Background
COVID-19 is a novel coronavirus first discovered in Wuhan, China, in late 2019 and declared a pandemic by the World Health Organization (WHO) in March 2020 [
1]. Two years later, several variants were detected, and over 5 million deaths were recorded [
2].
Epigenetics refers to the study of gene activity regulation and expression changes that are not dependent on the DNA sequence [
3]. DNA methylation, one of the hallmarks of epigenetics, involves the covalent addition of a methyl group to the 5′-carbon of a cytosine ring. Methylation is inversely correlated with gene expression [
4]. For instance, hypermethylation is often associated with the downregulation of genes, recently demonstrated in the ACE2 gene [
5]. Basic embryological and early developmental processes are controlled by DNA methylation in mammalians [
6]. Further, DNA methylation is also involved in disease and upon exposure to environmental factors [
7].
Patients with severe COVID-19 infection often suffer from respiratory failure and may require mechanical ventilation, associated with a mortality rate of up to 50% [
8,
9]. Several predictors of outcomes in critically ill patients have already been identified. They include primarily clinical variables, biochemical markers, and comorbidities [
10]. DNA methylation of host cells can be altered during infections, which modulates the immune response [
11]. It has been recently shown that DNA methylation regulates the activity of the immune system in COVID-19 infections and is associated with clinical outcomes, such as the severity of the disease, its association with respiratory failure, and ICU admission [
12‐
14]. However, data regarding death or recovery in COVID-19 patients is lacking. In this study, we report the presence of immune-related differentially methylated genes that predict survival in critically ill COVID-19 patients.
Discussion
To our knowledge, this is the first longitudinal study to investigate the methylation profile in critically ill COVID-19 patients with ARDS under mechanical ventilation and identify a methylome signature that predicts survival.
We showed that the epigenetic signature of critical COVID-19 infection is enriched for immune response pathways, particularly type I Interferon signaling, which is a key signature of the host response to this virus [
14,
33,
39,
40]. Interferon-driven response plays a vital role in shaping the fate of a viral infection, affecting the activation and differentiation of immune cells and the virus spread [
14,
33,
39,
40]. Other differentially methylated genes also contribute to immune-related functions and viral pathogenesis. For example, IFNAR1 and IFNAR2 genes partake in type I interferon-related pathways as main receptors for interferon-alpha and beta [
41]. Another gene is CLEC4M which encodes for the CD209L receptor and mediates the virus entry to epithelial and endothelial cells of various tissues [
42].
Compared to controls, critically ill COVID-19 patients showed a similar differential methylation pattern to previously reported studies [
12,
13,
30‐
35]. Konigsberg et al. recently identified 13033 differentially methylated CpGs, from which we have confirmed 3613 that represent 2290 genes [
40]. In particular, we found that the probes of robust predictors of COVID-19 were hypomethylated in our patients, including genes involved in interferon regulation and viral response. This may suggest an increased expression of those genes during critical COVID-19 infection, which has also been reported earlier [
14].
Interestingly, at inclusion, we did not observe any intra-differences in DNA methylation between dead and recovered groups. However, the same comparison showed significant differences at the last recorded time point, suggesting that most changes occurred as the disease progressed. Further, the inter-comparison of methylation changes between baseline and the last recorded time point revealed hypermethylation of pathways linked to host immune response such as interferon-alpha, TNF alpha, IL-6, and IL-2 signaling in patients who recovered, but not in those who died. Among the reported genes in patients who recovered, AIM plays a vital role in the immune response. It initiates the inflammatory cytokines release upon sensing exogenous nucleic acid inside the host cell, followed by pyroptosis (lytic cell death) [
43]. It has been associated with intensified immune responses to COVID-19 [
44]. CpGs in that gene are promoter-associated, and their hypermethylation suggests reduced AIM expression in patients just before recovery; thus, its reduced activity might be related to improvement and survival. Among the genes we reported in patients who died, LZTFL1 is known to inhibit epithelial-mesenchymal transition (EMT) in the lungs in the presence of inflammation or cancer [
45,
46]. EMT is a well-known pathway in fibrosis that is activated in prolonged lung inflammation and tissue injury [
47]. In-vitro studies showed that COVID-19 upregulates EMT pathway genes [
48]. In our study, LZTFL1 was hypermethylated, which would be translated by a decrease in its expression, less inhibition of EMT, and progressive lung injury.
Time course differential methylation analysis identified 49 CpGs, two of which are beta-defensin genes. Beta-defensins are antimicrobial peptides, modulators of microbiome diversity and host-microbe equilibrium in the mucosa of oropharyngeal and tracheal highways, and regulators of inflammatory responses secreted by neutrophils during infections [
49]. They are one of the primary arms of the innate immune system, contributing to immune cell activation and proliferation [
36]. In our study, the lower methylation of those genes in non-survivors suggests a higher expression of antimicrobial peptides throughout their ICU stay.
CD4 and CD8 T cells are critical elements in anti-viral immunity; they work harmoniously to recognize viral antigens, proliferate, kill infected cells, neutralize the virus, and memorize the viral print to respond faster in the case of future encounters [
50]. Our deconvolution analysis confirmed a lymphopenic profile (low CD4 and CD8 proportion) in COVID-19 patients upon admission to the ICU. This is consistent with previous reports and could be interpreted as a sign of dysfunctional or exhausted immune cells [
51]. At late stages, CD4 T cell proportion increased in survivors, indicating the restored function of the immune system [
51]. Neutrophils showed a sudden increase in patients who died at the last two time points, which could reflect a prolonged inflammatory response, contributing to severe conditions [
52]. One of the plausible theories behind the increase in neutrophils and hyper expression of the beta-defensins and other immune-related genes is the re-occurrence of a cytokine storm before death.
Among the genes that predict mortality, ROCK1 plays a crucial role in apoptosis by regulating membrane blebbing, a characteristic feature of apoptotic cells [
53], and H1F0 through apoptosis-induced DNA fragmentation and cellular component disassembly [
54]. It is well known that the apoptotic execution pathway initiates cell death once activated by an abnormal immune reaction [
55]. This finding was reported in cancer cells that resist the activation of this pathway to escape anti-cancer therapeutics in vitro [
56] but never reported in vivo in COVID-19 infections. On the other hand, higher methylation of PSMB9, MRPS2, MFHAS1, and MAT2B genes are known to be expressed in COVID-19 patients with high viral load or severe infection [
57‐
61], which could translate to a lower expression of those genes. It might be possible that a less severe infection at ICU admission predicts better survival.
Cumulative data suggest that epigenetics play an important role in the pathophysiology of several pathologies such as cardiovascular disease, diabetes, and cancer [
7,
62‐
63]. Recently, epigenetic markers were suggested as potential indicators and biomarkers for disease detection and progression [
64]. In acute Charcot disease, a rare diabetes complication characterized by bone destruction, we have previously shown the presence of differentially methylated genes involved in the migration process during monocyte differentiation into osteoclasts [
65]. Further, epigenetic-based therapy is increasingly used in several disciplines such as immunotherapy and cancer [
66,
67]. Current experimental approaches in infectious diseases in general and viral infections, mainly, are promising [
68,
69].
This study has a few limitations. The sample size of our patients was relatively moderate; hence a higher number of participants might have enabled us to detect more methylation calls knowing that power calculations for the sample size are not established for epigenetic analysis. We conducted the study in early 2020 during the first wave of COVID-19 when the Alpha variant was the only one universally reported. Therefore, we cannot ascertain that the same methylation changes exist with different variants in vaccination or the constant changes in drug therapies.
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