Increased plasma levels of extracellular mitochondrial DNA during HIV infection: A new role for mitochondrial damage-associated molecular patterns during inflammation

Abstract

HIV infection is characterized by a chronic inflammatory state. Recently, it has been shown that mitochondrial DNA (mtDNA) released from damaged or dead cells can bind Toll like receptor-9 (TLR9), an intracellular receptor that responds to bacterial or viral DNA molecules. The activation of TLR9 present within monocytes or neutrophils results in a potent inflammatory reaction, with the production of proinflammatory cytokines. We measured plasma levels of mtDNA in different groups of HIV⁺ patients, i.e., those experiencing an acute HIV infection (AHI), long term non progressors (LTNP), late presenters (LP) taking antiretroviral therapy for the first time, and healthy controls. We found that in AHI and LP mtDNA plasma levels were significantly higher than in healthy individuals or in LTNP. Plasma mtDNA levels were not correlated to peripheral blood CD4⁺ T cell count, nor to markers of immune activation, but had a significant correlation with plasma viral load, revealing a possible role for mtDNA in inflammation, or as a biomarker of virus-induced damage.

Introduction

The advent of combined antiretroviral therapy has deeply modified the prognosis of patients with human immunodeficiency virus type-1 (HIV-1) infection, but their life expectancy is still shorter than that of uninfected persons (Antiretroviral Therapy Cohort Collaboration, 2008). Several factors could account for this phenomenon, but the most recent data indicate that the vast majority of complications could be sustained, at least in part, by a chronic inflammatory state (Volberding and Deeks, 2010). This could be triggered and/or maintained by residual viral replication, persistent viral expression, loss of immunoregulatory cells that should dampen immune activation, increased fibrosis of lymph nodes, and microbial translocation (Deeks, 2009, Kuller et al., 2008).

Mitochondria are the main intracellular source of energy, in form of ATP, but can also produce dangerous molecules such as reactive oxygen species. Moreover, when cells are coping with an insult that is potentially harmful for the cell or for whole organism, they can release into the cytoplasm molecules that are able to trigger cell death such as cytochrome c or apoptosis inducing factor (Green and Kroemer, 2004).

Mitochondria contain several copies of a circular genome, the mitochondrial DNA (mtDNA), akin to a prokaryotic plasmid, codifying key proteins of the oxidative phosphorylation system, whose degradation origin formyl peptides. Recently, it has been shown that either mtDNA or degraded mitochondrial peptides can be involved in the pathogenesis of the systemic inflammatory response syndrome (SIRS), a condition that often affects patients who survive a trauma, and is characterized by the presence of shock and compromised function of several organs (Zhang et al., 2010). SIRS closely resembles sepsis, and is due, at least partially, to molecules called “damage-associated molecular patterns” (DAMPs). This novel, expanding family includes hyaluronan fragments, heat shock proteins, S100 proteins, amyloid-β, uric acid, interleukin (IL)-1α, IL-33, and the DNA-binding nuclear protein “high mobility group box 1” (Sims et al., 2010). DAMPs are conceptually similar to a set of conserved molecules defined “pathogen-associated molecular patterns” (PAMPs), which are produced and released by several microorganisms. PAMPs trigger a potent innate immune response, causing inflammation through the engagement of several Toll-like receptors (TLR), which can also be bound by DAMPs.

During trauma, mitochondrial DAMPs (MTDs) are released by damaged tissues, and found in high amounts in patients' plasma (Zhang et al., 2010). MTDs are represented by formyl peptides, that bind the formyl peptide receptor-1 (FPR-1), and mtDNA, that binds TLR-9, an intracellular receptor present in the endosomial system that responds to bacterial DNA, viral DNA and synthetic oligodeoxynucleotides containing unmethylated CpG motifs, like mtDNA. Injecting in rats MTDs equivalent to 5% of the liver causes a marked inflammation, with accumulation of albumin and elastase in lungs, influx of inflammatory cells in the airways and appearance of tumor necrosis factor (TNF)-α followed by IL-6 (Zhang et al., 2010).

It has been hypothesized that cells that are damaged during several pathological conditions such as chronic inflammations, ischemia–reperfusion injuries, or different infections can release mitochondrial structures (in particular: mtDNA, N-formyl peptides, cardiolipin, cytochrome c, ATP, carbamoyl phosphate synthetase-1, and finally reactive oxygen species) that trigger inflammation (Krysko et al., 2011, Manfredi and Rovere-Querini, 2010). As HIV-1 infection is characterized by a persistent chronic inflammation, we wondered whether MTDs could play a role in triggering or sustaining this phenomenon. For this purpose, we have studied plasma levels of mtDNA in HIV-1 positive patients showing a different course of the infection, and have correlated such levels to the activation of the immune system and to the plasma viremia. We analyzed individuals during acute primary infection (Cossarizza et al., 1995), patients with an advanced infection (including those with full blown acquired immunodeficiency syndrome, AIDS) but still naïve for antiretroviral therapy, and those defined “long term non progressors” (LTNPs), who had been infected since at least 8 years, always out of treatment, but with a normal number of CD4⁺ T cells, a low grade of apoptosis (Franceschi et al., 1997) and a good immunological control of the virus.