J Cell Mol Med. 2017 Feb;21(2):402-409. doi: 10.1111/jcmm.12985. Epub 2016 Oct 18.

HIV-1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion.

Morén C 1,2, González-Casacuberta I 1,2, Álvarez-Fernández C 3, Bañó M 1,2, Catalán-Garcia M 1,2, Guitart-Mampel M 1,2, Juárez-Flores DL 1,2, Tobías E 1,2, Milisenda J 1,2, Cardellach F 1,2, Gatell JM 3, Sánchez-Palomino S 3, Garrabou G 1,2.

1 Muscle Research and Mitochondrial Function Laboratory, Cellex-IDIBAPS, Faculty of Medicine-University of Barcelona, Internal Medicine Department-Hospital Clínic of Barcelona (HCB), Barcelona, Spain.
2 Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Raras (CIBERER), Madrid, Spain.
3 Cellex-IDIBAPS, Faculty of Medicine-University of Barcelona, Infectious Diseases Unit-Hospital Clínic of Barcelona (HCB), Barcelona, Spain.

 

Abstract

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV-1)-infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro-mitochondrial or anti-apoptotic strategies as in vitro cell platforms to deal with HIV-infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt-PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial-nuclear encoded subunits II-IV of cytochrome-c-oxidase (COXII-COXIV), respectively, as well as mitochondrial apoptotic events [voltage-dependent-anion-channel-1(VDAC-1)-content and caspase-9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1-assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC-1 and caspase-9 content were sharply decreased in both chronic HIV-1-infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV-chronicity, to test novel therapeutic options or to study HIV reservoirs.

PMID: 27758070

 

Supplement

A useful cell model

Human immunodeficiency virus‐1‐infected U1 and ACH2 cell lines are, respectively, promonocytic and lymphoid cell lines derived from uninfected U937 (same U1 cell line but uninfected) and CEM (same ACH2 cell line, but uninfected) precursor cells. U1 and ACH2 lines are characterized by harbouring one and two stable integrated copies of the HIV‐1 genome which replicates at a low rate, comparable to slow progression of the infection in patients. In these cell lines, HIV‐1 latently auto‐replicates itself, constituting a worldwide model to study chronic HIV infection. Accurate characterization of mitochondrial mechanisms and mitochondrial‐derived apoptosis has yet to be performed in these models of chronically infected cells.

We hypothesized that the mitochondrial and apoptotic damage initiated by HIV infection may contribute to the persistence of infection chronicity and progression. Given this context, mitochondrial genetics, function, expression and apoptotic levels are expected to be affected in both chronically HIV‐1‐infected promonocytic and lymphoid cell lines.

 

 

Figure 1. Cell models of the study. Chronically infected HIV‐1 promonocytic (U1) and lymphoid (ACH2) are cloned cell lines derived by limiting dilution cloning of U937 or CEM cells surviving an acute infection with HIV‐1 (LAV‐1 strain).

 

The mitochondrial phenotype and apoptotic profile of the HIV-infected cell lines was strikingly different with respect to the control counterparts, as regards either mitochondrial genome (mtDNA content), enzymatic activities (of complex IV, CIV) (Figure 2), mitochondrial subunit proteins cytochrome C oxidase II and IV (COXII and COXIV) (Figure 3), or by early and late apoptotic markers VDAC-1 and caspase-9 (Figure 4).

 

Figure 2. Mitochondrial DNA content and enzymatic activity of mitochondrial respiratory chain (CIV)

Figure 3. Mitochondrial protein subunits content (COX II and IV subunits of CIV and apoptotic marker VDAC)

Figure 4. Mitochondrial and apoptotic markers (VDAC-1 and caspase-9)

 

The significant differences observed in mitochondrial and apoptotic characterization point out to an altered mitochondrial phenotype as well as an apoptotic resistance in the infected cell lines. The mitochondrial and apoptotic damage induced by HIV infection may exacerbate these deleterious effects in feedback cycle, terminating in the loss of cell defense capability of the host and the progression of infection. Studies performed to date have highlighted the need to establish an in vitro model to further investigate possible therapeutic approaches to revert mitochondrial or apoptotic damage and determine novel tools to fight HIV progression.

Our findings suggest that the present model using promonocytic and lymphoid cell lines represent an adequate platform to test potential targets (such as mitochondrial or apoptotic therapeutic targets) to fight against HIV infection.