Immunological profiling in long COVID: overall low grade inflammation and T-lymphocyte senescence and increased monocyte activation correlating with increasing fatigue severity

Abstract

Background: Many patients with SARS-CoV-2 infection develop long COVID with fatigue as one of the most disabling symptoms. We performed clinical and immune profiling of fatigued and non-fatigued long COVID patients and age- and sex-matched healthy controls (HCs).

Methods: Long COVID symptoms were assessed using patient-reported outcome measures, including the fatigue assessment scale (FAS, scores ≥22 denote fatigue), and followed up to one year after hospital discharge. We assessed inflammation-related genes in circulating monocytes, serum levels of inflammation-regulating cytokines, and leukocyte and lymphocyte subsets, including major monocyte subsets and senescent T-lymphocytes, at 3-6 months post-discharge.

Results: We included 37 fatigued and 36 non-fatigued long COVID patients and 42 HCs. Fatigued long COVID patients represented a more severe clinical profile than non-fatigued patients, with many concurrent symptoms (median 9 [IQR 5.0-10.0] vs 3 [1.0-5.0] symptoms, p<0.001), and signs of cognitive failure (41%) and depression (>24%). Immune abnormalities that were found in the entire group of long COVID patients were low grade inflammation (increased inflammatory gene expression in monocytes, increased serum pro-inflammatory cytokines) and signs of T-lymphocyte senescence (increased exhausted CD8⁺ T_EMRA-lymphocytes). Immune profiles did not significantly differ between fatigued and non-fatigued long COVID groups. However, the severity of fatigue (total FAS score) significantly correlated with increases of intermediate and non-classical monocytes, upregulated gene levels of CCL2, CCL7, and SERPINB2 in monocytes, increases in serum Galectin-9, and higher CD8⁺ T-lymphocyte counts.

Conclusion: Long COVID with fatigue is associated with many concurrent and persistent symptoms lasting up to one year after hospitalization. Increased fatigue severity associated with stronger signs of monocyte activation in long COVID patients and potentially point in the direction of monocyte-endothelial interaction. These abnormalities were present against a background of immune abnormalities common to the entire group of long COVID patients.

Keywords: COVID-19; T-lymphocytes; fatigue; inflammation; long COVID; monocytes.

Figure 1

Prevalence of clinical symptoms across fatigued and non-fatigued long COVID patients assessed at 3, 6, and 12 months after hospital discharge. Symptoms are presented for groups of fatigued (total FAS score ≥22) and non-fatigued (total FAS score <22) long COVID patients at the time of collecting blood samples. In the non-fatigued long COVID group, some patients experienced fatigue at other follow-up visits. Symptoms were obtained from the Corona Symptom Checklist on the presence of new or worsened symptoms following SARS-CoV-2 infection (yes or no). * The fatigue symptom was obtained from the Fatigue Assessment Scale (FAS) questionnaire, a total FAS score ≥22 denotes fatigue. The dyspnea symptom was obtained from the modified medical research council dyspnea scale, grades ≥2 were used to denote dyspnea.

Figure 2

CD4⁺ T-lymphocyte and CD8⁺ T-lymphocyte subsets in fatigued and non-fatigued long COVID patients. Fatigue was defined as a total score of ≥22 on the Fatigue Assessment Scale (FAS) questionnaire. Data of CD4⁺ T-lymphocyte and CD8⁺ T-lymphocyte subsets did not differ significantly between groups of fatigued (n=37) and non-fatigued (n=33) long COVID patients. Significant group differences are presented for the entire group of long COVID patients as compared to healthy controls (n=42) using the Mann-Whitney U test, *p<0.05, **p<0.01, and ***p<0.001. (A) CD8⁺ T-lymphocyte counts across groups. The correlation between CD8⁺ T-lymphocyte counts and the fatigue severity (total FAS score) in long COVID patients was assessed using Spearman’s rank correlation coefficient. (B) The assessment of CD8⁺ T-lymphocyte subsets showed an increased percentages of CD8⁺ T_EMRA lymphocytes, particularly CD8⁺ T_EMRA-lymphocyte expressing CD279⁺CD57⁺, and CD8⁺ T_EMRA-lymphocyte expressing CD27^-CD28^-, in long COVID patients as compared to healthy controls. (C) The assessment of CD4⁺ T-lymphocyte subsets showed reduced percentages of CD4⁺ T_naive lymphocytes, regulatory CD4⁺ T-lymphocytes (CD4⁺CD25highFOXP3⁺), and CD4⁺ T_EM lymphocytes expressing CD279⁺CD57⁺ in long COVID patients as compared to healthy controls.

