TAP1 and LMP2 by IFN gamma. Gel shift analysis indicates that this site binds IRF-1. The expression of TAP1 and LMP2 are both greatly reduced in IRF-1-deficient mice. Surface class I MHC as well as CD8+ T cells are reduced in IRF-1-/- mice. The role of IRF-1 in the regulation of TAP1 and LMP2 suggests a mechanism for the antiviral properties of IRF-1 and the unexpected deficiency of CD8+ T cells observed in IRF-1-/- mice.

Differentiation of Monocytes to Macrophages Switches the Mycobacterium tuberculosis Effect on HIV-1 Replication from Stimulation to Inhibition: Modulation of Interferon Response and CCAAT/Enhancer Binding Protein β Expression

HIV-1 replication is inhibited in uninflamed lung macrophages and is stimulated during tuberculosis. Attempts to recapitulate activation of HIV-1 replication in primary monocytes and macrophages ex vivo and in the untreated and PMA-treated THP-1 cell line model in vitro have produced opposite results depending on the state of differentiation of the cells. After infection with Mycobacterium tuberculosis, monocytes enhanced HIV-1 replication and produced a stimulatory 37-kDa CCAAT/enhancer binding protein β (C/EBPβ) transcription factor, whereas macrophages suppressed HIV-1 replication and produced an inhibitory 16-kDa C/EBPβ transcription factor. IFN-β induced inhibitory 16-kDa C/EBPβ in macrophages, but had no effect on C/EBPβ expression in monocytes. Macrophages, but not monocytes, were able to activate IFN-stimulated gene factor-3 (ISGF-3), a transcription factor composed of STAT-1, STAT-2, and IFN regulatory factor (IRF)-9, after infection with M. tuberculosis or stimulation with type I IFN. Macrophages expressed IRF-9 DNA-binding activity, but monocytes did not, and addition of the IRF-9 component reconstituted ISGF-3 in extracts of IFN-treated monocytes. Modulation of IFN responsiveness upon differentiation occurred at least in part through a post-transcriptionally regulated increase in IRF-9 expression. Both monocytes and macrophages maintained IFN responsiveness, activating STAT-1 homodimer formation and transcription of the STAT-1 gene after IFN stimulation. In addition, both monocytes and macrophages were able to activate NF-κB upon infection with M. tuberculosis. These results show that induction of ISGF-3, expression of the inhibitory 16-kDa C/EBPβ, and suppression of HIV-1 replication via a transcriptional mechanism are macrophage-specific responses to infection with M. tuberculosis.

The SH2 Domain-containing Tyrosine Phosphatase PTP1D Is Required for Interferon α/β-induced Gene Expression

Interferons (IFNs) induce early response genes by stimulating Janus family (Jak) tyrosine kinases, leading to tyrosine phosphorylation of Stat (signal transducer and activator of transcription) proteins. Previous studies demonstrated that a protein-tyrosine phosphatase (PTP) is required for activation of the ISGF3 transcription complex by IFNα/β, but the specific PTP responsible remained unidentified. We now show that the SH2 domain containing tyrosine phosphatase PTP1D (also designated as SHPTP2, SHPTP3, PTP2C, or Syp) is constitutively associated with the IFNα/β receptor and becomes tyrosine-phosphorylated in response to ligand. Furthermore, transient expression of a phosphatase-inactive mutant or the COOH-terminal SH2 domain of PTP1D causes a dominant negative effect on IFNα/β-induced early response gene expression. These results provide strong evidence that PTP1D functions as a positive regulator of the IFNα/β-induced Jak/Stat signal transduction pathway.

