Alterations in Circulating Monocytes Predict COVID-19 Severity and Include Chromatin Modifications Still Detectable Six Months after Recovery

Abstract

An early analysis of circulating monocytes may be critical for predicting COVID-19 course and its sequelae. In 131 untreated, acute COVID-19 patients at emergency room arrival, monocytes showed decreased surface molecule expression, including low HLA-DR, in association with an inflammatory cytokine status and limited anti-SARS-CoV-2-specific T cell response. Most of these alterations had normalized in post-COVID-19 patients 6 months after discharge. Acute COVID-19 monocytes transcriptome showed upregulation of anti-inflammatory tissue repair genes such as BCL6, AREG and IL-10 and increased accessibility of chromatin. Some of these transcriptomic and epigenetic features still remained in post-COVID-19 monocytes. Importantly, a poorer expression of surface molecules and low IRF1 gene transcription in circulating monocytes at admission defined a COVID-19 patient group with impaired SARS-CoV-2-specific T cell response and increased risk of requiring intensive care or dying. An early analysis of monocytes may be useful for COVID-19 patient stratification and for designing innate immunity-focused therapies.

Keywords: COVID-19; HLA-DR; chromatin accessibility; circulating monocytes; transcriptome.

Figure 1

Circulating monocyte subsets, their surface molecule expression and their cytokine production after in vitro activation are altered in acute and post-COVID-19 patients. (A) Identification of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14−CD16+) monocytes. In HC, classical monocytes represented 77.7%, intermediate monocytes 5.7% and non-classical monocytes 6.8%. In acute COVID-19 those populations were 84.5%, 11.5% and 1.0% respectively, and in post-COVID-19 they were 75.5%, 6.9% and 7.6% respectively. (B) Principal component analysis (PCA) of HC (n= 45), acute (n = 131) and post-COVID-19 patients (n = 52) according to monocyte surface marker expression. Individual patient values (small points), mean (big point) and confidence interval of the mean (shadows) are represented (C) Examples and comparison of FSC, SSC, CD11b, CD16, CD33, CCR2, CCR5, CD86 and HLA-DR expression in monocytes from HC, acute and post-COVID-19 patients. (D) Cytokines secreted by isolated, LPS-stimulated monocytes. In unstimulated monocyte wells all cytokines were below the detection threshold: GM-CSF < 2.6 pg/mL; TNF-α < 6.4 pg/mL; IL1-RA < 1.6 pg/mL; IL1β < 1.6 pg/mL; IL-6 < 0.64 pg/mL; IL-8 < 0.64 pg/mL; IL-10 < 2.6 pg/mL; IL-18 < 0.64 pg/mL; CCL2 < 3 pg/mL; CCL3 < 3 pg/mL; CXCL10 < 2.6 pg/mL.*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant.

Figure 2

HLA-DR downregulation in monocytes correlates with higher plasma levels of IL-6, G-CSF, CXCL10, CCL2 and lower SARS-CoV-2-specific T cell response. (A) PCA of 20 cytokines in plasma from HC (n = 16), acute (n = 84) and post-COVID-19 (n = 10) patients. Individual patient values (small points), mean (big point) and confidence interval of the mean (shadows) are represented. (B) Contribution of each cytokine to PCA dimensions 1 and 2. Dashed line represents the mean of all cytokine contributions. (C) Fold changes of median log-transformed values of plasma cytokines analyzed in HC, acute and post-COVID-19 patients. Dotted line represents unchanged cytokine level. (D) Correlation analysis between plasma cytokine levels and monocyte surface expression of CD11b, CCR2, CCR5, CD86, HLA-DR, CD16 and CD33. (E) Proliferation of in vitro polyclonally stimulated CD4 and CD8 T cells from HC, acute COVID-19 and post-COVID-19. (F) SARS-CoV-2 S1, N and M specific cellular response in HC (n = 30), acute (n = 40) and post-COVID-19 (n = 41) patients. Dashed lines represent the established cut-off of positivity according to HC SFU means plus 3 standard deviations. (G) Correlation between specific T cell response and monocyte subsets and surface markers in acute and post-COVID-19 patients. Color of squares indicates direct (blue) or inverse (red) correlation. The size of the square is proportional to magnitude of correlation. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant.

Figure 3

The transcriptomic profiling of circulating monocytes reveals an anti-inflammatory program in early acute COVID-19 still detectable in post-COVID-19 subjects. (A) PCA based on 255 analyzed genes clearly separated monocytes from HC, acute and post-COVID-19 patients. DEG of monocytes from (B) HC and acute COVID19, (C) HC and post-COVID19, (D) acute and post-COVID19. In (B–D), top panels depict volcano plots showing the genes with significant differences, horizontal solid line indicates a p-value < 0.05 and horizontal dashed line indicates a p-value < 0.001; vertical lines indicate log2 fold change <−0.6 and >0.6; bottom panels depict heatmaps showing the individual expression of significant DEG in each subject.

Figure 4

Open chromatin profile is higher in acute, intermediate in post-COVID-19 and lower in HC circulating monocytes. (A) Venn diagram of open sites found in HC, acute and post-COVID-19 monocytes. (B) Global enrichment sites in HC, acute and post-COVID-19 monocytes. (C) MAFF-promoter region had greater opening in acute COVID-19 monocytes. (D) CD4-promoter region had greater opening in HC monocytes. (E) LBR-promoter region had greater opening in acute and post-COVID-19 monocytes.

Figure 5

Acute COVID-19 patients with low expression of surface molecules in monocytes have higher risk of ICU requirement or death. (A) K-means clustering heatmap of monocyte surface markers from acute COVID-19 patients at ER arrival identified two separate clusters (A,B) of acute COVID-19 patients. (B) Cluster A acute COVID-19 patients showed a significantly higher proportion of classical monocytes, and lower proportion of intermediate monocytes, when compared to cluster B. (C) Comparison of monocyte surface markers between clusters. (D) Differences in ICU requirement and death rate in cluster A versus cluster B. (E) Cumulative death Kaplan-Meier analysis of hospitalized cluster A and B acute COVID-19 patients. (F) Interval from hospitalization to death in cluster A and B patients. (G) Polyclonal CD4 and CD8 T cell proliferation in cluster A and B patients. (H) IFN-γ and IL-2 specific T cell response against S1, N and M peptides of SARS-CoV-2 in cluster A and cluster B patients. (I) Cluster B patients had higher number of S1 specific T cells at ER arrival. (J) Follow-up of HLA-DR expression in monocytes during hospitalization showed augmentation in surviving patients and decrease in patients who died. (K) Follow-up of specific S1, N and M T cell response and HLA-DR expression on monocytes in two representative patients, a survivor (top) and a non-survivor (down). X-axes represent total days (Days of symptoms: <0; days of hospitalization: >0; Day 0 represents ER arrival day). Vertical black, dashed lines represent the day of symptom onset. Vertical red, solid line represents the day of dying. Vertical green, dashed line represents the day of discharge. (L) Volcano plot of differently up and downregulated genes between cluster A (n = 10) and cluster B (n = 8) monocytes. (M) IRF1 was significantly downregulated in cluster A. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant.