Monocytes Contribute to Differential Immune Pressure on R5 versus X4 HIV through the Adipocytokine Visfatin/NAMPT

Background: The immune system exerts a diversifying selection pressure on HIV through cellular, humoral and innate mechanisms. This pressure drives viral evolution throughout infection. A better understanding of the natural immune pressure on the virus during infection is warranted, given the clinical interest in eliciting and sustaining an immune response to HIV which can help to control the infection. We undertook to evaluate the potential of the novel HIV-induced, monocyte-derived factor visfatin to modulate viral infection, as part of the innate immune pressure on viral populations. Results: We show that visfatin is capable of selectively inhibiting infection by R5 HIV strains in macrophages and resting PBMC in vitro, while at the same time remaining indifferent to or even favouring infection by X4 strains. Furthermore, visfatin exerts a direct effect on the relative fitness of R5 versus X4 infections in a viral competition setup. Direct interaction of visfatin with the CCR5 receptor is proposed as a putative mechanism for this differential effect. Possible in vivo relevance of visfatin induction is illustrated by its association with the dominance of CXCR4-using HIV in the plasma. Conclusions: As an innate factor produced by monocytes, visfatin is capable of inhibiting infections by R5 but not X4 strains, reflecting a potential selective pressure against R5 viruses. © 2012 Van den Bergh et al.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Mean infectivity of HIV strains in untreated MDM and PBMC.

<p>TCID50: 50% tissue culture infectious dose; MDM: monocyte-derived macrophage; PBMC: peripheral blood mononuclear cell.</p

Characterisation of visfatin-CCR5 interaction, as assessed by SPR.

<p>Different concentrations of visfatin were injected sequentially on immobilized CCR5. Vertical dashed lines indicate the start and end of injection. A) Raw data; B) fitting to a 1∶1 binding model and C) to a two binding site model, with respective K_D and X² values.</p

In vivo association of visfatin expression with coreceptor usage.

<p>mRNA expression of visfatin in monocytes of therapy-naïve HIV patients, normalised to GAPDH expression, as assessed by RT-qPCR; patients were grouped according to the coreceptor usage of the dominant plasma viral isolates, as assessed by in silico prediction algorithms (Support Vector Machines and Charge Rule algorithms) (triangles) or both in silico algorithms and U373.CCR5/CXCR4 infection assays (filled circles) and were matched for age, CD4 T Lymphocyte count and viral load. <i>p</i>-value calculated by nonparametric Mann-Whitney test.</p

IC50 values of visfatin in MDM for R5 HIV strains.

<p>IC50: 50% inhibitory concentration.</p

Modulation of viral infection and production of R5 and X4 HIV strains by visfatin.

<p>Infection of MDM and resting PBMC (pre-)treated with recombinant visfatin (200 ng/ml) by R5 HIV_BaL, HIV_968-2 and HIV_968-3 and X4 HIV_IIIB, HIV_968-1 and HIV_968-4: A-B) TCID50 values were determined using the method of Reed & Muench<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035074#pone.0035074-Reed1" target="_blank">[24]</a>, based on p24 measurement in culture supernatants of respectively MDM and PBMC. Infection in visfatin treated cells is expressed as a percentage of infection in untreated control cells from the same donor (a): an infection could not be established in absence of visfatin treatment; results for six independent donors are shown; C-D) viral production, as quantified by p24 secretion, 14 days after respectively MDM and PBMC infection with the different strains at 0.1 MOI; means ± SEM of six independent donors are shown; E) time of addition: MT-2 cells were infected with R5 HIV_BaL and HIV_968-2 and were treated with visfatin (200 ng/ml) after the indicated times; p24 production was assessed after 4 days of infection; F) viral production, as quantified by p24 secretion, 14 days after MDM infection with R5 HIV_BaL, HIV_968-2 and HIV_968-3 at 0.1 MOI, in the presence of different doses of visfatin (dose-response curve). NT: untreated control.</p

Relative fitness of R5 versus X4 HIV strains in presence and absence of visfatin and maraviroc.

<p>Flow cytometric evaluation of infection rates of Jurkat T cells by eGFP and DsRedExpress chimeric viruses at 0.1 MOI in presence and absence of visfatin (200 ng/ml) or maraviroc (5 nM): A) HIV_943-3 (R5) versus HIV_943-1 (X4) and B) HIV_BaL (R5) versus HIV_HxB2 (X4). Ratios are depicted of infection rates in double infections normalised to the mono-infection rates; all infections were performed in a colour-flipped setup. Mean and SEM were calculated based on the colour-flipped datasets within each experiment; representative results for three independent experiments are shown. NT: untreated control.</p

Optimization of relative fitness calculations for chimeric virus competitions.

<p>Infection of untreated Jurkat cells with BaL- and HxB2-Env-supplemented pBRNL43 IRES eGFP/DsRedExpress constructs; fluorescence levels were assessed by flow cytometry after 7 days of infection. A) Live gate; B) regions of eGFP+, DsRedExpress+ and double+; C) eGFP+ cells in BaL mono-infected cultures; D) DsRedEpress+ cells in HxB2 mono-infected cultures; E) eGFP+, DsRedExpress+ and double+ cells in BaL/HxB2 double-infected cultures.</p