Mycobacterium tuberculosis-Induced CXCR4 and Chemokine Expression Leads to Preferential X4 HIV-1 Replication in Human Macrophages

Opportunistic infections such as pulmonary tuberculosis (TB) increase local HIV-1 replication and mutation. As AIDS progresses, alteration of the HIV-1 gp120 V3 sequence is associated with a shift in viral coreceptor use from CCR5 (CD195) to CXCR4 (CD184). To better understand the effect of HIV/TB coinfection, we screened transcripts from bronchoalveolar lavage cells with high density cDNA arrays and found that CXCR4 mRNA is increased in patients with TB. Surprisingly, CXCR4 was predominately expressed on alveolar macrophages (AM). Mycobacterium tuberculosis infection of macrophages in vitro increased CXCR4 surface expression, whereas amelioration of disease reduced CXCR4 expression in vivo. Bronchoalveolar lavage fluid from TB patients had elevated levels of CCL4 (macrophage inflammatory protein-1β), CCL5 (RANTES), and CX3CL1 (fractalkine), but not CXCL12 (stromal-derived factor-1α). We found that M. tuberculosis infection of macrophages in vitro increased viral entry and RT of CXCR4, using HIV-1, but not of CCR5, using HIV-1. Lastly, HIV-1 derived from the lung contains CD14, suggesting that they were produced in AM. Our results demonstrate that TB produces a permissive environment for replication of CXCR4-using virus by increasing CXCR4 expression in AM and for suppression of CCR5-using HIV-1 by increasing CC chemokine expression. These changes explain in part why TB accelerates the course of AIDS. CXCR4 inhibitors are a rational therapeutic approach in HIV/TB coinfection.

Recombinant Gamma Interferon Stimulates Signal Transduction and Gene Expression in Alveolar Macrophages In Vitro and in Tuberculosis Patients

ABSTRACT Tuberculosis is the seventh leading cause of morbidity and mortality in the world, with eight million cases per year. Animal and human studies demonstrate an enrichment of CD4 cells at sites of disease, with a more favorable clinical course when there is a Th1 response with the presence of gamma interferon (IFN-γ). We previously treated patients who had multidrug-resistant tuberculosis with recombinant IFN-γ (rIFN-γ) in aerosol form and were able to convert smear-positive cases to smear negative with 12 treatments over 1 month. We hypothesized that rIFN-γ would induce signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) binding activity in alveolar macrophages (AM). AM treated in vitro showed clear upregulation of STAT-1 and IRF-1 by rIFN-γ. STAT-1 was not activated and IRF-1 was only weakly induced after 1 day of infection by Mycobacterium tuberculosis TN913. In bronchoalveolar lavage (BAL) cells obtained from 10 of 10 tuberculosis patients 10 ± 2 days post-antituberculosis treatment, there was no detectable STAT-1 or IRF-1 DNA-binding activity. After 4 weeks of treatment with rIFN-γ aerosol in addition to the antituberculosis drugs, 10 of 10 patients had increased STAT-1, IRF-1, and/or IRF-9 DNA-binding activity in BAL cells from lung segments shown radiographically to be involved and in those shown to be uninvolved. Symptoms and chest radiographs improved, and amounts of macrophage inflammatory cytokines and human immunodeficiency virus type 1 (HIV-1) viral loads (in five of five HIV-1-coinfected patients) declined in the second BAL specimens. rIFN-γ aerosol induces signal transduction and gene expression in BAL cells and should be evaluated for efficacy in a randomized, controlled clinical trial.

Aerosolized Gamma Interferon (IFN-γ) Induces Expression of the Genes Encoding the IFN-γ-Inducible 10-Kilodalton Protein but Not Inducible Nitric Oxide Synthase in the Lung during Tuberculosis

