New study findings shows that the SARS-CoV-2 coronavirus is also able to cause epigenetic changes and transcriptomic reprogramming in monocytes of COVID-19 patients.
The study findings were published on a preprint server and are currently being peer reviewed.
Our results reveal that peripheral blood monocytes from severe COVID-19 patients display aberrant DNA methylomes and transcriptomes associated with functions related to IFN type I signaling and antigen presentation, among others. The changes are significantly associated with organ damage and with DNA methylation changes occurring in bacterial sepsis.
Finally, our analysis suggests that pro-inflammatory cytokines, the release of immature or aberrant monocytes, and specific dysregulated immune cell–cell communication events may be responsible for some epigenetic changes.
To date, there have been very few DNA methylation studies addressing the involvement of COVID-19 DNA methylation in regulating the Angiotensin-converting Enzyme 2 (ACE2) type I membrane receptor gene, which is present in arterial, lung type II alveolar cells, where it acts as a SARS-CoV-2 receptor.
There is a suggestion that the host epigenome may represent a risk factor for COVID-19 infection. Very few studies have reported alterations in DNA methylation in relation to immune responses[73–75]. Our study aimed to explore the involvement of DNA methylation in relation to a severe COVID-19 outcome in the myeloid compartment, which is directly related to systemic inflammation.
We specifically studied monocytes because it is the cell type that undergoes the most dramatic transcriptomic reprogramming during COVID-19 infection[13,21,23,76]. In this regard, our study provides the first instance of DNA methylome profiling in a specific immune cell type in COVID-19 patients.
Our data revealed that most DNA methylation changes in monocytes derived from severe COVID-19 patients occurred in genomic sites enriched in PU.1 binding motifs, consistent with earlier studies showing its role as a pioneer TF directly recruiting TET2 and DNMT3b. In our case, most DNA methylation changes occurred in genes related to cytokines, MHC class II proteins and IFN signaling.
Similar results about the defective function of MHC-II molecules and activation of apoptosis pathways were obtained in single-cell atlas studies of PBMCs from severe COVID-19 patients[6,21,78,79] and in sepsis[80,81].
We found that DNA methylation changes in severe COVID-19 patients share some features with sepsis, especially those associated with the expression of tolerogenic cytokines like IL-10. The acute phase of these infections suggests a dysregulated inflammatory host response, resulting in an imbalance between pro-inflammatory and anti-inflammatory mediators.
Some studies have suggested that viral components induce STAT1 dysfunction and compensatory hyperactivation of STAT3 in SARS-CoV-2-infected cells. We noted the involvement of kinases like JNK, and earlier studies had shown that COVID-19 infection activates the JNK and ERK pathways that end in the AP-1-dependent gene expression of proinflammatory cytokines.
One of the most strongly affected TFs is STAT2, together with STAT6, which could be linked to the aberrant IFN signaling in monocytes in COVID-19. The presence of STAT2 downregulation also suggests a deficiency in the ability to cross-present to CD8+ T cells .
We also identified GRE binding sites in association with DNA methylation changes. Generally, the glucocorticoid receptor (GR) is activated when patients are treated with GC. However, we also noted significant GRE enrichment in patients who were not treated with GC, suggesting that endogenous production of GC in COVID-19 patients could regulate GR and affect DNA methylation at its genomic binding sites.
GC is also produced endogenously in sepsis patients in whom cytokines like IL-1β, TNFα and IL-6 induce its production from the adrenal cortex using cholesterol as a substrate to reduce inflammatory responses[86,87]. These cytokines were hypomethylated and overexpressed in our dataset, consistent with the results of other studies that have reported increased levels in the serum of COVID-19 patients[88,89]. GRE binding sites are enriched in the DMPs common to COVID-19 and sepsis.
GR is a nuclear receptor expressed in most cell types that can trigger the expression of anti-inflammatory genes through direct DNA binding. Furthermore, GRE represses the action of other inflammation-related TFs, including members of the NF-KB and AP-1 families[90,91], which are also known to be downregulated in our cohort.