Figure 3

Percentages of classical, intermediate, and non-classical monocytes in fatigued and non-fatigued long COVID patients. Fatigue was defined as a total score of ≥22 on the Fatigue Assessment Scale (FAS) questionnaire. (A) The percentages of classical monocytes (CD14⁺⁺CD16^-), intermediate monocytes (CD14⁺⁺CD16⁺), and non-classical monocytes (CD14⁺CD16⁺⁺) in groups of fatigued (n=35) and non-fatigued (n=34) long COVID patients and healthy controls (n=40). The percentage of monocyte subsets did not differ significantly between fatigued and non-fatigued long COVID patients. Significant group differences are presented for the entire group of long COVID patients as compared to healthy controls using the Mann-Whitney U test, *p<0.05. (B) The correlation between the percentage of monocyte subsets and the fatigue severity (total FAS score) in long COVID patients was assessed using Spearman’s rank correlation coefficient.

Figure 4

Gene expression levels in monocytes in fatigued and non-fatigued long COVID patients. The expression level of genes were normalized to the housekeeping gene ABL1 (ΔCT values) and expressed relative to the average ΔCT value of healthy controls (ΔΔCT values). (A) Three main clusters of mutually correlating monocyte genes can be identified. Cluster A comprises inflammation-regulation genes and genes related to adhesion, chemotaxis, apoptosis, and pyroptotic mechanisms of the cells. Cluster B comprises genes related to type 1 interferon driven inflammation. Cluster C comprises mainly genes related to mitochondrial anti-inflammatory action and cholesterol pump genes. Correlations between genes were assessed using Spearman’s rank correlation coefficient. (B) Fatigue was defined as a total score of ≥22 on the Fatigue Assessment Scale (FAS) questionnaire. Single gene expression levels in fatigued (n=35) and non-fatigued (n=34) long COVID patients, data are presented as mean values and are expressed relatively to the expression level of healthy controls (n=42); the intensity of red reflects higher expression (upregulation) and green reflects lower expression (downregulation). No statistically significant differences were found in the gene expression levels in monocytes between fatigued and non-fatigued long COVID patients (data not shown). Significant differences in single gene expression levels in long COVID groups as compared to healthy controls were assessed with a Wilcoxon signed rank test using Benjamini-Hochberg-method for multiple testing, *p<0.05, **p<0.01, and ***p<0.001. (C) The correlation between gene expression levels in monocytes and the fatigue severity (total FAS score) in long COVID patients was assessed using Spearman’s rank correlation coefficient.

Figure 5

Serum cytokine and soluble cell surface molecule levels in fatigued and non-fatigued long COVID patients. Fatigue was defined as a total score of ≥22 on the Fatigue Assessment Scale (FAS) questionnaire. (A) Serum cytokine and soluble cell surface molecule levels are presented for groups of fatigued (n=37) and non-fatigued (n=35) long COVID patients and healthy controls (n=42). These serum levels did not differ significantly between groups of fatigued and non-fatigued long COVID patients. Significant group differences are presented for the entire group of long COVID patients as compared to healthy controls using the Mann-Whitney U test, *p<0.05, **p<0.01, and ***p<0.001. (B) The correlation between Galectin-9 levels and the fatigue severity (total FAS score) in long COVID patients was assessed using Spearman’s rank correlation coefficient. CCL, C-C motif chemokine ligand; CXCL, C-X-C motif chemokine ligand; CD163, cluster of differentiation 163; IL, interleukin; IFN, interferon,; TNF-a, tumor necrosis factor-alpha.