Increased metallothionein gene expression, zinc, and zinc-dependent resistance to apoptosis in circulating monocytes during HIV viremia

Abstract

Circulating monocytes exhibit an apoptotic resistance phenotype during HIV viremia in association with increased MT expression. MTs are known to play an important role in zinc metabolism and immune function. We now show, in a cross-sectional study using peripheral monocytes, that expression of MT1 isoforms E, G, H, and X is increased significantly in circulating monocyte cells from HIV+ subjects during chronic viremic episodes as compared with uninfected subjects. This increase in expression is also observed during acute viremia following interruption of suppressive ART. Circulating monocytes from HIV+ donors were also found to have elevated zinc importer gene Zip8 expression in conjunction with elevated intracellular zinc levels in contrast to CD4(+)T-lymphocytes. In vitro HIV-1 infection studies with elutriated MDM confirm a direct relation between HIV-1 infection and increased MDM MT1 (isoform G) gene expression and increased intracellular zinc levels. A direct link between elevated zinc levels and apoptosis resistance was established using a cell-permeable zinc chelator TPEN, which reversed apoptosis resistance effectively in monocytes from HIV-infected to levels comparable with uninfected controls. Taken together, increases in MT gene expression and intracellular zinc levels may contribute directly to maintenance of an immune-activated monocyte by mediating an increased resistance to apoptosis during active HIV-1 viremia.

Figure 1.

Increased MT gene expression in circulating monocytes derived from HIV+ individuals with chronic or acute viremia. (A) Differential expression of MT1 isoforms in CD14⁺ monocytes derived from uninfected and HIV+ subjects during HIV viremia confirmed by real-time PCR [MT1E and MT1H, n=6 for uninfected and HIV+ donors; MT1G and MT1X results consisted of uninfected (n=6) and HIV+ donors (n=11)]. In t-test comparisons, ns indicates nonsignificant P; *, P < 0.05; **, P < 0.01. (B) Real-time PCR results using RNA isolated from CD14⁺ monocytes derived from cryopreserved PBMCs collected pre- and post-treatment interruption (n=3). *, P < 0.05, in ANOVA comparisons with monocytes from cryopreserved, uninfected PBMCs. Each symbol in group identifies same donors between panels.

Figure 2.

Intracellular zinc is increased in monocytes during chronic HIV viremia. (A) Intracellular zinc levels, measured via DCF uptake, were compared in monocytes and CD4⁺ T cells from three viremic HIV+ donors. Histograms of DCF flow cytometric reading, along with a composite of the three donors, are shown. MFI, Mean fluorescence intensity. (B) Intracellular zinc in CD14⁺ monocytes from uninfected (n=9) and HIV+-suppressed (n=5) or -viremic (n=6) donors measured by DCF uptake shows that active HIV replication increases intracellular zinc in monocytes. (C) Expression of zinc importer gene Zip8 assessed by real-time PCR. Groups (uninfected, n=9; HIV+, n=7) were analyzed using ANOVA or paired comparison tests. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Subject data for each group represented by circles and triangles.

Figure 3.

Monocyte apoptosis resistance in HIV viremic subjects associated with retention of higher intracellular zinc levels. (A) Monocytes derived from uninfected donors (•; n=10) or viremic HIV+ patients (○; n=8) were exposed to cadmium chloride (Cd²⁺; 100 μM) and FasL [100 ng/ml; uninfected (○; n=15) and HIV+ (•; n=10)] for 6 h. Cells were harvested, apoptosis was measured by staining for activated caspase-3, and samples were analyzed by flow cytometry. Totals shown were derived from CD14⁺ cells that were 7AAD-negative/caspase-3-positive and double-positive 7AAD/caspase-3 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Intracellular zinc was measured in monocytes from HIV+-infected (checkered bar; n=10) and uninfected (open bar; n=15) donors following apoptosis induction of FasL (100 ng/ml). *, P < 0.05, in ANOVA comparisons with monocytes from the untreated group.

Figure 4.

Higher threshold of monocyte apoptosis resistance in HIV infection upon an induced decrease of intracellular zinc levels. (A) Monocytes from uninfected (n=5) or HIV+ donors (n=5) were exposed to cadmium (100 μM) for 6 h in the presence or absence of TPEN. Cells were harvested and apoptosis assessed by staining for activated caspase-3 and 7AAD uptake. Shown is a representative cadmium chloride-induced apoptosis in an uninfected (left) and HIV donor (right). (B) Shown is a dose response of TPEN concentration and the induction of caspase-3 activation or intracellular zinc level in circulating monocytes from uninfected (n=5) or HIV+ subjects (n=5). Monocytes from HIV+ individuals appear to be less sensitive to TPEN treatment.