Mianda Wu

Increased Replication of HIV-1 at Sites of Mycobacterium tuberculosis Infection: Potential Mechanisms of Viral Activation

&NA; Tuberculosis (TB) enhances HIV‐1 replication and the progression to AIDS in dually infected patients. We employed pleural TB as a model to understand the interaction of the host with HIV‐1 during active TB, at sites of Mycobacterium tuberculosis (MTB) infection. HIV‐1 replication was enhanced both in the cellular (pleural compared with blood mononuclear cells) and acellular (pleural fluid compared with plasma) compartments of the pleural space. Several potential mechanisms for expansion of HIV‐1 in situ were found, including augmentation in expression of tumor necrosis factor (TNF)‐&agr; and the HIV‐1 noninhibitory &bgr;‐chemokine (MCP‐1), low presence of HIV‐1 inhibitory &bgr;‐chemokines (MIP‐1&agr;, MIP‐1&bgr;, and RANTES [regulated on activation, normal T expressed and secreted]), and upregulation in expression of the HIV‐1 coreceptor, CCR5, by pleural fluid mononuclear cells. Thus, at sites of MTB infection, conditions are propitious both for transcriptional activation cf HIV‐1 in latently infected mononuclear cells, and facilitation of viral infection of newly recruited cells. These mechanisms may contribute to enhanced viral burden and dissemination during TB infection.

Human Immunodeficiency Virus Type 1 (HIV-1) Quasispecies at the Sites of Mycobacterium tuberculosis Infection Contribute to Systemic HIV-1 Heterogeneity

ABSTRACT 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/μl, 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.

Activation of β-chemokines and CCR5 in persons infected with human immunodeficiency virus type 1 and tuberculosis

Tuberculosis (TB) in human immunodeficiency virus type 1 (HIV-1)-infected persons is associated with progression of HIV-1 disease. The expression of macrophage inflammatory protein (MIP)-1alpha and CCR5 was assessed in HIV-1-infected patients with pulmonary TB (HIV-1/PTB) and without PTB (HIV-1/C), PTB patients not infected with HIV-1 (PTB), and control subjects. Mycobacterium tuberculosis (MTB)-induced MIP-1alpha production was lower in peripheral blood mononuclear cells (PBMC) of HIV-1/PTB patients than in those of PTB patients (P< .05) and was lower in PBMC of HIV-1/C patients than in those of control subjects (P< .005). However, MIP-1alpha production was higher in PBMC of HIV/PTB patients than in those of HIV-1/C patients (P< .01). The pattern of MTB-induced RANTES production was similar to that of MIP-1alpha. However, MTB induced greater expression of mRNA for CCR5 in PBMC of HIV-1/PTB patients than in those of HIV-1/C patients (P< .04). Furthermore, the MTB-induced HIV p24 antigen level in PBMC of HIV-1/PTB patients with a CD4 cell count <500 cells/microL was higher (P< .05) than that in HIV-1/C patients. Thus, perturbations in chemokine pathways in HIV-1/PTB patients may accelerate HIV-1 disease.

Mechanisms of apoptosis of T-cells in human tuberculosis.

The role of TGF-β TNF-α 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-β or TNF-α decreased spontaneous (P ≤ 0.05) and MTB-induced (P≤ 0.02) T-cell apoptosis by 50–90%, but effects were not additive. Interestingly, only levels of TGF-β 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 ≤ 0.02), and MTB stimulation reduced intracellular Bcl-2 content in CD4 T-cells from patients only (P ≤ 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-β TNF-α and FasL.

Transcriptional activation of HIV by Mycobacterium tuberculosis in human monocytes

Recently it has been shown that infection with Mycobacterium tuberculosis increases the replication of HIV in mononuclear cells. The objective of this study was to investigate the mechanism(s) of up‐regulation of HIV in primary human monocytes. Monocytes from healthy subjects were infected with HIV in vitro and then cultured with purified protein derivative (PPD) of M. tuberculosis. Culture supernatants were assessed for HIV p24 and cytokines. HIV expression was assessed by reverse transcriptase‐polymerase chain reaction (RT‐PCR). PPD induced HIV‐infected monocytes to increased expression of HIV RNA and production of HIV p24. This effect correlated with production of tumour necrosis factor‐alpha (TNF‐α) in monocyte cultures. However, neutralizing antibody to TNF‐α only partly abrogated the PPD‐induced HIV p24 in these cultures. Also, PPD and culture filtrate of M. tuberculosis induced HIV mRNA expression. Further, using an adenovirus infection system containing an HIV long‐terminal repeat (LTR) reporter plasmid, we showed that M. tuberculosis and its PPD induced HIV LTR. Therefore, the effect of M. tuberculosis and its PPD on HIV replication in monocytes is primarily one of transcriptional activation.

Macrophage-Activating Cytokines in Human Immununodeficiency Virus Type 1–Infected and –Uninfected Patients with Pulmonary Tuberculosis

Tuberculosis (TB) is the most common opportunistic infection in human immunodeficiency virus type 1 (HIV-1)-infected patients globally and occurs throughout the course of HIV-1 disease. Here the production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by peripheral blood mononuclear cells (PBMC) of HIV-1-infected versus -uninfected patients with newly diagnosed pulmonary TB (PTB) was compared. Findings were correlated with cytokine profiles, clinical presentation, and expression of inducible nitric oxide (iNOS). Most HIV-1/PTB patients with a CD4 cell count of 200-500 cells/microL had high IFN-gamma production and radiographic evidence of atypical PTB. Low IFN-gamma production and radiographic evidence of reactivated PTB characterized both HIV-1/PTB patients with a CD4 cell count >or=500 cells/microL and HIV-1-uninfected patients. TNF-alpha levels were similar in all HIV-1/PTB patients, regardless of CD4 cell count. Induction of iNOS in PBMC was low and was associated with low IFN-gamma production. These data underscore the potential pathogenic role of macrophage-activating cytokines in TB in HIV-1-infected patients.

