A blood-based mRNA signature distinguishes people with Long COVID from recovered individuals

Abstract

Introduction: Long COVID is a debilitating condition that lasts for more than three months post-infection by SARS-CoV-2. On average, one in ten individuals infected with SARS CoV- 2 develops Long COVID worldwide. A knowledge gap exists in our understanding of the mechanisms, genetic risk factors, and biomarkers that could be associated with Long COVID.

Methods: In this pilot study we used RNA-Seq to quantify the transcriptomes of peripheral blood mononuclear cells isolated from COVID-recovered individuals, seven with and seven without Long COVID symptoms (age- and sex-matched individuals), on average 6 months after infection.

Results: Seventy genes were identified as significantly up- or down-regulated in Long COVID samples, and the vast majority were downregulated. The most significantly up- or downregulated genes fell into two main categories, either associated with cell survival or with inflammation. This included genes such as ICOS (FDR p = 0.024) and S1PR1 (FDR p = 0.019) that were both up-regulated, indicating that a pro-inflammatory state is sustained in Long COVID PBMCs compared with COVID recovered PBMCs. Functional enrichment analysis identified that immune-related functions were expectedly predominant among the up- or down-regulated genes. The most frequently downregulated genes in significantly altered functional categories were two leukocyte immunoglobulin like receptors LILRB1 (FDR p = 0.005) and LILRB2 (FDR p = 0.027). PCA analysis demonstrated that LILRB1 and LILRB2 expression discriminated all of the Long COVID samples from COVID recovered samples.

Discussion: Downregulation of these inhibitory receptors similarly indicates a sustained pro-inflammatory state in Long COVID PBMCs. LILRB1 and LILRB2 should be validated as prospective biomarkers of Long COVID in larger cohorts, over time and against clinically overlapping conditions.

Keywords: COVID-19; LILRB1; LILRB2; Long COVID; biomarker; inflammation; transcriptomics.

Figure 1

Symptoms affecting Long COVID participants and severity ratings. Seven of the participants with Long COVID completed questionnaires to list and rate the severity of their symptoms. Ratings of the symptoms were classified as 0 = no symptoms, 1-2 = mild, 3-4 = moderate, 5 = severe. Percentages are rounded to the nearest single decimal place.

Figure 2

Long COVID versus COVID recovered PBMC transcriptomes. (A) Venn diagram depicting the number of gene transcripts significantly up- or down-regulated in PBMCs from people with Long COVID versus PBMCs from COVID recovered individuals. The p-values which determined significance were corrected with FDR for multiple testing, (pFDR < 0.05 was used as the threshold for significance). Genes with an average reads per kilobase per million mapped reads (RPKM) lower than four in both cohorts were removed from the list of significant differentially expressed genes. This was to ensure that any identified genes were present in sufficient amounts to be detected via clinically appropriate methods. (B) Heatmap representation of seventy differentially expressed genes between Long COVID and COVID recovered PBMC samples. Red indicates higher expression while blue indicates lower expression. Each column is a different sample and each row is a different gene.

Figure 3

Principal component analysis (PCA) plot based on 70 differentially expressed genes in PBMCs efficiently distinguishes Long COVID (LC, red) PBMCs from most of the COVID recovered PBMCs (CR, blue) with one overlapping COVID recovered sample.

Figure 4

Elevated expression levels of S1PR1 and ICOS in blood-derived PBMCs from Long COVID patients versus COVID recovered individuals. (A) Comparison of S1PR1 expression between Long COVID and COVID recovered PBMCs. (B) Comparison of ICOS expression between Long COVID and COVID recovered PBMCs. (C) Scatter plot demonstrates the elevated expression of S1PR1 and ICOS in Long COVID (LC) samples. (D) PCA analysis demonstrates that S1PR1 and ICOS can discriminate all but one Long COVID (LC) sample from COVID recovered (CR), where the first component described 79.6% of variation in the expression data.

Figure 5

Reduced expression levels of LILRB1 and LILRB2 in blood-derived PBMCs effectively differentiate Long COVID patients from COVID recovered individuals. (A) Comparison of LILRB1 expression between Long COVID and COVID recovered PBMCs. (B) Comparison of LILRB2 expression between Long COVID and COVID recovered PBMCs. (C) Scatter plot demonstrates the reduced expression of LILRB1 and LILRB2 in Long COVID (LC) samples. (D) PCA analysis demonstrates the power of LILRB1 and LILRB2 to discriminate Long COVID (LC) samples from COVID recovered (CR) where the first component described 95.4% of variation in the expression data.