Systemic immune activation and microbial translocation in dual HIV/tuberculosis-infected subjects.

BACKGROUND: Systemic immune activation is a strong predictor of progression of human immunodeficiency virus type 1 (HIV-1) disease and a prominent feature of infection with Mycobacterium tuberculosis. OBJECTIVE: To understand the role of systemic immune activation and microbial translocation in HIV/tuberculosis dually infected patients over the full spectrum of HIV-1 immunodeficiency, we studied circulating sCD14 and lipopolysaccharide (LPS) and their relationship to HIV-1 activity. METHODS: Two cohorts of HIV/tuberculosis subjects defined by CD4 T-cell count at time of diagnosis of tuberculosis were studied: those with low (<350/µL) and those with high (≥ 350/µL) CD4 T-cell count. Circulating soluble CD14 (sCD14) and LPS were assessed. RESULTS: Levels of sCD14 were higher in HIV/tuberculosis with high (≥ 350/µL) as compared to low CD4 T-cell count (P < .001). Whereas sCD14 levels remained elevated in HIV/tuberculosis subjects with lower CD4 T-cell counts despite treatment of tuberculosis, in HIV/tuberculosis patients with higher CD4 T-cell count (≥ 350/µL), levels declined regardless of whether highly active antiretroviral therapy (HAART) was included with the anti-tuberculosis regimen. Circulating LPS levels in HIV/tuberculosis patients with CD4 T-cell count ≥ 350/µL were unaffected by treatment of tuberculosis with or without HAART. CONCLUSION: During HIV/tuberculosis, systemic immune activation is dissociated from microbial translocation. Changes in circulating sCD14 and LPS are dependent on CD4 T-cell count.

Induction of serine protease inhibitor 9 by Mycobacterium tuberculosis inhibits apoptosis and promotes survival of infected macrophages.

Our recent microarray analysis of infected human alveolar macrophages (AMs) found serine protease inhibitor 9 (PI-9) to be the most prominently expressed of a cluster of apoptosis-associated genes induced by virulent Mycobacterium tuberculosis. In the current study, we show that induction of PI-9 occurs within hours of infection with M. tuberculosis H37Rv and is maintained through 7 days of infection in both AMs and blood monocytes. Inhibition of PI-9 by small inhibitory RNA decreased M. tuberculosis-induced expression of the antiapoptotic molecule Bcl-2 and resulted in a corresponding increase in production of caspase 3, a terminal effector molecule of apoptosis. Further, PI-9 small inhibitory RNA mediated a significant reduction in the subsequent survival of M. tuberculosis within AMs. Thus PI-9 induction within human mononuclear phagocytes by virulent M. tuberculosis serves to protect these primary targets of infection from elimination by apoptosis and thereby promotes intracellular survival of the organism.

Dynamic variation in the cellular origin of HIV type 1 during treatment of tuberculosis in dually infected subjects.

HIV-1 replication remains elevated in dually infected HIV-1/TB subjects at completion of antituberculosis therapy. A viral immunocapture assay was used to examine the cellular origin of HIV-1 within plasma from HIV-1/TB subjects at time of diagnosis of pulmonary TB, at end of TB treatment, and 6 months after completion of treatment. Asymptomatic HIV-1-infected subjects without TB (HIV-1/C) served as controls. Both activated immature macrophage (CD36(+)) and CD4 T cell (CD26(+)) compartments contributed to viral load. Changes in the activation status of either cellular compartment paralleled their contribution to viral load. Levels of HIV-1 originating from activated (HLA-DR(+)) cells and from CD36(+) and CD26(+) mononuclear cells resolved to levels observed in HIV-1/C by the end of treatment. HIV-1 isolated by anti-CD3 immunocapture from HIV-1/TB patients remained significantly higher than from HIV-1/C patients at the end of TB treatment and at 12 months follow-up. Therefore, viral production by lymphocytes extends well beyond the completion of TB treatment.

Mycobacterium tuberculosis Induces Expansion of Foxp3 Positive CD4 T-cells with a Regulatory Profile in Tuberculin Non-sensitized Healthy Subjects: Implications for Effective Immunization against TB.

