Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women

Abstract

The placenta may play a critical role in inhibiting vertical transmission of HIV-1. Here we demonstrate that leukemia inhibitory factor (LIF) is a potent endogenous HIV-1-suppressive factor produced locally in placentae. In vitro, LIF exerted a potent, gp130-LIFRbeta-dependent, HIV coreceptor-independent inhibition of HIV-1 replication with IC50 values between 0.1 pg/ml and 0.7 pg/ml, depending on the HIV-1 isolate. LIF also inhibited HIV-1 in placenta and thymus tissues grown in ex vivo organ culture. The level of LIF mRNA and the incidence of LIF protein-expressing cells were significantly greater in placentae from HIV-1-infected women who did not transmit HIV-1 to their fetuses compared with women who transmitted the infection, but they were not significantly different from placentae of uninfected mothers. These findings demonstrate a novel pathway for endogenous HIV suppression that may prove to be an effective immune therapy for HIV infection.

Figure 1

Representative quantification and localization of LIF, IL-4, and IL-2 protein in placentae from transmitting and nontransmitting women using immunohistochemistry (a) and assisted computerized image analysis are seen (b). Cells staining with a brown precipitate express the protein indicated. Cells were counterstained blue with hematoxylin. Tissues from TNT placentae and normal placentae express significantly higher levels of IL-4 and LIF than TT placentae (^AP < 0.001; ^BP < 0.02). IL-2 was significantly elevated in TT placentae compared with TNT placentae (^CP < 0.05). IL-4, IL-2, and LIF are all expressed in placental lymphocytes (arrowheads), and LIF was also expressed in decidua and endothelial cells (arrow). Scale bar, 20 μm.

Figure 2

(a) Dose-dependent inhibition of HIV-1 infection by LIF is illustrated. Viral isolates were used at 1,000 TCID₅₀ for the R5-using HIV-1_Bal, 10,000 TCID₅₀ for the X4-using HIV-1_Lai, 3,000 TCID₅₀ for the X4-using HIV-1_Me40, and 2,000 TCID₅₀ for the primary isolates 33074, 33015, and 93US151. The IC₅₀ of LIF for all isolates tested was between 0.1 and 0.7 pg/ml. The mean p24 antigen produced in positive-control PBMCs (without addition of LIF) averaged 843 ± 412 pg/ml, including all isolates tested. (b) Effects of LIF on cell proliferation. LIF inhibited HIV-1 production without affecting cell proliferation in response to 0.25 μg/ml PHA.

Figure 3

Dose-dependent effects of LIF on reverse transcription in PBMCs infected with HIV-1 are illustrated. Quantitative real-time PCR results from cells treated with increasing concentrations of LIF and infected with HIV-1_Lai, HIV-1_Bal, or HIV-1_ME40. Both early (LTR U5/R) and late (LTR U3/gag) reverse transcripts were inhibited by LIF. Each determination was performed in triplicate.

Figure 4

Requirement of gp130-LIFRβ for HIV-1 inhibition by LIF is seen. (a) MT-2 cells do not express gp130-LIFRβ (CD130); however, (b) PBMCs express high levels of gp130-LIFRβ. Dotted lines represent PE-conjugated isotype controls for CD130-LIFRβ staining. (c) MT-2 cells are resistant to and PBMCs susceptible to the HIV-1–inhibitory effects of LIF. Anti-CD130 Ab’s abrogate the anti-HIV-1 effects of LIF. PBMCs and MT-2 cells were infected with 10,000 TCID₅₀ of HIV-1_Lai.

Figure 5

LIF inhibits HIV-1 by in placenta organ culture. Placenta tissue was infected with 3,000 TCID₅₀ HIV-1_Bal or 2,000 TCID₅₀ of the primary newborn isolate HIV-1_93US151 after treatment with 0, 0.1, 1.0, 10.0, or 100 pg/ml LIF (a). Inclusion of 5 μg/ml neutralizing, polyclonal anti-LIF Ab’s increased infection of the placenta organ culture relative to the untreated control. These data may reflect additional neutralization of endogenously produced LIF. (b) Representative laser confocal UFISH images demonstrate numerous productively infected cells (arrows) confirming infection of the organ culture in the absence of LIF. (c)Treatment with 1 pg/ml LIF greatly inhibits the number of productively infected cells. Autofluorescence was quenched, and cells were labeled with a trypan blue counterstain (red).

Figure 6

LIF inhibits HIV-1 infection in human thymus organ culture. Human thymus tissue was infected with 3,000 TCID₅₀ HIV-1_Lai or 3,000 TCID₅₀ HIV-1_Bal with and without pretreatment with 1 pg/ml LIF. Representative flow-cytometric dot plots of simultaneous immunophenotyping (CD4 + CD8)/UFISH performed on thymic cell suspensions on PI day 1 are presented (a and b). HIV-1_Lai infected predominantly the CD4, CD8 DP population of thymocytes and, to a lessor extent, the CD4 and CD8 SP populations (a). Treatment with LIF inhibited productive infection of the DP and SP thymocyte subsets with only a few (<0.1%) productively infected DP thymocytes identified (b).