Macrophage-Derived Factors with the Potential to Contribute to Pathogenicity of HIV-1 and HIV-2: Role of CCL-2/MCP-1

Abstract

Human immunodeficiency virus type 2 (HIV-2) is known to be less pathogenic than HIV-1. However, the mechanism(s) underlying the decreased HIV-2 pathogenicity is not fully understood. Herein, we report that β-chemokine CCL2 expression was increased in HIV-1-infected human monocyte-derived macrophages (MDM) but decreased in HIV-2-infected MDM when compared to uninfected MDM. Inhibition of CCL2 expression following HIV-2 infection occurred at both protein and mRNA levels. By microarray analysis, quantitative PCR, and Western blotting, we identified that Signal Transducer and Activator of Transcription 1 (STAT1), a critical transcription factor for inducing CCL2 gene expression, was also reduced in HIV-2-infected MDM. Blockade of STAT1 in HIV-infected MDM using a STAT1 inhibitor significantly reduced the production of CCL2. In contrast, transduction of STAT1-expressing pseudo-retrovirus restored CCL2 production in HIV-2-infected MDM. These findings support the concept that CCL2 inhibition in HIV-2-infected MDM is meditated by reduction of STAT1. Furthermore, we showed that STAT1 reduction in HIV-2-infected MDM was regulated by the CUL2/RBX1 ubiquitin E3 ligase complex-dependent proteasome pathway. Knockdown of CUL2 or RBX1 restored the expression of STAT1 and CCL2 in HIV-2-infected MDM. Taken together, our findings suggest that differential regulation of the STAT1-CCL2 axis may be one of the mechanisms underlying the different pathogenicity observed for HIV-1 and HIV-2.

Keywords: CCL2; CULLIN 2; HIV-1; HIV-2; RBX1; STAT1; macrophage.

Figure 1

Opposite effects of HIV-1 and HIV-2 infection on the expression of CCL2 by primary human MDM. (A–D) Differentiated primary human MDM from donor #8 were uninfected (Medium) or infected with a variety of HIV-1 and HIV-2 strains as indicated. The production of CCL2/MCP-1 following infection with HIV-1 (A) or HIV-2 (C) in primary human MDM was determined by ELISA at the indicated time points. Data shown are representative of results from 8 healthy donors. Replication of HIV-1 (B) and HIV-2 (D) was monitored by measuring RT activity in the harvested supernatants in duplicate. RT values differed by not more than 15%. (E) Distinct CCL2 induction following the infection with HIV-1 and HIV-2. The concentrations of CCL2 in the supernatants harvested from MDM infected with HIV-1 Ada, HIV-2 B5, B7, B8, or B9 strains at the peak viral replication time point were determined by ELISA and normalized to the expression levels of CCL2 in the supernatants harvested from uninfected MDM. Each symbol represents an individual donor. Data were shown as the mean ± SEM (n = 8), and the asterisks depict significant differences between HIV-1 and HIV-2 infection groups (n = 8), which were analyzed using the Student’s T-test. Asterisks *, ***, and **** depict p ≤ 0.05, p ≤ 0.005 and p ≤ 0.001, respectively. (F) HIV-2 infection inhibited CCL2 gene transcription. The expression levels of CCL2 mRNA were determined by quantitative RT-PCR. The abundance of the mRNA was calculated using the comparative CT method 2^−ΔΔCT, and the expression levels of the CCL2 gene were normalized to the expression levels of GAPDH. The fold changes in CCL2 mRNA expression levels for HIV-1- and HIV-2-infected primary human MDM were calculated/normalized relative to the mRNA levels in uninfected cells (Medium). Each experiment was carried out in duplicate and performed twice. Asterisks depict significant differences between uninfected (Medium) and HIV-2-infected groups; asterisks *, *** depict p ≤ 0.05 and p ≤ 0.005, respectively.

Figure 2

HIV-2 infection reduces the levels of STAT1 expression in primary human MDM. (A) Affymetrix microarray analysis of STAT1 gene expression in MDM infected with HIV-1 or HIV-2. Data shown represent the fold change of the microarray signal from HIV-infected MDM vs. the signal from uninfected MDM using two STAT-1 probes. (B,C) The levels of STAT1 mRNA (B) and protein (C) in uninfected (Medium) and HIV-1- or HIV-2-infected human primary MDM were assessed by quantitative RT-PCR and Western blotting, respectively. The expression levels of CCL2 mRNA were examined using cells harvested on day 15 post-infection, normalized by the levels of GAPDH mRNA, and presented as the amount relative to uninfected cells (B). STAT1 protein expression levels were also analyzed using cells harvested on day 15 post-infection, and the relative expression levels of STAT1 were quantified and normalized by the levels of β-actin (C). Asterisks *, **, and *** depict a statistically significant difference in the STAT1 mRNA levels between the untreated (Medium) MDM and HIV-2-infected MDM with p ≤ 0.05, p ≤ 0.01 and p ≤ 0.005, respectively.

Figure 3

Reduced levels of CCL2 observed following HIV-2 infection correlate with inhibition of STAT1 expression in primary human MDM. (A) STAT1 blockade reduces CCL2 expression in HIV-1- and HIV-2-infected MDM. Cell culture supernatants were harvested from uninfected (Medium), HIV-1- and HIV-2-infected MDM following treatment with 0.5 µg/mL of the STAT1 inhibitor, fludarabine, for 6 h and assessed for the expression levels of CCL2 by ELISA. Asterisks depict significant differences between groups treated with or without fludarabine. (B) Transduction of STAT1-expressing pseudo retrovirus increases CCL2 secretion by HIV-2-infected MDM. Uninfected (Medium) and HIV-2-infected MDM were transduced with pseudo retrovirus expressing STAT1 (pMIG-w-STAT1) or the empty vector-derived pseudo retrovirus (pMIG-w). CCL2 levels were determined 48 h post-transduction by ELISA. Asterisks *, **, and *** depict a statistically significant difference in the levels of CCL2 between the compared groups with p ≤ 0.05, p ≤ 0.01 and p ≤ 0.005, respectively.

Figure 4

Knockdown of CUL2/RBX1 restores STAT1 and CCL2 expression in HIV-2-infected MDM. (A–D) Uninfected (Medium) and HIV-2-infected MDM were transfected with control siRNA or siRNA specifically targeting human RBX1, CUL1, CUL2, CUL3, CUL4, CUL5 or CUL7. CCL2 levels in the cell culture supernatants were determined 72 (A) and 96 (B) hours post-transfection by ELISA. Each symbol represents an individual donor. Data were shown as the mean ± SEM of the results from 5 donors (siCUL4 and siCUL5), 6 donors (siCUL1, siCUL3, and siCUL7), or 11 donors (siCTRL and siRBX1). Asterisks depict significant differences between the compared groups as indicated, which were determined by Student’s t-test analysis. The expression levels of STAT1 protein in uninfected and HIV-2-infected MDM following siRNA transfection for 96 h were determined by Western blotting and quantified by normalization to the levels of β-actin used as a loading control (C,D). Asterisks *, **, ***, and **** depict a statistically significant difference in the levels of CCL2 between the compared groups with p ≤ 0.05, p ≤ 0.01, p ≤ 0.005 and p ≤ 0.001, respectively.