Elevated levels of macrophage migration inhibitory factor (MIF) in the plasma of HIV-1-infected patients and in HIV-1-infected cell cultures: a relevant role on viral replication

Abstract

The cytokine macrophage migration inhibitory factor (MIF) is involved in the pathogenesis of inflammatory and infectious diseases, however its role in HIV-1 infection is unknown. Here we show that HIV-1-infected patients present elevated plasma levels of MIF, that HIV-1-infected peripheral blood mononuclear cells (PBMCs) release a greater amount of MIF, and that the HIV-1 envelope glycoprotein gp120 induces MIF secretion from uninfected PBMCs. The HIV-1 replication in PBMCs declines when these cells are treated with anti-MIF antibodies, and exposure of HIV-1-infected cells to the ABC-transporter inhibitor probenecid results in inhibition of MIF secretion. The addition of recombinant MIF (rhMIF) to HIV-1-infected PBMCs enhances viral replication of CCR5- or CXCR4-tropic HIV-1 isolates. Using a T CD4(+) cell lineage containing an HIV long terminal repeats (LTR)-Luciferase construct, we detected that rhMIF promotes transcription from HIV-1 LTR. Our results show that HIV-1 induces MIF secretion and suggest that MIF influences the HIV-1 biology through activation of HIV-1 LTR.

Figure 1

HIV-1-infected patients present higher plasma levels of MIF relative to uninfected individuals. Plasmas from HIV-1-infected individuals (HIV-1 pos), and from healthy volunteers (control plasmas; HIV-1 neg) were assessed for MIF concentrations by ELISA. Bars represent means ± SEM of 10 healthy volunteers (HIV-1 neg) or 30 HIV-1-infected patients (HIV-1 pos). * p<0.0001

Figure 2

HIV-1 infection and HIV-1 gp120 induce MIF release by PBMCs. (A) PBMCs were infected by an R5-tropic HIV-1 isolate (Ba-L), and after 7 days MIF contents were evaluated in the culture supernatants by ELISA (* p<0.04). Individual values and medians are represented for each group. (B) Lines show individual MIF release in the uninfected and in HIV-1-infected PBMC depicted in figure 2-A. (C) MIF contents in culture supernatants of PBMCs infected by an X4-tropic HIV-1 isolate (Tybe) (* p<0.04). Mean of MIF values by cells cultured only with medium (Nill): 757.8 pg/mL. (D) Macrophages were infected by the HIV-1 isolate Ba-L and MIF release was assessed by ELISA at 7 (7d) and 14 (14d) days after infection. Individual values and medians are represented for each group. (E) Uninfected PBMCs were exposed to recombinant HIV-1 envelope protein gp120 (5 μg/mL) derived from the HIV-1 isolate Ba-L, and MIF secretion was evaluated in culture supernatants by ELISA after 16 hours (* p<0.006). Mean of MIF values by cells cultured only with medium (Nill): 427 pg/mL. In (C) and (E) bars show means ± SEM for 3 and 5 different donors, respectively, and all experiments were done in triplicates per donor.

Figure 2

HIV-1 infection and HIV-1 gp120 induce MIF release by PBMCs. (A) PBMCs were infected by an R5-tropic HIV-1 isolate (Ba-L), and after 7 days MIF contents were evaluated in the culture supernatants by ELISA (* p<0.04). Individual values and medians are represented for each group. (B) Lines show individual MIF release in the uninfected and in HIV-1-infected PBMC depicted in figure 2-A. (C) MIF contents in culture supernatants of PBMCs infected by an X4-tropic HIV-1 isolate (Tybe) (* p<0.04). Mean of MIF values by cells cultured only with medium (Nill): 757.8 pg/mL. (D) Macrophages were infected by the HIV-1 isolate Ba-L and MIF release was assessed by ELISA at 7 (7d) and 14 (14d) days after infection. Individual values and medians are represented for each group. (E) Uninfected PBMCs were exposed to recombinant HIV-1 envelope protein gp120 (5 μg/mL) derived from the HIV-1 isolate Ba-L, and MIF secretion was evaluated in culture supernatants by ELISA after 16 hours (* p<0.006). Mean of MIF values by cells cultured only with medium (Nill): 427 pg/mL. In (C) and (E) bars show means ± SEM for 3 and 5 different donors, respectively, and all experiments were done in triplicates per donor.