ABSTRACT Gamma interferon (IFN-γ) is critical in the immune response against Mycobacterium tuberculosis. In an ongoing trial of aerosol IFN-γ in conjunction with standard drug therapy, we have observed activation of IFN signaling in bronchoalveolar lavage (BAL) cells from tuberculosis (TB) patients. We hypothesized that aerosol IFN-γ treatment of pulmonary TB would increase expression of genes important for the control of TB. We investigated the expression of downstream genes by measuring inducible nitric oxide synthase (iNOS) and the chemokine IFN-inducible 10-kDa protein (IP-10) by real-time quantitative reverse transcription-PCR. In vitro, M. tuberculosis induced IP-10, and IFN-γ stimulated this further, with no effect on iNOS expression. We studied 21 patients with pulmonary TB and 7 healthy subjects. Similar to the in vitro model, IP-10 mRNA was increased in BAL cells from TB patients and was augmented after treatment with aerosolized IFN-γ. TB was also associated with elevated iNOS mRNA, but aerosolized IFN-γ did not further enhance expression. Genomic analysis identified 1,300 of 4,058 genes expressed in BAL cells from six TB patients before and after 1 month of therapy, including aerosolized IFN-γ. However, only 15 genes were differentially regulated by IFN-γ. We conclude that iNOS and IP-10 mRNA expression is increased in TB but that aerosol IFN-γ treatment increases expression of few genes in the human lung.

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.

Inhibition of Response to Alpha Interferon by Mycobacterium tuberculosis

ABSTRACT We previously reported that infection by Mycobacterium tuberculosis, the causative agent of tuberculosis, leads to secretion of alpha/beta interferon (IFN-α/β). While IFN-α/β ordinarily stimulates formation of signal transducer and stimulator of transcription-1 (STAT-1) homodimers and IFN-stimulated gene factor-3 (ISGF-3), only ISGF-3 is found in infected human monocytes and macrophages. We have now investigated the basis for this unusual profile of transcription factor activation and its consequences for regulation of transcription, as well as the impact of infection on response to IFN-α. After infection, IFN-α stimulation of STAT-1 homodimers is inhibited in monocytes and macrophages, while stimulation of ISGF-3 increases in monocytes but tends to decline in macrophages. Effects of infection on the abundance of ISGF-3 subunits, STAT-1, STAT-2, and interferon regulatory factor 9, and on tyrosine phosphorylation of STAT-1 and STAT-2 explain the observed changes in DNA-binding activity, which correlate with increased or inhibited transcription of genes regulated by ISGF-3 and STAT-1. Infection by Mycobacterium bovis BCG does not inhibit IFN-α-stimulated tyrosine phosphorylation of STAT-1, formation of homodimers, or transcription of genes regulated by STAT-1 homodimers, suggesting that inhibition of the response to IFN-α/β by M. tuberculosis is an aspect of pathogenicity. Thus, this well-known feature of infection by pathogenic viruses may also be a strategy employed by pathogenic bacteria.

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.

Proteasome- and p53-dependent Masking of Signal Transducer and Activator of Transcription (STAT) Factors

Hepatoma Hep3B cell lines stably expressing a temperature-sensitive p53 species (p53-Val-135) displayed a reduced response to interleukin-6 (IL-6) when cultured at the wild-type (wt) p53 temperature (Wang, L., Rayanade, R., Garcia, D., Patel, K., Pan, H., and Sehgal, P. B. (1995) J. Biol. Chem. 270, 23159-23165). We now report that in such cultures IL-6 caused a rapid (20-30 min) and marked loss of cellular immunostaining for STAT3 and STAT5, but not for STAT1. The loss of STAT3 and STAT5 immunostaining was transient (lasted 120 min) and tyrosine kinase-dependent, and even though the loss was blocked by the proteasome inhibitors MG132 and lactacystin it was not accompanied by changes in cellular levels of STAT3 and STAT5 proteins suggesting that IL-6 triggered a rapid masking but not degradation of these transcription factors. STAT3 and STAT5 masking was accompanied by a reduction in IL-6-induced nuclear DNA-binding activity. The data suggest that p53 may influence Jak-STAT signaling through a novel indirect mechanism involving a wt p53-dependent gene product which upon cytokine addition is activated into a “STAT-masking factor” in a proteasome-dependent step.