OBJECTIVE: Infection by MTB or exposure to MTB constituents is associated with intense microbial stimulation of the immune system, through both antigenic and TLR components, and induction of a milieu that is rich in pro-inflammatory/anti-inflammatory cytokines. Here, we addressed the basis of induced regulatory T-cell (iT-reg) expansion in response to MTB stimulation, in the absence of prior T cell antigen responsiveness. METHODS: PBMC from HIV-1 un-infected TST negative and TST positive control subjects were stimulated by virulent MTB H37Rv lysate (L), a French press preparation of MTB that includes all bacterial components. Phenotype of MTB H37RvL induced iT-reg was assessed using immunostaining and flow cytometry. Functional capacity of iT-reg was assessed using 3H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in the presence or absence of iT-reg in corresponding culture supernatants in response to TCR stimulation. Realtime PCR was used to assess IDO and FoxP3 mRNA expression. RESULTS: The capacity of MTB H37RvL to induce CD4+CD25hi+ Foxp3+ T-cells in PBMC from TST negative subjects was robust (p<0.001), and in fact comparable to induction of iT-reg in PBMC from TST positive subjects. MTB-induced CD4+CD25hi+ T-reg were TGFß positive (p<0.05). Further, MTB H37RvL induced CD4+CD25hi+ Foxp3+ iT-reg suppressed 3H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in response to TCR stimulation. MTB H37RvL induction of iT-reg was significantly stronger (p<0.01) than that by TLR-2, TLR-4, TLR-9 ligands, or combination of all TLR ligands. MTB H37RvL inducted indoleamine 2,3-dideoxygenase (IDO) mRNA expression in monocytes (p<0.001), and co-culture with the IDO inhibitor, D-1MT, decreased frequencies of T-reg (p<0.05). Inhibition of TGFß by siRNA reduced Foxp3 mRNA expression in CD4 T cells (p<0.05). CONCLUSION: Therefore, MTB and its components expand functional iT-reg in human mononuclear cells from MTB non-sensitized subjects. Also, MTB-induced iT-reg expansion depends on mononuclear phagocyte expression of both TGFß and IDO.

Expansion and productive HIV-1 infection of Foxp3 positive CD4 T cells at pleural sites of HIV/TB co-infection.

BACKGROUND: CD4 T-cells expressing Foxp3 are expanded systemically during active tuberculosis (TB) regardless of HIV-1 co-infection. Foxp3+ CD4 T cells are targets of HIV-1 infection. However, expansion of HIV-1 infected Foxp3+ CD4 T cells at sites of HIV/TB co-infection, and whether they contribute to promotion of HIV-1 viral activity is not known. METHODS: Pleural fluid mononuclear cells (PFMC) from HIV/TB co-infected patients with pleural TB were characterized by immune-staining and FACS analysis for surface markers CD4, CD127, CCR5, CXCR4, HLA-DR and intracellular expression of Foxp3, HIVp24, IFN-γ and Bcl-2. Whole PFMC and bead separated CD4+CD25+CD127- T cells were assessed for HIV-1 LTR strong stop (SS) DNA by real-time PCR, which represents viral DNA post cell entry and initiation of reverse transcription. RESULTS: High numbers of HIV-1 p24 positive Foxp3+ and Foxp3+CD127- CD4 T cells were identified in PFMC from HIV/TB co-infected subjects. CD4+Foxp3+CD127- T cells displayed high expression of the cellular activation marker, HLA-DR. Further, expression of the HIV-1 co-receptors, CCR5 and CXCR4, were higher on CD4+Foxp3+T cells compared to CD4+Foxp3- T cells. Purified CD4+CD25+CD127- T cells isolated from PFMC of HIV/TB co-infected patients, were over 90% CD4+Foxp3+T cells, and exhibited higher HIV-1 SS DNA as compared to whole PFMC, and as compared to CD4+CD25+CD127- T cells from an HIV-infected subject with pleural mesothelioma. HIV-1 p24+ Foxp3+ CD4+T cells from HIV/TB patients higher in Bcl-2 expression as compared to both HIV-1 p24+ Foxp3- CD4 T cells, and Foxp3+ CD4+T cells without HIV-p24 expression. CONCLUSION: Foxp3+ CD4 T cells in PFMC from HIV/TB co-infected subjects are predisposed to productive HIV-1 infection and have survival advantage as compared to Foxp3 negative CD4 T cells.

Short Communication: Expression of APOBEC3G and Interferon Gamma in Pleural Fluid Mononuclear Cells from HIV/TB Dual Infected Subjects.

