Investigating mitochondrial dysfunction and inflammation in long-COVID and primary mitochondrial disease

Abstract

SARS-CoV-2, identified in 2019, is a multisystemic illness that has resulted in ~780M infections worldwide. A subgroup of COVID-19 patients experiences lingering symptoms, including fatigue, shortness of breath, post-exertional malaise, and cognitive impairment. This chronic, debilitating disorder, herein referred to as “long COVID” (LCOV), has several phenotypic similarities to primary mitochondrial disorders (MITO). Of note, both populations experience an exacerbation of symptoms during physical and mental activity. Primary mitochondrial diseases result in a dysfunction of the mitochondrial respiratory chain, leading to energy-related impairments. Recent evidence has implicated mitochondrial dysfunction in the pathogenesis of LCOV; however, few studies have explored the relationship between mitochondrial dysfunction, respiration, and cells of the adaptive immune response. Since lymphopenia has been reported to affect CD4⁺ and CD8⁺ cells in COVID-19, and resident T cells adapt their utilization of major energy pathways in response to immune challenge, we sought to investigate immune metabolism in LCOV and MITO by analyzing mitochondrial respiration and dynamics in these T cell subsets. Blood samples (~40 mL) were collected from the antecubital vein of healthy control (CON), MITO, and LCOV individuals. PBMCs were separated from whole-blood by density-gradient centrifugation and further isolated into CD4⁺ and CD8⁺ cells by magnetic-activated cell sorting (MACS). Metabolic variables including oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed with a Seahorse XF metabolic analyzer. MitoSOX and TOMM20 stains were also performed to detect superoxide levels and quantify mitochondrial volume, respectively. Finally, a multiplex cytokine analysis was used to assess plasma inflammatory markers. We found that CD8⁺ but not CD4⁺ T cell metabolism, was dysregulated in LCOV and MITO. Both clinical populations had lower basal and maximal OCR. Reactive oxygen species (i.e., the by-products of oxidative phosphorylation) appeared to be the proximate cause of mitochondrial dysfunction in MITO as mutations in mtDNA impair ETC function, causing electrons to leak and react with oxygen. LCOV exhibited lower total mitochondrial volume compared to healthy controls, resulting in lower mitochondrial respiration in specific immune cells. Additionally, systemic biomarkers of mitochondrial stress, such as plasma lactate, GDF-15, and FGF-21, remained within normal or non-indicative ranges in LCOV, suggesting no systemic mitochondrial impairment. The circulating cytokine profile in LCOV did not mirror the characteristic inflammatory pattern of MITO observed in the current study, which involved consistent, directional changes in key innate immune cytokines. Therefore, while there remain some clinical similarities with MITO, LCOV has a distinct underlying mechanism, marked by cell-specific mitochondrial changes without systemic dysfunction.