International Study Finds That SARS-Cov-2 Causes Epigenetic Changes And Transcriptomic Reprogramming In Monocytes Of COVID-19 Patients!
: Alarmingly. 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!
Such effects by the virus could be associated with the release of aberrant immature monocytes, increased systemic levels of pro-inflammatory cytokines, and changes in immune cell crosstalk in these individuals.
The COVID-19 Research
was conducted by researchers from Josep Carreras Research Institute (IJC) - Spain, Wellcome Sanger Institut e- United Kingdom, University of Cambridge - United Kingdom, Universitat Autònoma de Barcelona - Spain, Vall d'Hebron University Hospital – Spain and the East China Normal University (ECNU), Shanghai – China.
COVID-19 manifests with a wide spectrum of clinical phenotypes, ranging from asymptomatic and mild to severe and critical.
At present, severe and critical COVID-19 patients are characterized by marked changes in the myeloid compartment, especially monocytes.
Little is known however about the epigenetic alterations that occur in these cells during hyperinflammatory responses in severe COVID-19 patients.
The study team obtained the DNA methylome and transcriptome of peripheral blood monocytes from severe COVID-19 patients. DNA samples extracted from CD14+CD15-monocytes of 48 severe COVID-19 patients and 11 healthy controls were hybridized on MethylationEPIC BeadChip arrays. In parallel, single-cell transcriptomics of 10 severe COVID-19 patients were generated. CellPhoneDB was used to infer changes in the crosstalk between monocytes and other immune cell types.
The study findings indicated DNA methylation changes in CpG sites associated with interferon-related genes and genes associated with antigen presentation, concordant with gene expression changes.
It was noted that these changes significantly overlapped with those occurring in bacterial sepsis, although specific DNA methylation alterations in genes specific to viral infection were also identified.
The study team also found these alterations to comprise some of the DNA methylation changes occurring during myeloid differentiation and under the influence of inflammatory cytokines.
A progression of DNA methylation alterations in relation to the Sequential Organ Failure Assessment (SOFA) score was found to be related to interferon-related genes and T-helper 1 cell cytokine production.
CellPhoneDB analysis of the single-cell transcriptomes of other immune cell types suggested the existence of altered crosstalk between monocytes and other cell types like NK cells and regulatory T cells.
The study findings show the occurrence of an epigenetic and transcriptional reprogramming of peripheral blood monocytes, which could be associated with the release of aberrant immature monocytes, increased systemic levels of pro-inflammatory cytokines, and changes in immune cell crosstalk in these patients.
The study findings were published on a preprint server and are currently being peer reviewed.
SARS-CoV-2 coronavirus, the etiological agent of COVID-19, attacks the nasopharyngeal mucosa. In response, the host (humans) mounts an immune response at the local mucosa and systemic level, whose delicate balance determines the course of illness.
Though immune responses to COVID-19 are diverse, they could range between asymptomatic to mild and severe pneumonia, acute respiratory distress syndrome, and death.
Various past studies have elucidated the impact of exacerbated immune responses associated with severe COVID-19. Yet, many aspects of hyperinflammatory responses in severe COVID-19 that occur at a systemic level remain unclear. For instance, epigenetic alterations in the myeloid compartment, especially monocytes of critical COVID-19 patients.
It has been noted that inflammatory monocytes can induce a cytokine storm in severe COVID-19 patients. For instance, their DNA methylation profiles, among other epigenetic marks, vary in response to inflammatory cytokines e.g., interleukin (IL)-6 and interferon-gamma (IFNγ).
Also, single-cell omics studies have also shown that monocytes from severe COVID-19 patients show reduced expression of class II major histocompatibility complex (MHC-II) antigens.
Importantly, the differentiation and activation of monocytes and other myeloid cells are directly associated with epigenetic mechanisms. Thus, profiling the epigenetic and transcriptomic reprogramming in monocytes could help understand why some immune pathways get dysregulated during severe COVID-19.
The study team obtained peripheral blood mononuclear cells (PBMCs) of 58 patients with reverse transcription-polymerase chain reaction (RT-PCR) confirmed COVID-19 for examining transcriptomic changes in this specialized cell population. All these patients contracted COVID-19 between October and November 2020 and had taken admission to the intensive care unit (ICU) of Vall d’Hebron University Hospital in Barcelona.
The study team utilized 48 of the 58 samples for DNA methylation analysis and PBMCs from 10 of the 58 patients for droplet-based single-cell ribonucleic acid sequencing (scRNA-seq) data. The researchers used 14 additional samples from other patients for DNA methylation and expression validation, of which nine, five, and six were from severe and mild COVID-19 patients, respectively, and six were healthy donors (HDs). The control population for the DNA methylation analysis comprised 11 HDs.
The study team utilized flow cytometry (FC) to isolate the monocyte population, followed by DNA isolation. Next, the researchers hybridized 500 nanograms of this DNA on Infinium Methylation EPIC BeadChip arrays enabling the assessment of over 850,000 methylation sites at single-nucleotide resolution per sample.
The study team also obtained the Cy3 and Cy5 fluorescent intensities from the methylated and unmethylated alleles.
Lastly, the researchers assessed beta (b) and M values. The former is the ratio of methylated probe intensity to the sum of the methylated and unmethylated probe intensities. The latter is the log2 ratio of the intensities of the methylated and unmethylated probes.
The study findings showed that in a healthy individual, the proportions of monocytes can shift between classic (CM), intermediate (IM), and non-classic (NCM) monocytes. The monocyte subpopulations in the study cohort had an average purity of 98% and showed a significant increase and a decrease in the CM and NCM populations, respectively.
In order to avoid neutrophil contamination, the study team isolated singlets and CD14+CD15- cells.
Interestingly, when compared to health donors (HDs), severe COVID-19 patients had 2211 differentially methylated positions (DMPs) in CpG sites. The CpG sites in DNA are rich in cytosine and guanine nucleotides separated by a phosphate.
Also, the study team noted that 1773 and 438 of these 2211 DMPs were hypermethylated and hypomethylated, respectively. Furthermore, the principal component analysis (PCA) of these DMPs showed that the two groups of monocytes (COVID-19 and HD) separated along the first PCA axis.
These study findings did not vary with the pre-existing conditions of these COVID-19 patients or their treatment with dexamethasone. The PCA also showed the overlap of patients with different clinical parameters.
The study team noted an enrichment in promoters and enhancers in the DMPs of both hyper- and hypo-methylated clusters. Furthermore, the team noted a progression of DNA methylation alterations concerning the Sequential Organ Failure Assessment (SOFA) score, which, in turn, was related to IFN-related genes and T helper 1 (Th1) cell cytokine production.
The CellPhoneDB database is a public repository of ligands, receptors, and their interactions to enable understanding of cell-cell communications. This analysis of the single-cell transcriptomes of other immune cells suggested the changes in crosstalk between monocytes, natural killer (NK) cells, and regulatory T cells (Treg).
The study findings showed that the alterations in immune cell crosstalk contributed to transcriptional reprogramming in monocytes in severe COVID-19 patients, which, in turn, was governed by IFN-related genes and genes associated with antigen presentation and chemotaxis.
The study findings demonstrated a complex relationship between DNA methylation changes in COVID-19 patients and changes that occur during myeloid differentiation and are inducible by pro-inflammatory cytokines. Some of such DNA methylation changes perpetuated dysregulated immune responses that occurred parallelly with changes in expression levels of the closest genes. Also, these changes significantly overlapped with the epigenetic changes found in patients with sepsis, and others showed an association with organ dysfunction.
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