Fibroblast growth factor 21 and Growth and differentiation factor 15 as potential systemic biomarkers of mitochondrial toxicity and associations with disease severity and immune suppression in HIV-1 infected patients in Tshwane, South Africa

Abstract

South Africa has the highest prevalence of human immunodeficiency virus (HIV) in the world with approximately 7.77 million people infected. The wide usage of antiretroviral therapy (ART) has been credited with making HIV a manageable chronic disease. Nucleoside reverse-transcriptase inhibitors (NRTIs) and nucleotide reverse-transcriptase inhibitors (NtRTIs) belong to a class of HIV drugs that act by preventing the conversion of viral ribonucleic acid (RNA) into a complementary deoxyribonucleic acid (cDNA) molecule which is later integrated into the host genome. Nucleoside reverse-transcriptase inhibitors provide the backbone of first-line HIV regimens, however, chronic use of this class of drug has been associated with several deleterious side-effects which manifest as conditions such as cardiomyopathy, hepatic steatosis (with or without lactic acidosis), myopathy and neurological disorders such as Alzheimer’s and Parkinson’s disease. A decrease in mitochondrial (mt) DNA and mtDNA-encoded proteins used for processes like oxidative phosphorylation (OXPHOS) may result in the accumulation of reactive oxygen species (ROS) which could, in turn, result in mitochondrial damage and affect cellular functions.

However, not all mitochondrial toxicity (MT) stems from the chronic use of NRTI/NtRTI-based ART. Several in vitro studies have shown that the interaction between HIV and the mitochondria could also contribute to mitochondrial dysfunction via inflammatory conditions which increase the expression of pro-inflammatory cytokines, including tumour necrosis factor-alpha, that inhibit mitochondrial function and promote cellular apoptosis.

The measurement of MT is complex. Currently accepted methods, which rely on measuring the mtDNA relative to nuclear (n) DNA (mtDNA/nDNA) ratio, require costly specimens that are difficult to obtain, such as muscle biopsies. While several studies have suggested that peripheral blood mononuclear cells (PBMCs) should reflect what is found in muscle biopsies, other concerns with the current approach include; 1) the possible amplification of homologues nDNA pseudogenes by mtDNA specific-primers, due to duplication of the mitochondrial genome within the nuclear genome; 2) errors caused by using repetitive and/or highly variable regions from genes such as beta-actin and 18S ribosomal (r) RNA, and; 3) the size differences between mitochondrial and nuclear genomes contributing to a dilution bias which makes interpreting the results difficult. Recently, two biomarkers; serum fibroblast growth factor 21 (FGF-21) and serum growth and differentiation factor 15 (GDF-15), have been identified by several studies as possible alternatives to the current method of MT detection. To the best of our knowledge, these biomarkers have, however, not been assessed in the study of MT in HIV-infected individuals.

The present study investigated the possible associations between disease severity and use of combination (c) ART, and mitochondrial damage. The associations between the cluster of differentiation 4 positive (CD4+) T-cell count, viral load (VL), and the use of cART and the extent of MT before initiation of cART, 12-months post-cART, and the change over time were also assessed. In addition, the concentration of the systemic biomarkers (FGF-21 and GDF-15) was compared with the changes in mtDNA/nDNA ratio, as a measure of MT, in HIV-infected individuals.

Stored, remnant plasma and PBMC samples that had previously been collected from HIV-positive participants before initiation of NRTI-based cART and again after 12-months of treatment, were used in the present study. In addition, 15 healthy, HIV-negative volunteers were recruited as control subjects. Plasma samples and PBMCs had been stored at -80 degrees Celsius (℃) following collection. The extent of MT was determined by detecting changes in mtDNA content, measured as the mtDNA/nDNA ratio by utilizing a quantitative polymerase chain reaction (qPCR) assay. The systemic levels of FGF-21 and GDF-15 were assessed by measuring the concentrations of these biomarkers present in plasma samples using suspension bead array technology.

From the results obtained, it was found that there was a depletion in mtDNA copy number in the HIV-infected individuals following a 12-month NRTI-based cART regimen. In addition, when compared to the HIV-negative controls, the HIV-positive participants displayed lower expression of mtDNA. Furthermore, there was also evidence of a significant association between the age of the participant and the levels of mtDNA, in that older participants displayed a reduction in mtDNA levels. While not significant, tobacco use was found to influence mtDNA levels, with most of the tobacco users in the current study displaying a decrease in mtDNA expression. From these findings, it would be recommended that the influence of both age and tobacco use on the expression of mtDNA be studied further. In contrast, no associations between the expression of mtDNA and the participants’ CD4+ T-cell count (baseline and 12-months following treatment) and VL (baseline), were found.

It was also observed that GDF-15 was a more sensitive biomarker than FGF-21, which showed negligible differences in concentration in either the HIV-positive (baseline and 12-months post-cART) or the healthy, HIV-negative participants. The systemic levels of GDF-15 were higher in the HIV-infected participants, at both baseline and 12-months following cART, when compared to the HIV-uninfected participants.

Furthermore, the GDF-15 concentrations had a stronger associated with lower baseline CD4+ T-cell count and immune recovery, rather than with the viral load of the individuals. However, no correlation between the biomarkers FGF-21 and GDF-15 and the standard measure of MT, the mtDNA/nDNA ratio, was found. This would indicate that the biomarkers may not be a suitable alternative for determining changes in mtDNA in HIV-infected individuals placed on NRTI-based ART regimens. Growth and differentiation factor 15 levels may, however, be indicative of disease progression, particularly in relation to the CD4+ T-cell count.