Figure 2

HIV-1 infection and HIV-1 gp120 induce MIF release by PBMCs. (A) PBMCs were infected by an R5-tropic HIV-1 isolate (Ba-L), and after 7 days MIF contents were evaluated in the culture supernatants by ELISA (* p<0.04). Individual values and medians are represented for each group. (B) Lines show individual MIF release in the uninfected and in HIV-1-infected PBMC depicted in figure 2-A. (C) MIF contents in culture supernatants of PBMCs infected by an X4-tropic HIV-1 isolate (Tybe) (* p<0.04). Mean of MIF values by cells cultured only with medium (Nill): 757.8 pg/mL. (D) Macrophages were infected by the HIV-1 isolate Ba-L and MIF release was assessed by ELISA at 7 (7d) and 14 (14d) days after infection. Individual values and medians are represented for each group. (E) Uninfected PBMCs were exposed to recombinant HIV-1 envelope protein gp120 (5 μg/mL) derived from the HIV-1 isolate Ba-L, and MIF secretion was evaluated in culture supernatants by ELISA after 16 hours (* p<0.006). Mean of MIF values by cells cultured only with medium (Nill): 427 pg/mL. In (C) and (E) bars show means ± SEM for 3 and 5 different donors, respectively, and all experiments were done in triplicates per donor.

Figure 2

HIV-1 infection and HIV-1 gp120 induce MIF release by PBMCs. (A) PBMCs were infected by an R5-tropic HIV-1 isolate (Ba-L), and after 7 days MIF contents were evaluated in the culture supernatants by ELISA (* p<0.04). Individual values and medians are represented for each group. (B) Lines show individual MIF release in the uninfected and in HIV-1-infected PBMC depicted in figure 2-A. (C) MIF contents in culture supernatants of PBMCs infected by an X4-tropic HIV-1 isolate (Tybe) (* p<0.04). Mean of MIF values by cells cultured only with medium (Nill): 757.8 pg/mL. (D) Macrophages were infected by the HIV-1 isolate Ba-L and MIF release was assessed by ELISA at 7 (7d) and 14 (14d) days after infection. Individual values and medians are represented for each group. (E) Uninfected PBMCs were exposed to recombinant HIV-1 envelope protein gp120 (5 μg/mL) derived from the HIV-1 isolate Ba-L, and MIF secretion was evaluated in culture supernatants by ELISA after 16 hours (* p<0.006). Mean of MIF values by cells cultured only with medium (Nill): 427 pg/mL. In (C) and (E) bars show means ± SEM for 3 and 5 different donors, respectively, and all experiments were done in triplicates per donor.

Figure 2

HIV-1 infection and HIV-1 gp120 induce MIF release by PBMCs. (A) PBMCs were infected by an R5-tropic HIV-1 isolate (Ba-L), and after 7 days MIF contents were evaluated in the culture supernatants by ELISA (* p<0.04). Individual values and medians are represented for each group. (B) Lines show individual MIF release in the uninfected and in HIV-1-infected PBMC depicted in figure 2-A. (C) MIF contents in culture supernatants of PBMCs infected by an X4-tropic HIV-1 isolate (Tybe) (* p<0.04). Mean of MIF values by cells cultured only with medium (Nill): 757.8 pg/mL. (D) Macrophages were infected by the HIV-1 isolate Ba-L and MIF release was assessed by ELISA at 7 (7d) and 14 (14d) days after infection. Individual values and medians are represented for each group. (E) Uninfected PBMCs were exposed to recombinant HIV-1 envelope protein gp120 (5 μg/mL) derived from the HIV-1 isolate Ba-L, and MIF secretion was evaluated in culture supernatants by ELISA after 16 hours (* p<0.006). Mean of MIF values by cells cultured only with medium (Nill): 427 pg/mL. In (C) and (E) bars show means ± SEM for 3 and 5 different donors, respectively, and all experiments were done in triplicates per donor.

