In their recent study, the authors from the Netherlands investigated the expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-2 (IDO2) in patients with post-acute sequelae of COVID-19 (PASC), and its correlation with PASC symptoms and pathophysiology.
COVID-19 patients often experience prolonged symptoms that can last for months, a condition known as long COVID or PASC. Patients with long COVID experience a wide range of organ dysfunctions and clinical symptoms, and the most disabling are severe fatigue, cognitive dysfunction, post-exertional malaise, shortness of breath, and autonomic dysfunction.
Previous studies revealed enhanced degradation of an essential amino acid tryptophan to kynurenine and further breakdown products in SARS-CoV-2 infected individuals, likely caused by the interferon-induced tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO)1, which is typically induced by viral infections. The authors of this study noted that otherwise rarely expressed tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-2 (IDO2) degrades the essential amino acid tryptophan similar to isotype IDO1. In their previous work, the same research group used immunohistochemical analyses to show that the tryptophan-catabolizing enzyme IDO2 is abundantly expressed and active in the lung, heart and brain cells, monocytes and lymphocytes of patients with fatal COVID-19. These findings were associated with reduced systemic tryptophan and elevated breakdown products (kynurenine, quinolinic acid and 3OH-kynurenine), as well as with autophagy and apoptosis. These results indicate that IDO2 contributes to the pathophysiology of fatal COVID-19.
Additionally, the authors stated that IDO2 expression can be induced by a ligand-activated transcription factor, the aryl hydrocarbon receptor (AHR). Intriguingly, infection with coronaviruses has been shown to activate AHR in mice. Moreover, AHR was found in the nucleus of IDO2-expressing cells in fatal COVID-19, indicating its transcriptional activity, and likely driving the expression of IDO2.
These findings led scientists to investigate the role of IDO2 expression in patients with PASC.
About the study
Researchers analyzed samples from four distinct study cohorts, with 12–15 samples of each patient group. PASC patients had symptoms for at least three months and reported a wide variety of severe symptoms (mainly fatigue, post-exertional malaise and cognitive problems). None of the PASC patients were hospitalized during SARS-CoV-2 infection, in order to avoid a bias in PASC symptoms induced by hospitalization. The presence of SARS-CoV-2 infection was determined by PCR or a rapid antigen test. Controls were participants with SARS-CoV-2 who had not been hospitalized, and who did not have any remaining symptoms. The patients and controls were not vaccinated before the infection.
The results showed that IDO2 was expressed in peripheral blood mononuclear cells (PBMC) from PASC patients long after the initial infection by SARS-CoV-2. Monocytes and lymphocytes from PASC patients that were collected 294 (median) days after the SARS-CoV-2 infection showed IDO2 activity. IDO2 was found in monocytes (CD14+) and lymphocytes (CD3+), mostly CD4+ and CD20+ lymphocytes. In addition, IDO2-expressing PBMC from patients with PASC had lower cellular levels of tryptophan and higher levels of kynurenine compared to those from fully recovered patients. This was paralleled by higher levels of kynurenine metabolites in the circulation, particularly xanthurenic acid, and for some patients by higer levels of 3OH-anthranilic acid.
Patients in all groups had elevated levels of systemic kynurenine and downstream metabolites, even though the infection was at least two months prior to blood sampling. The plasma samples from fatal/severe COVID-19 cases had an abundance of 3OH-kynurenine, quinolinic acid and kynurenic acid, as well as the lowest levels of tryptophan and 3OH-anthranilic acid in comparison to the other groups. It is noteworthy that 3-hydroxykynurenine (3-HK) and quinolinic acid have neurotoxic effects. In patients with PASC, elevated levels of xanthurenic acid were found, which has been reported to affect mitochondrial activity. On the other hand, elevated median levels of 3OH-anthranilic acid were observed even in individuals who were never hospitalized or who were hospitalized with no or only mild residual symptoms.