Sites of HIV/TB coinfection are characterized by increased HIV-1 replication and a TH1 profile. However, expression of HIV-1 restriction factors, such as APOBEC3G (A3G) in situ, is unknown. Using an RT-profiler focused on genes related to HIV-1 expansion, we examined pleural fluid mononuclear cells (PFMCs) from patients with HIV/TB coinfection in comparison to HIV-uninfected patients with TB disease. Significant expression of interferon (IFN)-γ and restriction factors A3G and A3F and TRIM5α in PFMCs was found. Genes correlating significantly with the expression of IFN-γ included A3G and A3F. However, pleural fluid HIV-1 viral load and HIV-1 gag/pol mRNA in PFMCs did not correlate with A3G activity.

Aspiration pneumonia. Recognizing and managing a potentially growing disorder.

Gross aspiration of liquid or particulate matter into the lung can result in severe hypoxemia, pulmonary infiltrates in dependent lung regions, fever, and leukocytosis. The initial lung injury is primarily due to inflammatory mediators rather than infection. The responsible bacterial pathogens differ between community-acquired and nosocomial aspiration pneumonia. Many aspiration pneumonias are mixed aerobic-anaerobic infections. Enteric gram-negative bacilli and S aureus are more common in nosocomial aspiration pneumonia. Current treatment guidelines support initial empirical antibiotic therapy in patients with severe aspiration pneumonia pending culture results. Appropriate initial treatment improves outcome. Antimicrobial therapy for aspiration pneumonia is often empirical and should be based on patient characteristics, the setting in which aspiration occurred, the severity of pneumonia, and available information regarding local pathogens and resistance patterns.

Short communication: circulating plasma HIV-1 viral protein R in dual HIV-1/tuberculosis infection.

Circulating free HIV-1 viral protein R (Vpr) is found in up to one third of subjects with HIV-1 infection. Free Vpr presumably shares some of the immunopathogenic effects of cell-associated Vpr. Here we assessed Vpr in plasma and pleural fluid from HIV/tuberculosis (TB) dually infected subjects with pleural TB and from plasma of patients with pulmonary HIV/TB. Vpr was assessed by western blot analysis. In plasma from HIV/TB subjects with pulmonary TB free Vpr could be detected in 47%. Only one subject, among 26 tested, with HIV monoinfection showed plasma Vpr activity. The majority (87.5%) of patients with pleural HIV/TB demonstrated free Vpr reactivity in their plasma. However, no Vpr activity was found in autologous pleural fluid samples from pleural HIV/TB patients. Standard (s) Vpr reactivity was reduced markedly by the addition of sVpr to pleural fluid from HIV-uninfected subjects. A high incidence of plasma Vpr reactivity in HIV/TB patients implies heightened processing and release of this HIV-1 accessory protein during HIV/TB coinfection. The contribution of free Vpr to HIV-1 immunopathogenesis during HIV/TB needs to be studied.

Role of protease inhibitor 9 in survival and replication of Mycobacterium tuberculosis in mononuclear phagocytes from HIV-1-infected patients.

OBJECTIVE AND DESIGN: Predisposition to opportunistic infections by Mycobacterium tuberculosis (MTB) is a concomitant of HIV-1 infection and occurrence of tuberculosis is independent of circulating CD4(+) T-cell count in HIV-1-infected patients. Infection of mononuclear phagocytes from healthy individuals by virulent MTB is associated with expression of the antiapoptotic molecule protease inhibitor 9 (PI-9), and PI-9 contributes to successful parasitism of macrophages by MTB. Here we studied the contribution of PI-9 to successful MTB infection of monocytes from HIV-1-infected patients. METHODS: Blood monocytes obtained from HAART-treated HIV-1-infected patients (HIV+) and healthy controls were assessed for support of MTB H37Rv growth by assessment of MTB 16S ribosomal (r)RNA in cell lysates on day 1 and day 7 by real-time reverse transcription-PCR. PI-9 expression in monocyte cell lysates was assessed by ELISA and by reverse transcription-PCR. Inhibition of intracellular PI-9 was achieved by siRNA to PI-9 and compared to control constructs. RESULTS: Monocytes from HIV-infected patients supported higher MTB growth [MTB 16S rRNA (d7/d1)] as compared with monocytes from healthy controls. Both PI-9 protein and mRNA were significantly higher in monocytes from HIV-infected patients as compared with healthy controls. PI-9 protein levels prior to MTB infection correlated with MTB replication on day 7, and with plasma soluble CD14 levels. Silencing of PI-9 by transfection of monocytes from HIV-1-infected patients with PI-9-specific siRNA prior to infection improved intracellular containment of MTB. CONCLUSION: Increased intracellular PI-9 activity in mononuclear phagocytes from HIV-infected patients contributes to successful intracellular infection by virulent MTB.

