Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature upon SARS-CoV-2 sensing by monocytes in COVID-19

Abstract

Alterations in the myeloid immune compartment have been observed in COVID-19, but the specific mechanisms underlying these impairments are not completely understood. Here we examined the functionality of classical CD14 ⁺ monocytes as a main myeloid cell component in well-defined cohorts of patients with mild and moderate COVID-19 during the acute phase of infection and compared them to that of healthy individuals. We found that ex vivo isolated CD14 ⁺ monocytes from mild and moderate COVID-19 patients display specific patterns of costimulatory and inhibitory receptors that clearly distinguish them from healthy monocytes, as well as altered expression of histone marks and a dysfunctional metabolic profile. Decreased NFκB activation in COVID-19 monocytes ex vivo is accompanied by an intact type I IFN antiviral response. Subsequent pathogen sensing ex vivo led to a state of functional unresponsiveness characterized by a defect in pro-inflammatory cytokine expression, NFκB-driven cytokine responses and defective type I IFN response in moderate COVID-19 monocytes. Transcriptionally, COVID-19 monocytes switched their gene expression signature from canonical innate immune functions to a pro-thrombotic phenotype characterized by increased expression of pathways involved in hemostasis and immunothrombosis. In response to SARS-CoV-2 or other viral or bacterial components, monocytes displayed defects in the epigenetic remodelling and metabolic reprogramming that usually occurs upon pathogen sensing in innate immune cells. These results provide a potential mechanism by which innate immune dysfunction in COVID-19 may contribute to disease pathology.

SciScore for 10.1101/2022.04.03.486830: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: COVIDITY study is a prospective observational serial sampling study of whole blood to observe the evolution of SARS-CoV-2 infection to characterize the host response to infection over time in peripheral blood (ethics approval obtained from the Health Research Authority, South Central Oxford C Research Ethics Committee). Consent: Samples from patients with moderate COVID-19 admitted to hospitals in London (Hammersmith Hospital, Charing Cross Hospital, Saint Mary’s Hospital) and eligible to participate in the MATIS trial61 provided consent (ethics approval by the Health Research Authority,
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The antibodies used in the stainings were the following: CD14 (61D3, eBioscience), CD3 (UCHT1, BD), CD19 (HIB19, BD), CD1c (L161, Biolegend),	CD14 suggested: None CD3 suggested: None CD19 suggested: None CD1c suggested: None L161 suggested: None
The antibodies used for intracellular staining were the following: H3K27Ac, H3K9Me2, H3K4Me3, H3K27Me3 (all from Cell Signaling Technology), TNF (Mab11, Biolegen) and IL-10 (JES3-907, Thermo Fisher Scientific)	H3K4Me3 suggested: None H3K27Me3 suggested: None Mab11 suggested: None IL-10 ( JES3-907 suggested: None
Intracellular staining of puromycin was performed using the anti-puromycin monoclonal antibody (1:600 dilution, clone R4743L-E8) for 45 minutes at 4 ºC.	anti-puromycin suggested: (Rafael Jose ArgÃ¼ello - Dendritic cell laboratory, Centre dÂ´Immunology de Marseille Luminy Cat# R474, RRID:AB_2827926)
Experimental Models: Cell Lines
Sentences	Resources
Generation of virus stocks: SARS-CoV-2 virus (SARS-CoV-2/England/IC19/2020 isolate, kindly provided by Wendy S Barclay) was expanded in Vero-E6 cells.	Vero-E6 suggested: None
Titration of virus stocks: For SARS-CoV-2 titration, samples were serially diluted in OptiPRO SFM, 2X GlutaMAX (1:10) and added to Vero cell monolayers for 1 hour at 37 °C, 5% CO2.	Vero suggested: None
For CCCoV titration, viral supernatants were serially diluted in DMEM, non essential amino acids (1:10) and added to MRC-5 (229E strain),	MRC-5 suggested: None
BSC-1 (OC43 strain) or LLCMK2 (NL63 strain) cell monolayers for 1 hour at 37 °C, 5% CO2.	BSC-1 suggested: None
Software and Algorithms
Sentences	Resources
Samples were run on a Fortessa instrument (BD Biosciences) and analyzed using FlowJo v.10.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Briefly, reads were aligned to the reference genome (GRCh38.99) using STAR v2.7.366 in the two-pass mode (ENCODE recommended parameters) and gene expression was quantified using featureCounts67.	STAR suggested: (STAR, RRID:SCR_004463)
Mapping statistics and quality control metrics from FastQC and RNA-SeQC68 indicated high data quality for all samples with no outliers detected.	FastQC suggested: (FastQC, RRID:SCR_014583)
Principal component analysis (PCA) with the prcomp function was used to explore the relationship between samples, after the filtered gene counts were transformed using a regularized log transformation from the DESeq270 package.	DESeq270 suggested: None
Differential gene expression analysis was carried out using DESeq2, comparing unstimulated monocytes from COVID-19 patients (n=10) to unstimulated monocytes from healthy controls (HC) (n=6), and SARS-CoV-2-stimulated monocytes from COVID-19 patients (n=14) to stimulated monocytes from HC (n=12).	DESeq2 suggested: (DESeq, RRID:SCR_000154)
Pheatmap package was used to draw heatmaps illustrating variation in gene expression across samples.	Pheatmap suggested: (pheatmap, RRID:SCR_016418)
We tested for enrichment of this gene set in the COVID-19 versus healthy contrasts using the geneSetTest function and barcodeplot functions from limma.	limma suggested: (LIMMA, RRID:SCR_010943)
Cultures were subsequently stained with CD3 (UCHT1, BD Biosciences), CD20 (H1, BD Biosciences), CD14 (M5E2, Biolegend), CD16 (B73.1, BD Biosciences), phospho-IRF3 (Ser 396, Bioss), phospho-NFkB p65 (Ser 529, BD Biosciences) in PBS for 1 hour at room temperature, washed with PBS and resuspended in 250 µl PBS.	BD Biosciences suggested: (BD Biosciences, RRID:SCR_013311)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

Read the original source

Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature upon SARS-CoV-2 sensing by monocytes in COVID-19

This article has been Reviewed by the following groups

Listed in

Abstract

Article activity feed