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.

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.

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.

Characterizing traditionally defined periodontal disease in HIV+ adults.

BACKGROUND: Results have varied from previous studies examining the level and extent of periodontal disease (PD) in HIV-1 infected (HIV+) adults. These studies used different methodologies to measure and define PD and examined cohorts with divergent characteristics. Inconsistent methodological approaches may have resulted in the underestimation of traditionally-defined PD in HIV+ individuals. OBJECTIVES: To characterize the level, extent and predictors (i.e. immunologic, microbiologic, metabolic and behavioral) of PD in an HIV+ cohort during the era of highly active antiretroviral therapy (HAART). STUDY DESIGN: Cross-sectional study. SETTING: HIV+ adults receiving outpatient care at three major medical clinics in Cleveland, OH. Subjects were seen from May, 2005 to January, 2008. MEASUREMENTS: Full-mouth periodontal examinations included periodontal probing depth (PPD), recession (REC) and clinical attachment level (CAL). Subgingival plaque was assessed for DNA levels of Porphyromonas gingivalis (Pg), Tannerella forsythia, and Treponema denticola by real-time DNA PCR assays developed for each pathogen. Rather than using categories, we evaluated PD as three continuous variables based on the percent of teeth with >or=1 site per tooth with PPD >or= 5mm, REC > 0 mm and CAL >or= 4mm. RESULTS: Participants included 112 HIV+ adults. Each subject had an average 38% (+/-24%) of their teeth with at least one site of PD >or= 5 mm, 55% (+/-31%) of their teeth with at least one site of REC > 0 mm, and 50% (+/-32%) of their teeth with at least one site of CAL >or= 4 mm. CD4+ T-cell count <200 cells/mm(3) was significantly associated with higher levels of REC and CAL, but not PPD. Greater levels of Pg DNA were associated with PPD, REC and CAL.By regression analysis, CD4+ T-cell count <200 cells /mm3 had approximately twice thedeleterious effect on CAL as did smoking (standardized beta coefficient 0.306 versus 0.164) [corrected]. Annual dental visit compliance remained an independent predictor for lower levels of PD. CONCLUSIONS: The level and extent of PD were high in this cohort even though most patients were being treated with HAART. The definition of periodontal disease used and cohort characteristics examined can influence the level of periodontal disease reported in studies of persons with HIV. Traditional periodontal pathogens are associated with PD in this cohort. Those with CD4+ T-cell counts <200 cells/mm(3) are at greater risk for PD. Therefore, earlier HAART initiation may decrease exposure to immunosuppression and reduce PD morbidity. Continuity of dental care remains important for HIV+ patients even when they are being treated with HAART.

Induction of HIV type 1 expression correlates with T cell responsiveness to mycobacteria in patients coinfected with HIV type 1 and Mycobacterium tuberculosis.

An in vitro mononuclear cell system to model the microenvironment of coinfection with HIV-1 and Mycobacterium tuberculosis (MTB) was developed. This cellular system was used to assess the interaction of MTB-infected monocytes and T cells from dually infected HIV-1/TB patients with pulmonary tuberculosis (TB). Subjects with higher induction of HIV-1 gag/pol mRNA expression after MTB stimulation had increased MTB-specific T cell IFN-gamma and TNF-alpha production. Lack of HIV-1 mRNA induction did not correlate with increased induction of regulatory T cells (T-reg) as measured by MTB-induced Foxp3 mRNA. HIV-1 induction did not significantly correlate with clinical parameters including plasma HIV-1 viral load or CD4(+) T cell count. These data model MTB-induced HIV-1 replication at the microenvironment of MTB reactivation/infection. The data suggest that the magnitude of MTB-specific T cell responses drives local viral pathogenesis regardless of the stage of HIV-1 disease as reflected by plasma viral load or CD4(+) T cell count.

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.

Transactivation of human immunodeficiency virus-1 in T-cells by Mycobacterium tuberculosis-infected mononuclear phagocytes.

Worldwide, tuberculosis (TB) is the most frequent coinfection with human immunodeficiency virus-1 (HIV-1), and active TB enhances the progression of HIV-1 disease in dually infected subjects. In the microenvironment of Mycobacterium tuberculosis (MTB)-infected foci, where HIV-1-infected CD4 T-cells come into contact with MTB-infected macrophages, the direct interaction of the two cell types in the activation of latent HIV-1 may be important. In this study we sought to determine whether MTB-infected human primary mononuclear phagocytes-namely, alveolar macrophages (AMs) and their less mature blood precursors, monocytes-activate HIV-1 in a T-cell line stably transfected with an HIV-1 long terminal repeat (LTR) reporter construct (1G5 cells) and induce HIV-1 expression in T-cells from HIV-1-infected subjects. MTB-infected monocytes and AMs, and not Mycobacterium avium-infected cells, activated HIV-1 LTR in 1G5 cells in the presence and absence of HIV-1 tat. Transactivation of HIV-1 LTR by MTB-infected mononuclear phagocytes was mediated mainly by tumor necrosis factor-alpha. In AMs, but not monocytes, membrane tumor necrosis factor-alpha contributed to transactivation of HIV-1 LTR. MTB-infected MNs from 60% of HIV-infected subjects induced HIV-1 LTR in 1G5 cells as well. Furthermore, HIV-1 transcription was induced in autologous T-cells from 30% of the HIV-1-infected subjects. We therefore conclude that MTB-infected mononuclear phagocytes can transactivate HIV-1 in CD4 cells. Transactivation of latent HIV-1 in CD4 T-cells by MTB-infected mononuclear phagocytes may in part be responsible for increased HIV activity at sites of MTB infection during dual infection in vivo.

Human immunodeficiency virus type 1 (HIV-1) quasispecies at the sites of Mycobacterium tuberculosis infection contribute to systemic HIV-1 heterogeneity.

We have recently reported an increased heterogeneity in the human immunodeficiency virus type 1 (HIV-1) envelope gene (env) in HIV-1-infected patients with pulmonary tuberculosis (TB) compared to patients with HIV-1 alone. This increase may be a result of dissemination of lung-derived HIV-1 isolates from sites of Mycobacterium tuberculosis infection and/or the systemic activation of the immune system in response to TB. To distinguish between these two mechanisms, blood and pleural fluid samples were obtained from HIV-1-infected patients with active pleural TB in Kampala, Uganda (CD4 cell counts of 34 to 705 cells/microl, HIV-1 plasma loads of 2,400 to 280,000 RNA copies/ml, and HIV-1 pleural loads of 7,600 to 4,500,000 RNA copies/ml). The C2-C3 coding region of HIV-1 env was PCR amplified from lysed peripheral blood mononuclear cells and pleural fluid mononuclear cells and reverse transcriptase-PCR amplified from plasma and pleural fluid HIV-1 virions of eight HIV-1 patients with pleural TB. Phylogenetic and phenetic analyses revealed a compartmentalization of HIV-1 quasispecies between blood and pleural space in four of eight patients, with migration events between the compartments. There was a trend for a greater genetic heterogeneity in the pleural space, which may be the result of an M. tuberculosis-mediated increase in HIV-1 replication and/or selection pressure at the site of infection. Collectively, these findings suggest that HIV-1 quasispecies in the M. tuberculosis-infected pleural space may leak into the systemic circulation and lead to increased systemic HIV-1 heterogeneity during TB.