J Antimicrob Chemother. 2015 Aug;70(8):2330-6. doi: 10.1093/jac/dkv101.

Mitochondrial and apoptotic in vitro modelling of differential HIV-1 progression and antiretroviral toxicity.

 

Morén C1, Bañó M1, González-Casacuberta I1, Catalán-Garcia M2, Guitart-Mampel M1, Tobías E1, Cardellach F1, Pedrol E3, Peraire J4,Vidal F4, Domingo P5, Miró Ò6, Gatell JM7, Martínez E7, Garrabou G1.

  • 1Muscle Research and Mitochondrial Function Laboratory, Cellex-IDIBAPS, Faculty of Medicine, University of Barcelona, Hospital Clinic of Barcelona (HCB), Barcelona, Spain Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
  • 2Muscle Research and Mitochondrial Function Laboratory, Cellex-IDIBAPS, Faculty of Medicine, University of Barcelona, Hospital Clinic of Barcelona (HCB), Barcelona, Spain Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain macatala@clinic.ub.es.
  • 3Internal Medicine Department, Hospital of Figueres, Girona, Spain.
  • 4Infectious Diseases Unit, Department of Internal Medicine, Hospital Universitari Joan XXIII, IISPV, Universitat Rovira i Virgili, Tarragona, Spain.
  • 5Infectious Diseases Unit, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain.
  • 6Muscle Research and Mitochondrial Function Laboratory, Cellex-IDIBAPS, Faculty of Medicine, University of Barcelona, Hospital Clinic of Barcelona (HCB), Barcelona, Spain.
  • 7Infectious Diseases Unit, Faculty of Medicine, University of Barcelona, Hospital Clinic of Barcelona (HCB), Barcelona, Spain.

 

Abstract 

OBJECTIVES:

Ex vivo analysis of mitochondrial function may reveal HIV progression and the impact of ART. We propose a mitochondrial and apoptotic in vitro model using Jurkat T cells incubated with plasma. The objectives of this study were to evaluate mitochondrial and apoptotic lesions in this model in relation to HIV progression, and to assess the effect of >1 year of standard non-thymidine-containing therapy. 

METHODS:

This was a cross-sectional comparison among three age- and gender-matched groups (n = 19 × 3): healthy non-HIV-infected participants, HIV-infected long-term non-progressors (LTNPs) and standard antiretroviral-naive chronically infected patients [standard progressors (Sps)], longitudinally evaluated before (Sp1) and after (Sp2) >1 year of efavirenz + tenofovir + emtricitabine therapy. We analysed mitochondrial DNA content by RT-PCR, mitochondrial function by spectrophotometry, mitochondrial protein synthesis by western blot analysis, mitochondrial dynamics by western blot analysis (MFN2), apoptotic transition pore formation by western blot analysis (VDAC-1) and mitochondrial membrane potential and annexin V/propidium iodide fluorescence by flow cytometry. 

RESULTS:

There was a decreasing non-significant trend towards lower mitochondrial parameters for HIV-infected values with respect to uninfected control reference values. HIV progression (LTNP versus Sp1) was associated with decreased mitochondrial genetic, functional and translational parameters, which partially recovered after treatment intervention (Sp2). Mitochondrial fusion showed a trend to decrease non-significantly in Sp patients compared with LTNP patients, especially after therapy. All apoptotic parameters showed a trend to increase in Sp1 with respect to LTNP, followed by recovery in Sp2. 

CONCLUSIONS:

We proposed an in vitro model for mitochondrial and apoptotic assessment to test the effects of HIV infection and its therapy, resembling in vivo conditions. This model could be useful for clinical research purposes.

KEYWORDS: HIV progressors; apoptosis; in vitro model; mitochondrial function

PMID: 25921514

 

SUPPLEMENTS:

We propose a simple and useful in vitro model resembling in vivo conditions using cultured cells incubated with patient’s serum. To assess differential molecular pathways potentially affected by a pathological agent which is present in serum, a feasible assay consists of incubating the cell line of interest with standard complete media (except for the FBS) supplemented by 10% of the patient/control serum that is either containing or lacking the deleterious agents, to further search for differential cell damage (Figure 1a). After a certain period of time, which may vary depending on the condition willing to be tested, it is possible to monitorize the biological pathways of interest in the cells which have been exposed to either “healthy” or “damaging” serum, in order to assess whether the exposure to the pathologic serum has prompted any observable alteration. This experimental approach may increase the knowledge towards the elucidation of pathological processes triggered by the exposition to a plasmatic soluble agent and the possibility to test potential therapeutic approaches. It is possible to use fresh or cryopreserved serum for the incubation steps, and the use of serum is recommended over plasma, due to the clotting process of the latter, in case of testing plasma against hematologic-derived cell lines. In our study, due to the lack of the possibility to obtain serum, we incubated cells in plasma and promptly proceeded to perform the experimental monitoring, prior to the coagulation processes initialization. In all cases, but especially in case of plasma to avoid background interferences coming from coagulation system, ideally there should be parallel assays to compare a control group (incubated with healthy serum), and the studied group (incubated with the pathologic serum) (Figure 1b).

 

AO FIG1

 

In our study, we took profit of our retrospective biobank containing plasma of the study population groups. Commercial immortalized Jurkat cells were cultured and grown to 250 000–500 000 cells/mL. Afterwards the cells were incubated in complete medium [RPMI + 1% (v/v) antibiotics], containing 10% (v/v) plasma from either a group of uninfected controls, a group of naïve (not undergoing antiretroviral treatment) HIV-infected patients presenting a slow progression of the disease and a group of naïve HIV-infected patients presenting a standard progression of the disease, either before or after one year of antiretroviral treatment. Incubation of the cells with the different types of plasma of each group was performed simultaneously. Thus, the experimental procedures always included a sample from each group of study to be analyzed in parallel. Immediately after this incubation, fresh samples were used to test biomarkers of differential HIV progression or antiretroviral toxicity by flow cytometry, and the remaining material was cryopreserved for further molecular and biochemical analysis.

In our study, mitochondrial trends towards a worse mitochondrial condition in terms of mitochondrial genetics, mitochondrial translation and function parameters, of mitochondrial dynamics (mitochondrial fusion) were observed in those cells incubated with plasma of HIV-infected patients, compared to the controls. Furthermore some differential trends depending on the type of the progression of the disease or the concomitant presence of antiretrovirals were found. Further studies should be addressed to test incubations with fresh and cryopreserved serum. The feasibility of this in vitro model represents a potential useful tool for the assessment of mitochondrial and apoptotic pathways, or any other potential biomarker, during the exposition of cell lines to plasmatic and serologic pathogens under different conditions over time.

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann