Minocycline attenuates HIV infection and reactivation by suppressing cellular activation in human CD4+ T cells

Abstract

Treatment of human immunodeficiency virus (HIV) infection with highly active antiretroviral therapy (HAART) is effective but can be associated with toxic effects and is expensive. Other options may be useful for long-term therapy. The immunomodulatory antibiotic minocycline could be an effective, low-cost adjunctive treatment to HAART. Minocycline mediated a dose-dependent decrease in single-cycle CXCR4-tropic HIV infection and decreased viral RNA after infection of CD4+ T cells with HIV NL4-3. Reactivation from latency was also decreased in a primary CD4+ T cell-derived model and in resting CD4+ T cells from HIV-infected patients. Minocycline treatment resulted in significant changes in activation marker expression and inhibited proliferation and cytokine secretion of CD4+ T cells in response to activation. This study demonstrates that minocycline reduces HIV replication and reactivation and decreases CD4+ T cell activation. The anti-HIV effects of minocycline are mediated by altering the cellular environment rather than directly targeting virus, placing minocycline in the class of anticellular anti-HIV drugs.

Figure 1.

Human immunodeficiency virus (HIV) replication in CD4⁺ T cells using a single-cycle replication model. CD4⁺ T cells were stimulated with soluble anti-CD3/CD28 antibodies (1 replicate with phytohemagglutinin) and then pretreated with or without minocycline. Subsequently, cells were infected with an HIV X4 pseudovirus (multiplicity of infection, 0.1). After 72 h, cells were fixed with formaldehyde, and infection was assessed by fluorescence-activated cell sorting analysis of green fluorescent protein (GFP) expression, normalized to untreated infected control. Cells treated with up to 50 µg/mL minocycline had viability between 75% and 100% compared with untreated infected control cells (Trypan blue exclusion). Values represent means and ranges (n⩾3). Lines of best fit were calculated by linear regression analysis.

Figure 2.

Intracellular human immunodeficiency virus (HIV) DNA and RNA levels after in vitro HIV NL4-3 infection of CD4⁺ T cells. CD4⁺ T cells were pretreated with minocycline (diamonds) or not pretreated (squares), followed by activation with anti-CD3/CD28 antibodies with minocycline maintenance. Cells were subsequently infected with HIV NL4-3 (multiplicity of infection, 0.01). Total cellular DNA and RNA were isolated 24 h after infection. A, HIV DNA levels normalized to interferon β DNA (n=5). B, HIV gag RNA levels normalized to 18S ribosomal RNA (n=5). Lines represent means. *Significant difference compared with untreated control (paired t test).

Figure 3.

Human immunodeficiency virus (HIV) reactivation in a primary CD4⁺ T cell-derived model of HIV latency. Cells from a primary CD4⁺ T cell-derived model of HIV latency were pretreated with minocycline (diamonds and circles) or not pretreated (squares), followed by stimulation with anti-CD3/CD28 antibodies. After 72 h, level of reactivation was determined by fluorescence-activated cell sorting analysis of the mean fluorescence intensity of green fluorescent protein (GFP) cells (A) or the percentage of viable GFP⁺ cells (B), normalized to those in untreated, activated controls. Lines represent means (n=2).

Figure 4.

Ex vivo reactivation of latent human immunodeficiency virus (HIV) in resting CD4⁺ T cells from HIV-infected patients with suppressed viremia during highly active antiretroviral therapy. Highly purified resting (>99%) CD4⁺DR⁻ T cells were pretreated with minocycline (filled diamonds) or not pretreated (open squares). Two replicates were maintained with antiretrovirals in culture (shaded squares and shaded diamonds). Cells were then activated for 7 days with anti-CD3/CD28 antibodies and cultured with minocycline and antiretrovirals. A, Viral RNA isolated from pooled supernatants was quantified using real-time reverse-transcription polymerase chain reaction targeting HIV-1 gag RNA (n=6) (P values calculated by Wilcoxon matched-pairs test). B, Cells at day 7 after activation were stained with antibodies to CD25 (n=6), CD69 (n=4), and Ki-67 (n=6), and marker expression was analyzed by fluorescence-activated cell sorting. Viability in minocycline-treated cultures remained >80% until day 5. By day 7, viability was typically 60%–80% (relative to untreated control, as shown by Trypan blue staining). Lines represent means (P values calculated by paired t tests).

Figure 5.

Phenotypes of minocycline-treated CD4⁺ T cells after CD3/CD28 costimulation. CD4⁺ T cells were pretreated with minocycline or not pretreated, followed by activation with anti-CD3/CD28 antibodies. Fluorescence-activated cell sorting was used to analyze longitudinal expression of CD25, CD71, CD69, and CD11a (mean fluorescence intensity) (A) and Ki-67, CD45RA, HLA-DR, and chemokine (C-C motif) receptor 5 (CCR5) (percentage of positive cells) (B) up to day 5 after activation; values represent means ± standard deviations [SDs] (n⩾3 where error bars are shown). C, Cytokines were quantified in cell culture supernatants by multiplex enzyme-linked immunosorbent assay. Secretion of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α from minocycline-treated cells is shown, relative to that in untreated, activated controls; values represent means ± SDs (n=4). P values for comparisons with controls were calculated by paired (A and B) or 1-sample (C) t test; *20 µg/mL, ^#10 and 20 µg/mL, ⁺all doses.