Activation of P-TEFb at sites of dual HIV/TB infection, and inhibition of MTB-induced HIV transcriptional activation by the inhibitor of CDK9, Indirubin-3'-monoxime.

At sites of Mycobacterium tuberculosis (MTB) infection, HIV-1 replication is increased during tuberculosis (TB). Here we investigated the role of positive transcription elongation factor (P-TEFb), comprised of CycT1 and CDK9, as the cellular cofactor of HIV-1 Tat protein in transcriptional activation of HIV-1 in mononuclear cells from HIV-1-infected patients with pleural TB. Expression of CycT1 in response to MTB was assessed in mononuclear cells from pleural fluid (PFMC) and blood (PBMC) from HIV/TB patients with pleural TB, and in blood monocytes (MN) from singly infected HIV-1-seropositive subjects. We then examined whether the CDK9 inhibitor, Indirubin 3'-monoxime (IM), was effective in inhibition of MTB-induced HIV-1 mRNA expression. We found higher expression of CycT1 mRNA in PFMCs as compared to PBMCs from HIV/TB-coinfected subjects. MTB induced the expression of CycT1 and HIV-1 gag/pol mRNA in both PFMCs from HIV/TB subjects and MN from HIV-1-infected subjects. CycT1 protein was also induced by MTB stimulation in PFMCs from HIV/TB patients, and both MN and in vitro-derived macrophages. Inhibition of CDK9 by IM in both PFMCs from HIV/TB and MN from HIV-1-infected subjects in response to MTB led to inhibition of HIV-1 mRNA expression. These data imply that IM may be useful as an adjunctive therapy in control of HIV-1 replication in HIV/TB dually infected subjects.

Mechanisms of apoptosis of T-cells in human tuberculosis.

The role of TGF-beta TNF-alpha FasL and Bcl-2 in apoptosis of CD4 T-cells during active TB was studied. Coculture of PBMC from TB patients with neutralizing antibodies to TGF-beta or TNF-alpha decreased spontaneous (P < or = 0.05) and MTB-induced (P < or = 0.02) T-cell apoptosis by 50-90%, but effects were not additive. Interestingly, only levels of TGF-beta in supernatants correlated with rates of spontaneous and MTB-induced apoptosis. FasL surface and mRNA expression were higher in unstimulated and MTB-stimulated PBMC from patients than controls, and neutralization of FasL abrogated apoptosis of T-cells from patients only. Intracellular Bcl-2 protein was lower among unstimulated CD4 T-cells from patients than those from controls (P < or = 0.02), and MTB stimulation reduced intracellular Bcl-2 content in CD4 T-cells from patients only (P < or = 0.001). These findings may indicate that, during TB, predisposition of CD4 T-cells to apoptosis may involve both low expression of Bcl-2, and excessive expression of TGF-beta TNF-alpha and FasL.

Role of cellular activation and tumor necrosis factor-alpha in the early expression of Mycobacterium tuberculosis 85B mRNA in human alveolar macrophages.

BACKGROUND: Infection of alveolar macrophages (AMs), which constitute the first line of defense against Mycobacterium tuberculosis, initiates an intense interaction between the host's innate immune response and mycobacteria that may assist in the successful intracellular parasitism of M. tuberculosis. METHODS: Expression of tumor necrosis factor (TNF)- alpha and M. tuberculosis 85B mRNA was studied in M. tuberculosis-infected AMs, to better delineate the role of macrophages in the early events in initiation of infection. RESULTS: Both TNF- alpha mRNA and M. tuberculosis 85B were induced in AMs; at 24 h, the time point of maximum TNF- alpha induction, the mRNA levels for TNF- alpha and M. tuberculosis 85B correlated with one another, and induction of either gene correlated strongly with their protein levels. Inhibition of endogenous TNF- alpha by soluble (s) TNF receptor (R) I and sTNFRII reduced expression of both TNF- alpha and M. tuberculosis 85B. The activation of nuclear factor- kappa B was found to underlie expression of both TNF- alpha and M. tuberculosis 85B. Exogenous TNF- alpha was slightly more potent than interleukin (IL)-6 and granulocyte-macrophage colony-stimulating factor and was significantly stronger than IL-1 in inducing expression of M. tuberculosis 85B. Interestingly, inhibition of bactericidal mediators, reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs), reduced expression of TNF- alpha and M. tuberculosis 85B genes in M. tuberculosis-infected AMs. CONCLUSION: Activation of AMs by M. tuberculosis initiates a cascade of events whereby TNF- alpha, ROI, and RNI enhance the expression of the M. tuberculosis 85B gene.

