BY: SHAN AND BITA

Regis Eduardo G. et al. (2010)

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

Journal of Virology, Vol. 399. pages: 31-38

Background:

MIF= cytokine macrophage migration inhibitory factor

gp120= HIV-1 envelop glycoproteinp24= HIV-1 antigenPBMCS= peripheral blood mononuclear

cells

• HIV-1 leads to immunosupression and makes the individual more susceptible to opportunistic infections and tumors.

• Pro-inflammatory cytokines such as TNF-α and IL-6 are considered to up-regulate HIV-1 replication while anti-inflammatory cytokines like IFN, IL-10, IL-27 decrease it.

• LPS is a strong inducer of MIF secretion and it can be found in the serum of HIV-1 infected individuals

• MIF is an upstream activator of innate immunity and triggers release of TNF-α and IL-6.

• Elevated level of MIF in serum has been observed in other diseases such as west Nile virus, Dengue fever, rheumatoid arthritis.

• So far no studies has been done for the role of MIF in HIV-1 infection

biology.kenyon.edu/.../ Lentiviral/hiv_image.jpg

Objective: Study whether MIF is involved in the pathogenesis of HIV-1 and if it influences HIV-1 replication

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Main materials used: •CCR5-dependent isolate HIV-1 Ba-L and CXCTR-4-dependent isolate HIV-1 type

•PBMCS derived from healthy donors cultured in human serum, antibiotics, and IL-2•Macrophages derived from the PBMC cell cultures•CD4+T Jurkat cell line transfected with luciferase

Methods:•Looked for the presence of MIF in HIV-1 infected patients•Looked for the presence of MIF in PBMC (and macrophages) amongst uninfected and infected cultures•Looked for the presence of MIF in HIV-1 infected PBMC treated with probenecid•Looked for the presence of viral replication in HIV-infected PBMC treated with anti-MIF antibodies, and the presence of HIV-1 transcription in CD4+T cells treated with PHA, Tat, or rhMIF

Main Findings and Experimental approach:

Fig.1: HIV- infected present higher plasma level of MIFPlasma collected from a selected group of HIV infected individuals (n=30), and a control group of healthy volunteers (n=10). Evaluation of MIF levels by Elisa

The HIV-1-infected patients who were not under anti-retroviral treatment and did not present clinical signs of co-infections had higher plasma levels of MIF than the healthy individuals

Main Findings and Experimental approach:

Fig. 2A: Infection with HIV-1 doubled the release of MIFInfection of human primary PBMCs with R5-tropic HIV-1 isolate. Evaluation of MIF secretion in the culture supernatants by Elisa, 7 days after infectionExperiments done in triplicates per donor

PBMCs infected with R5-tropic HIV-1 isolate, X4-tropic HIV-1, or HIV-1 gp120 secreted MIF

Fig. 2D: HIV-1 infection of macrophages didn’t change secretion of MIFInfection of macrophages with Ba-L HIV-1 isolate. MIF secretion assessed by Elisa, 7 and 14 days after infectionExperiments done in triplicates per donor

Main Findings and Experimental approach:

Fig. 3. MIF release upon HIV-1 infection is dependent of ABC transporterspre-treatment of cells with probenecid (10 μM, for 45 min), infection with HIV-1, and reapplication of probenecid to infected cells. Measurement of MIF at day 7 post- infectionExperiments done in triplicates of three donors

HIV-1 infected PBMC cells, when treated with probenecid (an ABC transporter inhibitor) had less MIF secretion

Fig. 4: MIF inhibition down-regulates HIV-1 replicationTreatment of HIV-1 infected PBMCs with neutralizing anti-MIF polyclonal antibodies immediately after infection. Evaluation of viral replication by Elisa, 7 days after infectionExperiments done in triplicates of 3, 5, and 5 donors.

Main Findings and Experimental approach:Infected PBMCs, when treated with anti-MIF antibodies, had a

reduction in HIV-1 replication, while infected CD4+T cells, when treated with rhMIF, had an increase in HIV-1 transcription

Fig. 6: rhMIF increases HIV-1 replication via induction of direct HIV-1 transcriptionAddition of rhMIF to cultures of Jurkat CD4+ T derivative cell line containing integrated HIV-LTR luciferase construct. Evaluation of luminescence by luminometer

HIV-1 (gp120)

Greater MIF releaseVia ABC

transporters

macrophagesHIV-1

transcription

Via HIV-1 LTR

HIV-1 replication

Anti-MIF antibodies

decrease

Discussion:

Critiques:•Experiments could have been done beyond 7 days.•Small sample of volunteers; the number of HIV positive patients may be 20 instead of 30.•There are limitations to an in vivo study, and no cultures came from HIV positive patients.•Overall a good clear research paper; many controls were made.

•MIF is mainly restricted to secretion by HIV-1 infected PBMCS cells, and can contribute to viral replication. Soluble viral protein gp120 may contribute to MIF secretion at a systemic level.

•Opportunistic pathogens may induce high levels of MIF by secondary LPS stimulation. •Anti-MIF antibodies decrease viral replication; therefore, MIF could be used as a target molecule for anti-HIV-1 therapy.

PBMCs

NO yes