Bioactivation of Latent Transforming Growth Factor β1 by Mycobacterium tuberculosis in Human Mononuclear Phagocytes

Biologically active transforming growth factor beta 1 (TGFβ1) has been identified at sites of Mycobacterium tuberculosis (MTB) infection in the lung; however, the underlying mechanism(s) for its activation is not clear. Here using an enzyme‐linked immunospot assay for TGFβ1, we show that human blood monocytes (MN) and alveolar macrophages (AM) produce bioactive TGFβ1 upon stimulation by MTB. However, only MTB‐stimulated MN increased TGFβ1 production on a per cell basis. The frequency of TGFβ1‐producing MN was reduced by an inhibitor of plasmin, bdellin, indicating a role for plasmin pathways in the bioactivation of cytokine. The expression of urokinase plasminogen activator receptor (uPAR) mRNA and both surface and soluble uPAR (CD87) was increased in MTB‐activated MN. However, antibody neutralization of uPAR suppressed bioactive TGFβ1 in MN alone. Thus, the more immature MN, which are continuously recruited to the lung during tuberculosis (TB), have a higher capacity to bioactivate TGFβ1 by expression of components of the plasmin pathway. Excess production and bioactivation of TGFβ1 at sites of MTB infection may undermine host immune responses during TB.

Distinct cytokine and regulatory T cell profile at pleural sites of dual HIV/tuberculosis infection compared to that in the systemic circulation

Pleural tuberculosis (TB) remains a common presentation of Mycobacterium tuberculosis (MTB) infection in HIV/TB dually infected subjects, and both cellular and acellular components of the pleural milieu promote HIV‐1 replication; however, they remain uncharacterized. Using cytokine array of pleural fluid and real‐time reverse transcription–polymerase chain reaction (RT–PCR) and immunophenotype analysis, pleural fluid mononuclear cells (PFMC) were compared to systemic counterparts [i.e. plasma and peripheral blood mononuclear cells (PBMC)]. Significant increases in pleural fluid cytokines compared to plasma were limited to interleukin (IL)‐6, IL‐8, interferon (IFN)‐γ and transforming growth factor (TGF)‐β, and did not include other T helper type 1 (Th1) (IL‐2, IL‐15), Th2 or Th17 cytokines. Patterns and levels of cytokines were indistinguishable between pleural fluid from HIV/TB and TB patients. Forkhead box P3 (FoxP3) mRNA in PFMC was increased significantly and correlated highly with levels of IL‐6 and IL‐8, less with TGF‐β, and not with IFN‐γ. Among CD4 T cells, FoxP3‐reactive CD25hi were increased in HIV/TB dually infected subjects compared to their PBMC, and up to 15% of FoxP3+ CD25hi CD4 T cells were positive for IL‐8 by intracellular staining. These data implicate a dominant effect of MTB infection (compared to HIV‐1) at pleural sites of dual HIV/TB infection on the local infectious milieu, that include IL‐6, IL‐8, IFN‐γ and TGF‐β and regulatory T cells (Treg). A correlation in expansion of Treg with proinflammatory cytokines (IL‐6 and IL‐8) in pleural fluid was shown. Treg themselves may promote the inflammatory cytokine milieu through IL‐8.

Inhibition of human immunodeficiency virus-1 (HIV-1) by β-chemokine analogues in mononuclear cells from HIV-1-infected patients with active tuberculosis

Tuberculosis (TB) enhances human immunodeficiency virus‐1 (HIV‐1) activity in patients with dual HIV‐1/TB infection. Therapies that control augmentations of HIV‐1 activity at sites of Mycobacterium tuberculosis (MTB) infection may be useful in inhibition of viral expansion. Regulated upon activation, normal T‐cell expressed and secreted (RANTES) analogues (AOP and NNY) are potent in inhibiting the entry of primary HIV‐1 isolates into host mononuclear cells. These analogues were used to inhibit MTB‐induced HIV‐1 entry in blood monunuclear cells (PBMC) from patients with pulmonary TB, and pleural fluid mononuclear cells (PFMC) from patients with pleural TB. PBMC or PFMC were cultured with and without MTB in presence and absence of RANTES analogues. HIV‐1 strong stop DNA was assessed by real‐time polymerase chain reaction (PCR) as a measure of infection. CCR5 mRNA was assessed by real‐time reverse transcription (RT)‐PCR and by immunostaining and FACS analysis. HIV‐1 infection was induced by MTB in vitro in PBMC from the majority (14 of 20) of HIV‐1/TB subjects, and new infection was inhibited by AOP‐ or NNY‐RANTES. HIV‐1 infection was also inhibited by these reagents in MTB‐induced PFMC from three of three patients with pleural TB. Expression of CCR5 mRNA was significantly induced by MTB in PBMC from patients with pulmonary TB. Further, expression of CCR5 was higher in PFMC compared to PBMC from patients with pleural TB. Also, CCR5 was fourfold higher on CD14+ pleural mononuclear cells than on CD4+ lymphocytes. Blocking new HIV‐1 infection of mononuclear cells may be useful in control of HIV‐1 during dual HIV‐1/TB infection.

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.