Figure 3

MIF release upon HIV-1 infection is dependent of ABC transporters. PBMCs were treated with probenecid (Prob; 10 μM, 45 minutes), then infected or not by an R5-tropic HIV-1 isolate (BaL), and probenecid was re-applied. MIF contents were measured by ELISA in culture supernatants 7 days after infection (* p<0.0002; ** p<0.004). Mean of MIF values by HIV-1-infected cells cultured only with medium (Nill): 1.6 ng/mL. Data represent means ± SEM for 3 different donors done in triplicates.

Figure 4

MIF neutralization diminishes HIV-1 replication. PBMCs were infected by an R5-tropic HIV-1 isolate (BaL) and treated with anti- human MIF polyclonal antibodies (20 μg/mL) or isotype control. Viral replication was measured after 7 days in culture supernatants by ELISA (* p<0.0001). Mean of p24 values by HIV-1-infected cells cultured only with medium (Nill): 147 ng/mL. Data represent means ± SEM for 5 different donors, done in triplicates.

Figure 5

MIF addition exacerbates HIV-1 replication. (A) PBMCs were infected by an R5-tropic HIV-1 isolate (Ba-L), and treated with rhMIF at indicated concentrations. HIV-1 replication was measured after 7 days by a p24 Ag ELISA in culture supernatants (12 ng/mL, * p<0.009; 25 ng/mL, ** p<0.03, both relative to untreated cells). Mean of p24 values by HIV-1-infected cells cultured only with medium (Nill): 123 ng/mL; (B) PBMCs were infected by an X4-tropic HIV-1 (Tybe) and exposed to rhMIF (25 ng/mL). Viral replication was measured 7 days after infection, as described (* p<0.04). Mean of p24 values by HIV-1-infected cells cultured only with medium (Nill): 73 ng/mL. Data represent means ± SEM for 3 (6 ng/ml), 5 (12 ng/mL) and 9 (25 ng/mL) (A), or 5 (B) different donors, done in triplicates.

Figure 5

MIF addition exacerbates HIV-1 replication. (A) PBMCs were infected by an R5-tropic HIV-1 isolate (Ba-L), and treated with rhMIF at indicated concentrations. HIV-1 replication was measured after 7 days by a p24 Ag ELISA in culture supernatants (12 ng/mL, * p<0.009; 25 ng/mL, ** p<0.03, both relative to untreated cells). Mean of p24 values by HIV-1-infected cells cultured only with medium (Nill): 123 ng/mL; (B) PBMCs were infected by an X4-tropic HIV-1 (Tybe) and exposed to rhMIF (25 ng/mL). Viral replication was measured 7 days after infection, as described (* p<0.04). Mean of p24 values by HIV-1-infected cells cultured only with medium (Nill): 73 ng/mL. Data represent means ± SEM for 3 (6 ng/ml), 5 (12 ng/mL) and 9 (25 ng/mL) (A), or 5 (B) different donors, done in triplicates.

Figure 6

rhMIF promotes HIV-1 transcription from LTR. CD4⁺ T Jurkat derivative cell lineage (1G5) containing stably transfection of luciferase under control of HIV-1 LTR was left untreated (control) or treated with PHA (2 μg/mL), Tat (100 ng/mL) or MIF (25 ng/mL). After 20hs, cells were washed and lyzed for luminescence evaluation. Bars represent one representative of 3 independent experiments with similar results.