Characterization of HIV-1 infection and innate sensing in different types of primary human monocyte-derived macrophages

Abstract

Macrophages play an important role in human immunodeficiency virus (HIV) pathogenesis and contribute to establishment of a viral reservoir responsible for continuous virus production and virus transmission to T cells. In this study, we investigated the differences between various monocyte-derived macrophages (MDMs) generated through different differentiation protocols and evaluated different cellular, immunological, and virological properties. We found that elevated and persistent HIV-1 pWT/BaL replication could be obtained only in MDMs grown in RPMI containing macrophage colony-stimulating factor (M-CSF). Interestingly, this MDM type was also most responsive to toll-like receptor stimulation. By contrast, all MDM types were activated to a comparable extent by intracellular DNA, and the macrophage serum-free medium-(Mac-SFM-)differentiated MDMs responded strongly to membrane fusion through expression of CXCL10. Finally, we found that HIV infection of RPMI/M-CSF-differentiated MDMs induced low-grade expression of two interferon-stimulated genes in some donors. In conclusion, our study demonstrates that the differentiation protocol used greatly influences the ability of MDMs to activate innate immune reactions and support HIV-1 replication. Paradoxically, the data show that the MDMs with the strongest innate immune response were also the most permissive for HIV-1 replication.

Figure 1

Morphology of MDMs differentiated by four distinct protocols. Representative images of MDMs differentiated for 7 days with (a) RPMI GM-CSF, (b) RPMI M-CSF, (c) Mac-SFM GM-CSF, or (d) Mac-SFM M-CSF.

Figure 2

Evaluation of HIV-1 infection and replication in different MDM populations. MDMs were produced during a 7-day period by four different differential protocols and infected with HIV-1 pWT/BaL (5000 × TCID50). Supernatants were harvested at the indicated time points after infection, and p24 levels were evaluated by ELISA. (a) RPMI GM-CSF, (b) RPMI M-CSF, (c) Mac-SFM GM-CSF, and (d) Mac-SFM M-CSF. Data shown are from two different experiments including 6 donors in total.

Figure 3

Innate sensing of PAMPs in human MDMs via TLR4 and TLR7/8. MDMs were produced during a 7-day period through stimulation by four differentiation protocols. The different MDM types were stimulated with either LPS (100 ng/ml), R848 (5 μg/mL), or left untreated (UT) for 6 hours before isolation of supernatants. Levels of CCL5 and CXCL10 were measured by ELISA. Data shown are mean values ± SEM from 10 donors from 3 separate experiments.

Figure 4

Innate sensing of DNA in human MDMs via intracellular DNA sensors. MDMs were produced during a 7-day period through stimulation by four differentiation protocols. The MDMs were transfected with poly(dA : dT) (1.7 μg/mL) using lipofectamine2000, treated with lipofectamine alone, or left untreated (UT) for 6 hours before harvest of supernatants. Levels of CCL5 and CXCL10 were measured by ELISA. Data shown are means ± SEM from 6 donors from 2 separate experiments.

Figure 5

Induction of ISGs by HIV-1 in human MDMs. MDMs were produced during a 7-day period through two differentiation protocols (RPMI M-CSF and Mac-SFM GM-CSF). MDMs were treated with IFN-α (100 U/mL), Sendai virus (SeV) (0.5 MOI), HIV-1 pWT/BaL (5000 × TCID50), or left untreated for 6 hours before total RNA was harvested and analysed for viperin (a) and (c) and ISG56 (b) and (d) expression by PCR. Data are shown from two donors. Data are normalized to GAPDH and shown as fold induction relative to untreated.