Serum proteomics hint at an early T-cell response and modulation of SARS-CoV-2-related pathogenic pathways in COVID-19-ARDS treated with Ruxolitinib

Abstract

Background: Acute respiratory distress syndrome (ARDS) in corona virus disease 19 (COVID-19) is triggered by hyperinflammation, thus providing a rationale for immunosuppressive treatments. The Janus kinase inhibitor Ruxolitinib (Ruxo) has shown efficacy in severe and critical COVID-19. In this study, we hypothesized that Ruxo's mode of action in this condition is reflected by changes in the peripheral blood proteome.

Methods: This study included 11 COVID-19 patients, who were treated at our center's Intensive Care Unit (ICU). All patients received standard-of-care treatment and n = 8 patients with ARDS received Ruxo in addition. Blood samples were collected before (day 0) and on days 1, 6, and 10 of Ruxo treatment or, respectively, ICU admission. Serum proteomes were analyzed by mass spectrometry (MS) and cytometric bead array.

Results: Linear modeling of MS data yielded 27 significantly differentially regulated proteins on day 1, 69 on day 6 and 72 on day 10. Only five factors (IGLV10-54, PSMB1, PGLYRP1, APOA5, WARS1) were regulated both concordantly and significantly over time. Overrepresentation analysis revealed biological processes involving T-cells only on day 1, while a humoral immune response and complement activation were detected at day 6 and day 10. Pathway enrichment analysis identified the NRF2-pathway early under Ruxo treatment and Network map of SARS-CoV-2 signaling and Statin inhibition of cholesterol production at later time points.

Conclusion: Our results indicate that the mechanism of action of Ruxo in COVID-19-ARDS can be related to both known effects of this drug as a modulator of T-cells and the SARS-CoV-2-infection.

Keywords: COVID-19; Ruxolitinib; SARS-CoV-2; acute respiratory distress syndrome; proteomics.

FIGURE 1

Serum proteomes of critically ill COVID-19 patients with and without Ruxo treatment. (A) Left panel: Volcano plot of MS data indicating differential protein expression between COVID-19 patients with or without ARDS [Ruxo group (green) vs. control group (red)]. “p” indicates the raw p-value, “bon” indicates the Bonferroni-corrected p-value. Right panel: Time trajectory from principal component analysis for the protein PGLYRP1. (B) Principal component analysis (PCA) score plot derived from mass spectrometry (MS) data of different patients and sampling time points using treatment as a design-factor. Each individual is color-coded. Additionally, for each subject, “Treatment” is coded by symbol shape and “ApproxDay” by size. (C) Partial least square regression analysis (PLS) derived from the MS data. Coding as in panel (B).

FIGURE 2

Changes in serum proteomes of COVID-19 patients upon Ruxo treatment over time. (A) Partial least square regression analysis (PLS) derived from the MS data of different patients and sampling time points using time as a design-factor. Each individual is color-coded. Additionally, for each subject, “Treatment” is coded by symbol shape and “ApproxDay” by size. Samples from the same subject are connected by a line. (B) Volcano plot of MS data indicating differential protein expression in COVID-19 patients under treatment with Ruxo at day 1 (green) compared to day 0 (red). “p” indicates the raw p-value, “bon” indicates the Bonferroni-corrected p-value. (C) General linear modeling of protein expression as a function of sampling day.

FIGURE 3

Overrepresentation analysis of differentially regulated serum proteins in COVID-19 patients under Ruxo treatment. ORA was performed on differentially regulated proteins (raw p-value < 0.05) as detected by MS on (A) day 1, (B) day 6, and (C) day 10. The top 20 GO terms of the category biological process from analyses using the clusterProfiler package were plotted. Note that direction of regulation (up or down) was not considered in this analysis. The barplots indicate the level of significance and the number of included genes for each term. See Supplementary Tables 6–8 in Supplementary Material 2 for complete ORA results.

FIGURE 4

Pathway enrichment analysis of differentially regulated serum proteins in COVID-19 patients under Ruxo treatment. Wiki-Pathway enrichment analysis was performed on differentially regulated proteins (raw p-value < 0.05) as detected by MS on (A) day 1, (B) day 6 and (C) day 10. Heatmap-like plots indicate expression of individual genes involved in each pathway.

FIGURE 5

Serum cytokine levels in treated and untreated COVID-19 patients. Cytometric bead array assay performed with serum samples collected at different time points (day 0, day 1, day 6, and day 10) from three COVID-19 patients under Ruxo treatment and two control patients, without Ruxo treatment. One patient in the Ruxo group also received steroids (marked by #).