Randomized trial of adjunctive interleukin-2 in adults with pulmonary tuberculosis.

Interleukin (IL)-2 has a central role in regulating T cell responses to Mycobacterium tuberculosis. Adjunctive immunotherapy with recombinant human IL-2 was studied in a randomized, placebo-controlled, double-blinded trial in 110 human immunodeficiency virus-seronegative adults in whom smear-positive, drug-susceptible pulmonary tuberculosis was newly diagnosed. Patients were randomly assigned to receive twice-daily injections of 225, 000 IU of IL-2 or placebo for the first 30 days of treatment in addition to standard chemotherapy. Subjects were followed for 1 year. The primary endpoint was the proportion of patients with sputum culture conversion after 1 and 2 months of treatment. After 1 month, the proportion of patients for whom sputum culture converted to negative was 17% for the IL-2 group compared with 30% in the control group (p = 0.14; chi2). After 2 months, 77% in the IL-2 group were culture negative compared with 85% of those receiving placebo (p = 0.29, chi2). Results were similar when patients with isoniazid monoresistance were included in the analysis. There were no differences in weight gain and no improvement in fever, cough, and chest pain between groups. One patient in each arm relapsed. IL-2 did not enhance bacillary clearance or improvement in symptoms in human immunodeficiency virus-seronegative adults with drug-susceptible tuberculosis.

Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity.

Tuberculosis (TB) vaccine development is hindered by the lack of clear surrogate markers of protective human immunity to Mycobacterium tuberculosis. This study evaluated the hypothesis that immune-mediated inhibition of mycobacterial growth would more directly correlate with protective TB immunity than other immunologic responses. Bacille Calmette-Guérin (BCG) vaccination, known to induce partial protection against TB, was used as a model system to investigate mechanistic relationships among different parameters of antigen-specific immunity. Effects of primary and booster intradermal BCG vaccinations were assessed in 3 distinct assays of mycobacterial inhibition. Correlations between vaccine-induced growth inhibition and other immune responses were analyzed. BCG significantly enhanced all antigen-specific responses. Peak responses occurred at 2 months after boosting. Statistical analyses suggested that each assay measured unique aspects of mycobacterial immunity. Despite previous evidence that type 1 immune responses are essential for TB immunity, interferon-gamma production did not correlate with mycobacterial inhibition. These results have important implications for TB vaccine development.

Sputum cytokine levels in patients with pulmonary tuberculosis as early markers of mycobacterial clearance.

Sputum and serum from patients with active pulmonary tuberculosis (TB), healthy purified protein derivative-positive adults, and patients with bacterial pneumonia were collected to simultaneously assess local immunity in the lungs and peripheral blood. To determine whether cytokine profiles in sputum from TB patients and control subjects were a reflection of its cellular composition, cytospin slides were prepared in parallel and assessed for the presence of relative proportions of epithelial cells, neutrophils, macrophages, and T cells. Gamma interferon (IFN-gamma) in sputum from TB patients was markedly elevated over levels for both control groups. With anti-TB therapy, IFN-gamma levels in sputum from TB patients decreased rapidly and by week 4 of treatment were comparable to those in sputum from controls. Further, IFN-gamma levels in sputum closely followed mycobacterial clearance. Although detected at fourfold-lower levels, IFN-gamma immunoreactivities in serum followed kinetics in sputum. TNF-alpha, interleukin 8 (IL-8) and IL-6 also were readily detected in sputum from TB patients at baseline and responded to anti-TB therapy. In contrast to IFN-gamma, however, TNF-alpha and IL-8 levels also were elevated in sputum from pneumonia controls. These data indicate that sputum cytokines correlate with disease activity during active TB of the lung and may serve as potential early markers for sputum conversion and response to anti-TB therapy.