Mycobacterium tuberculosis and Human Immunodeficiency Virus Type 1 Cooperatively Modulate Macrophage Apoptosis via Toll Like Receptor 2 and Calcium Homeostasis

Abstract

The emergence of drug resistant strains of Mycobacterium tuberculosis (M. tuberculosis) together with reports of co-infections with the human immunodeficiency virus (HIV) has renewed interest to better understand the intricate mechanisms prevalent during co-infections. In this study we report a synergistic effect of M. tuberculosis and HIV-1, and their antigens Rv3416 and Nef, respectively, in inhibiting apoptosis of macrophages. This inhibition involves the TLR2 pathway and second messengers that play complementing and contrasting roles in regulating apoptosis. Interestingly, the route of calcium influx into cells differentially regulates apoptosis during antigenic co-stimulation. While calcium released from intracellular stores was anti-apoptotic, calcium influx from the external milieu was pro-apoptotic. Further, molecular sensors of intracellular calcium release aid in antigen mediated inhibition of apoptosis. A cross-regulation between oxidative burst and differential routing of calcium influx governed apoptosis. Interestingly, the HIV-1 Nef supported anti-apoptotic responses in macrophages whereas Vpu had no significant effect. These results point to a synergistic liaison between M. tuberculosis and HIV-1 in regulating macrophage apoptosis.

Suppression of Toll-like receptor 2–mediated proinflammatory responses by Mycobacterium tuberculosis protein Rv3529c

“…The human monocyte-Mf cell line THP-1 was maintained in RPMI-1640 medium supplemented with 10% FBS and 2 mM L-glutamine and was used throughout the study. Consistent with previous reports from our laboratory, THP-1 monocytes were differentiated into Mfs with PMA [9]. Unless otherwise mentioned, cells were stimulated with 15 mg/ml of recombinant Rv3529c, with or without ligands to various TLRs (Pam 3 Csk 4 for TLR2 and LPS for TLR4 at 1 mg/ml, imiquimod for TLR7 at 5 mg/ml, and CpG DNA for TLR9 at 5 mM), each for indicated times.…”

“…T4 DNA ligase and ligation buffer (Thermo Fisher Scientific) were used for overnight ligation of the amplicons to the pGEMT and pET28b vector (Sigma-Aldrich). The histidine-tagged protein Rv3529c was purified with Ni-NTA agarose beads (Qiagen) followed laboratory standardized procedures [9,10]. Sequencing of the Rv3529c gene was confirmed after cloning.…”

“…Rv3529c, Rv3416, and BSA were biotinylated using published protocols from the laboratory [9,10]. PMA-treated THP-1 cells were stimulated with biotinylated Rv3529c conjugated to PE-streptavidin (15 mg/ml) for 1 h. Z-stack images were collected at 1-mm intervals to score for internalization of the protein using confocal microscopy (Nikon C2).…”

“…S1C, Rv3529c was completely internalized within 1 h of incubation, indicating that it is readily taken up by Mfs. Next, THP-1-derived Mfs were stimulated with Pam 3 Csk 4 , a specific ligand for TLR2 [9], in the presence or absence of Rv3529c, and ROS was measured by FACS. As shown in Fig.…”

Expressions of toll-like receptors 2 and 4, and relative cellular factors in HIV patients with tuberculosis infection

“…It is still unclear how TLR2 influences HIV and MTB infection. Mehto et al [23] reported that MTB and HIV-1 promote apoptosis in macrophages through the TLR2 signal pathway. In addition, Qin et al [24] found that the inhibitory immune reaction of TLR2 is damaged by inducing over-expression of regulatory T-cell (Treg), which causes viral infection.…”