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. 2014 Nov;58(11):6444-53.
doi: 10.1128/AAC.03270-14. Epub 2014 Aug 18.

Effects of tenofovir on cytokines and nucleotidases in HIV-1 target cells and the mucosal tissue environment in the female reproductive tract

Nabanita Biswas  1 , Marta Rodriguez-Garcia  2 , Zheng Shen  2 , Sarah G Crist  2 , Jack E Bodwell  2 , John V Fahey  2 , Charles R Wira  1
Affiliations

Effects of tenofovir on cytokines and nucleotidases in HIV-1 target cells and the mucosal tissue environment in the female reproductive tract

Nabanita Biswas et al. Antimicrob Agents Chemother. 2014 Nov.

Abstract

Tenofovir (TFV) is a reverse transcriptase inhibitor used in microbicide preexposure prophylaxis trials to prevent HIV infection. Recognizing that changes in cytokine/chemokine secretion and nucleotidase biological activity can influence female reproductive tract (FRT) immune protection against HIV infection, we tested the hypothesis that TFV regulates immune protection in the FRT. Epithelial cells, fibroblasts, CD4(+) T cells, and CD14(+) cells were isolated from the endometrium (Em), endocervix (Cx), and ectocervix (Ecx) following hysterectomy. The levels of proinflammatory cytokines (macrophage inflammatory protein 3α [MIP-3α], interleukin 8 [IL-8], and tumor necrosis factor alpha [TNF-α]), the expression levels of specific nucleotidases, and nucleotidase biological activities were analyzed in the presence or absence of TFV. TFV influenced mRNA and/or protein cytokines and nucleotidases in a cell- and site-specific manner. TFV significantly enhanced IL-8 and TNF-α secretion by epithelial cells from the Em and Ecx but not from the Cx. In contrast, in response to TFV, IL-8 secretion was significantly decreased in Em and Cx fibroblasts but increased with fibroblasts from the Ecx. When incubated with CD4(+) T cells from the FRT, TFV increased IL-8 (Em and Ecx) and TNF-α (Cx and Ecx) secretion levels. Moreover, when incubated with Em CD14(+) cells, TFV significantly increased MIP-3α, IL-8, and TNF-α secretion levels relative to those of the controls. In contrast, nucleotidase biological activities were significantly decreased by TFV in epithelial (Cx) and CD4(+) T cells (Em) but increased in fibroblasts (Em). Our findings indicate that TFV modulates proinflammatory cytokines, nucleotidase gene expression, and nucleotidase biological activity in epithelial cells, fibroblasts, CD4(+) T cells, and CD14(+) cells at distinct sites within the FRT.