The IDO2 expression was decreased in PBMC from patients with PASC upon administration of the AHR antagonist. The AHR was found to be localized in the nucleus of IDO2-positive cells, consistent with the concept that the AHR is driving IDO2 expression. IDO2-expressing cells displayed the marker for autophagy (L3CB), but not, or minimally, that of apoptosis (cleaved-caspase 3). The incubation with the AHR antagonist inhibited autophagy and apoptosis, but, AHR remained in the nucleus despite treatment with the AHR antagonist.
The IDO2-expressing PBMC had high levels of kynurenine and low levels of tryptophan and an aberrant cellular metabolism with autophagy and reduced mitochondrial function. The basal and maximal oxygen consumption rates were reduced in PBMC from PASC patients compared to those of recovered patients and healthy individuals, according to mitochondrial stress tests in PBMC. The spare respiratory capacity was reduced in PBMC from PASC patients and recovered patients in comparison to healthy controls. These findings indicate that monocytes and lymphocytes expressing IDO2 from patients with PASC display a diminished capacity to respond to stress conditions. The pathophysiological mechanisms that result in these aberrant mitochondrial activities are not clear, although it is possible that xanthurenic acid may be involved.
Additionally, scientists analyzed PBMC from patients with PASC and fully recovered patients for 219 metabolites covering most of the central carbon metabolic pathways, including glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and NAD metabolism. PBMC from PASC patients showed elevated kynurenine and lower tryptophan compared to those from fully recovered patients. In addition to tryptophan, 14 amino acids were reduced in PBMC from patients with PASC. Also, the reduced levels of pyruvate, acetyl-CoA, citric acid and nicotinamide riboside may be related to altered activity of the Krebs cycle and mitochondria in general.
Researchers then performed the immunohistochemical analysis on brain samples from two deceased PASC patients. The first patient was a 52-year-old male with a history of a stroke (7 years ago) and symptomatic epilepsy. He displayed symptoms of neuro long COVID for four months after he was diagnosed with the SARS-CoV-2 infection. The patient died from status epilepticus. The second patient was an 80-year-old male with PASC symptoms for five months after he was diagnosed with SARS-CoV-2 infection. He died of palliative sedation. The immunohistochemical analysis of brain tissue revealed abundant IDO2 expression, which was spatially related to 3OH-anthranilic acid and quinolinic acid, and with nuclear, transcriptionally active AHR. The IDO2 expression was associated with abundant autophagy and limited apoptosis in both brains. Unexpectedly, the results showed a significant overlap between the IDO1 stain and IDO2 stain in both brain samples. The N-protein of SARS-CoV-2 was absent.
Overall, this study indicates that the AHR-IDO2-kynurenine pathway is involved in SARS-CoV-2-induced pathology, including PASC. The expression and activity of IDO2 was observed in PBMC and brain tissue from patients with PASC long after the SARS-CoV-2 infection. IDO2 expression was driven by the AHR and coincided with an altered cellular metabolism, which was reflected by autophagy, and reduced mitochondrial functions in blood cells. Ex vivo, an AHR antagonist stopped IDO2 expression and autophagy.
The SARS-CoV-2 infection induces IDO2 expression, but the resulting pathology appears to be related to the specific kynurenine catabolites. Whether IDO2 activity indeed determines PASC pathophysiology may be addressed in clinical trials focusing on the IDO2 pathway. The authors suggested that an AHR antagonist might be a therapeutic option because there are no potent inhibitors of IDO2 available for humans. They noted that some pharmaceutical companies have an AHR antagonist in their portfolio, that are nearing completion or have completed phase 1 trials.
This article was published in eBioMedicine (part of the LANCET Discovery Science)
Guo L et al. Prolonged indoleamine 2,3-dioxygenase-2 activity and associated cellular stress in post-acute sequelae of SARS-CoV-2 infection. EBioMedicine 2023;94: 104729. (Open Access) https://doi.org/10.1016/j.ebiom.2023.104729