Taken together, our results suggest the existence of a relationship between extracellular factors associated with the cytokine storm occurring in severe COVID-19 and DNA methylation changes. Several studies have shown an increase in the levels of inflammatory cytokines in severe COVID-19, which may contribute to the severity of the disease.
However, it is also possible that the DNA methylation changes are partly due to the release of immature or altered monocytes from myelopoiesis, as reported for severe COVID-19[93–96] and sepsis. Release of immature myeloid cells from the bone marrow in severe COVID-19 is reminiscent of emergency myelopoiesis.
This is a well-known phenomenon, characterized by the mobilization of immature myeloid cells to restore functional immune cells, and by its contribution to the dysfunction of innate immunity. In fact, a proportion of the hypermethylated CpGs in monocytes from severe COVID-10 patients overlap with regions that become demethylated during myeloid differentiation.
This suggests that part of the hypermethylated CpG sites in isolated peripheral blood CD14+ might be associated with aberrantly differentiated monocytes released into the bloodstream in severe COVID-19 patients. However, the small numbers of CD34+ cells in the PBMC fraction of COVID-19 patients and the lack of CD14+ cells in this subset suggest no interference with our results for CD14+CD15-cells, isolated with our method.
The relationship between DNA methylation and gene expression is complex. DNA methylation patterns are cell-type-specific and are established during dynamic differentiation events by site-specific remodeling at regulatory regions. In general, methylation of CpGs located in gene promoters, first exons, and introns is negatively correlated with gene expression.
The analysis of our data shows that there is an inverse correlation between the CpG methylation changes and the expression levels of the closest genes. The comparison of the inferred TFs associated with DNA methylation changes and gene expression changes shows common factors like IRF2 and IRF3, which regulate downregulated genes and hypermethylated CpGs. In this context, it is possible that reduced levels of IFN regulatory factor IRF3 or defective IRF7 function reduces the level of IFNα/β gene expression, increasing the sensitivity to viral infection[12,101].
Finally, analysis of cell–cell communication has revealed potential relationships between DNA methylation changes and altered communication of monocytes and other immune cells (e.g., T, plasma B and NK cells). Our data suggest the potential reduction of interactions between monocytes and NK cells through CD160, which mediates the antibody-dependent cell-mediated cytotoxicity that it is essential for IFNγ production.
The potentially greater interaction between monocytes and Treg through multiple ligand and receptor pairs is an interesting finding, since Tregs are immunosuppressive cells responsible for maintaining immune homeostasis. In any case, the use of CellPhone DB is useful for inferring cell-cell communications events; however additional validation experiments would be necessary to validate interactions and activation of downstream signaling pathways.
In our study, we could not determine whether the observed DNA methylation alterations in COVID-19 were the cause or the consequence of the changes in gene expression. The analysis of mild COVID-19 cases, in which the DNA methylation and expression level of a few genes showed differences in their similarities with severe COVID-19 cases, suggests that there are cases where expression changes might anticipate DNA methylation changes. In any case, it is reasonable to propose that some DNA methylation changes help perpetuate dysregulated immune responses.
Some limitations of our study include the size of the cohort, and the unequal numbers of individuals administered particular drugs in the different patient groups, which could have affected the COVID-19 data. However, despite these limitations, we found no significant differences among severe COVID-19 patients with respect to the time they were admitted to the ICU or began to receive treatment. This suggests that DNA methylation is quite a general occurrence in the context of COVID-19. Another limitation concerns the cell population analyzed, since the method for monocyte isolation comprises two populations, CM and IM, one of which (CM) is expanded in the patient group. However, the analysis including the monocyte subsets as a covariate indicates that there are no major differences. Finally, in the comparison with DNA methylation of progenitor cells, it is important to note that the DMPs were overlapped with genomic regions, and not single-base data, and further analyses would be required.
Future studies would benefit from having access to a wider cohort in which it is possible to identify significant links between alterations and drug treatments. Incorporating mild and asymptomatic cases would improve our ability to dissect drug- and severity-related specificity in relation to DNA methylation changes. As is the case for other medical conditions, the analysis of DNA methylation changes would be very likely to help predict disease severity, progression and recovery.