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Figures

FIG 1
FIG 1
Effects of TFV on cytokine gene expression in epithelial cells and fibroblasts from the FRT. Epithelial cells were isolated from endometrium, and fibroblasts were isolated from endometrium, endocervix, and ectocervix. Cells were treated with 1 mg/ml of TFV. RNA was isolated, and RT-PCR was performed to compare the levels of mRNA of MIP-3α, IL-8, and TNF-α at different time points. Each TFV-treated sample was compared with its own control at the same time point, and fold changes in mRNA expression were calculated by converting each control to 1. Bars represent means ± SEM from 6 to 8 independent experiments with different patients. *, P < 0.05; **, P < 0.01. Shown are expression levels of the MIP3α gene (A), IL-8 gene (B), or TNF-α gene (C) from Em epithelial cells and Em, Cx, and Ecx fibroblasts.
FIG 2
FIG 2
Effect of TFV on MIP-3α, IL-8, and TNF-α secretion from FRT epithelial cells and fibroblasts. Epithelial cells and fibroblasts were isolated from endometrium, endocervix, and ectocervix. Cells were treated with 1 mg/ml of TFV for 24 h. Secretions of MIP-3α, IL-8, and TNF-α were analyzed from the apical and basolateral surfaces of Em, Cx, and Ecx epithelial cells and from culture supernatants of Em, Cx, and Ecx fibroblasts by ELISA. Bars represent means ± SEM from 6 to 8 independent experiments with different patients. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (A) Secretion of MIP3α, IL-8, and TNF-α by apical surface of FRT Em, Cx and Ecx; (B) secretion of MIP3α, IL-8, and TNF-α by basolateral surface of FRT Em, Cx and Ecx; (C) secretion of IL-8 by Em, Cx and Ecx fibroblasts. C, control.
FIG 3
FIG 3
Effect of TFV on MIP-3α, IL-8, and TNF-α in CD4+ T cells and CD14+ cells. CD4+ T cells were isolated from Em, Cx, and Ecx, and CD14+ cells were isolated from Em. Cells were treated with 1 mg/ml of TFV for 24 h. RNA was isolated, and RT-PCR was performed to compare the levels of mRNA of MIP-3α, IL-8, and TNF-α. mRNA was expressed as a relative expression compared to untreated samples. Secretions of MIP-3α and IL-8 were analyzed from culture supernatants by ELISA. Each TFV-treated sample was compared with its own control, and fold changes in protein secretion were calculated by converting each control to 1. Bars represent means ± SEM from 6 to 8 independent experiments with different patients. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (A) Expression of MIP3α, IL-8, and TNF-α from Em, Cx, and Ecx CD4+ T cells in the presence of TFV; (B) secretion of IL-8 and TNF-α from Em, Cx, and Ecx CD4+ T cells; (C) expression of MIP3α, IL-8, and TNF-α from CD14+ cells from Em in the presence of TFV; (D) secretion of MIP3α, IL-8, and TNF-α from CD14+ cells from Em in the presence of TFV.
FIG 4
FIG 4
Site-dependent effect of TFV on 5′-nucleotidase gene expression in FRT CD4+ T cells. CD4+ T cells were isolated from Em, Cx, and Ecx, and cells were treated with 1 mg/ml of TFV for 24 h. RNA was isolated, and RT-PCR was performed to compare the levels of mRNA of the nucleotidase genes NT5E, NT5C2, NT5C3L, NT5C1A, NT5C, and NT5M in the presence of TFV from CD4+ T cells in Em (A), Cx (B), and Ecx (C). mRNA was expressed as a relative expression compared to untreated samples. Five to 9 independent experiments with different patients were done. *, P < 0.05; **, P < 0.01.
FIG 5
FIG 5
Effect of TFV on 5′-nucleotidase gene expression in CD14+ cells. CD14+ cells were isolated from Em and treated with 1 mg/ml of TFV for 24 h. RNA was isolated, and RT- PCR was performed to compare the levels of mRNA of the nucleotidase genes NT5E, NT5C2, NT5C3L, NT5C1A, NT5C, and NT5M. Six to 9 independent experiments with different patients were done. *, P < 0.05. Relative mRNA expression (A) and fold change in the mRNA expression (B) of NT5E, NT5C2, NT5C3L, NT5C1A, NT5C, and NT5M gene expression in the presence of TFV from CD14+ cells in Em. Each TFV-treated sample was compared with its own control, and fold changes in mRNA expression were calculated by converting each control to 1.
FIG 6
FIG 6
Effect of TFV on 5′-nucleotidase activity in epithelial cells, fibroblasts, CD4+ T cells, and CD14+ cells. 5′-nucleotidase biological activity was measured (see Materials and Methods) in epithelial cells, fibroblasts, and CD4+ T cells from Em, Cx, and Ecx and from CD14+ cells from Em after 24 h with 1 mg/ml of TFV treatment. Values are expressed as milli-enzyme units (mEU) per 1 million cells. Bars represent means ± SEM from 5 or 6 separate experiments with different donors. *, P < 0.05. Epithelial cells (A) fibroblasts (B), CD4+ T cells (C), and CD14+ cells (D) from Em, Cx, and Ecx after 24 h with 1 mg/ml of TFV treatment.
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