MTb increases NFAT5 mRNA levels in human MDM
Given that macrophages, which are the primary target of MTb infection, are also a major reservoir of HIV-1 as infection progresses [40], we next investigated whether MTb is able to directly enhance NFAT5 mRNA expression in primary human MDM. We prepared MDM from five normal donors, stimulated the cells with the MTb lysate or left them unstimulated, and measured NFAT5 gene expression levels by quantitative real-time PCR at 24 and 48 hours. We also investigated whether HIV-1 infection is capable of inducing NFAT5 mRNA synthesis by infecting MDM with live or heat-inactivated R5-tropic representatives of subtype B (HIV-1Bal), C (HIV-198IN22), or E (HIV-192TH64). As shown in Figure 2A, stimulation with MTb lysate significantly increased NFAT5 mRNA levels at 24 (p<0.05) and 48 (p<0.01) hours, whereas infection with viable or heat-inactivated HIV-1 isolates did not increase NFAT5 mRNA levels (Figure 2A). Thus, MTb specifically enhances NFAT5 mRNA expression in human MDM, and this response continues to increase for at least 48 hours post-stimulation (Figure 2A).
10.1371/journal.ppat.1002620.g002 Figure 2  MTb induces NFAT5 gene expression and knockdown of NFAT5 impairs HIV-1 replication during MTb co-infection.
(A) MTb but not HIV-1 increases NFAT5 mRNA synthesis in human macrophages. MDM were isolated from PBMC obtained from four normal donors. The cells were incubated for 24 or 48 hours with heat-inactivated or live R5-tropic HIV-1 subtype B, C, or E isolates, live MTb strain CDC1551 (1∶1 MDM∶bacilli), or left untreated. Quantitative real-time PCR analysis of NFAT5 mRNA expression levels revealed that the low, constitutively present level of NFAT5 mRNA expressed in untreated cells was not affected by exposure to live or inactivated HIV-1, but was significantly increased at both 24 (*, p<0.05) and 48 (**, p<0.01) hours following infection with MTb. (B) NFAT5-specific siRNA effectively suppresses NFAT5 expression in MDM in both the absence and presence of MTb co-infection. MDM (1×106 cells/well in a 6-well plate) from four normal donors were transfected with siRNA specific for NFAT5 or, as a control, GFP, and were then infected with MTb (1∶1 MDM∶bacilli) or left uninfected. In the cells transfected with control siRNA, MTb infection significantly increased NFAT5 expression in comparison to uninfected cells (open bars). When MDM were transfected with NFAT5-specific siRNA, both constitutively expressed (p = 0.048) and MTb-induced (p = 0.021) NFAT5 mRNA were significantly suppressed (grey bars). (C) HIV-1 subtype B replication is significantly suppressed in the presence of MTb co-infection when NFAT5 expression is abrogated in human MDM. MDM from four normal donors were transfected with NFAT5-specific and control siRNAs as described in 2B. Cells were then infected with HIV-1Lai/Bal-env followed by co-infection with MTb CDC1551. Virus replication was measured at days 6, 9, and 12 post-infection. At day 12, virus replication was significantly reduced in cells transfected with NFAT5-specific siRNA in comparison to cells transfected with control siRNA (*, p<0.05). We monitored the morphology of cells within the infected cultures and did not observe noticeable macrophage necrosis even at the final timepoint of viral harvest.