key: cord-347512-veavzt6d
authors: Ueland, Thor; Heggelund, Lars; Lind, Andreas; Holten, Aleksander R.; Tonby, Kristian; Michelsen, Annika E.; Jenum, Synne; Jørgensen, Marthe J.; Barratt-Due, Andreas; Skeie, Linda G.; Nordøy, Ingvild; Aanensen Fraz, Mai Sasaki; Quist-Paulsen E, Else; Pischke, Søren E.; Johal, Simreen K.; Hesstvedt, Liv; Bogen, Mette; Fevang, Børre; Halvorsen, Bente; Müller, Fredrik; Bekken, Gry Kloumann; Mollnes, Tom E.; Dudman, Susanne; Aukrust, Pål; Dyrhol-Riise, Anne M.; Holter, Jan C.
title: Elevated plasma sTIM-3 levels in severe Covid-19 patients
date: 2020-09-21
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2020.09.007
sha: 
doc_id: 347512
cord_uid: veavzt6d

Background The pathogenesis of COVID-19 is still incompletely understood, but seems to involve immune activation and immune dysregulation. Objective We examined parameters of activation of different leukocyte subsets in COVID-19 infected patients in relation to disease severity. Methods We analyzed plasma levels of myeloperoxidase (MPO, neutrophil activation), soluble (s) CD25 and soluble T cell immunoglobulin mucin domain-3 (sTIM-3) (markers of T cell activation and exhaustion) and sCD14 and sCD163 (markers of monocyte/macrophage activation) in 39 COVID-19 infected patients at hospital admission and two additional times during the first 10 days in relation to the need for ICU treatment. Results Our major findings were: (i) Severe clinical outcome (ICU) was associated with high plasma levels sTIM-3 and MPO suggesting activated and potentially exhausted T cells and activated neutrophils, respectively. (ii) In contrast, sCD14 and sCD163 showed no association with need for ICU treatment. (iii) sCD25, sTIM-3 and MPO were inversely correlated with the degree of respiratory failure as assessed by P/F ratio and positively correlated with the cardiac marker N-terminal pro-B-type natriuretic peptide. Conclusion Our findings suggest that neutrophil activation and in particular activated T cells may play an important role in the pathogenesis of COVID-19 infection, suggesting that T cell targeted treatment options and downregulation of neutrophil activation could be of importance in this disorder.

patients with severe disease, as compared to those with mild to moderate disease [8] [9] [10] . There are also reports of lymphopenia, and in particular T cell lymphopenia, in those with severe disease 10, 11 . However, so far the role of the different leukocyte subsets in the pathogenesis of COVID-19 infection has not been fully elucidated.

In the present study we examined plasma parameters of activation of different J o u r n a l P r e -p r o o f

Informed consents were obtained from all patients or next-of-kin if patients were incapacitated of giving consent. The study was approved by the South-Eastern Norway

Regional Health Authority (reference number: 106624).

Outcome was defined as the need for treatment at the ICU during hospitalization. Indication for admission to ICU was respiratory failure that required mechanical ventilation support or non-invasive ventilation support that could not be given at the hospital ward.

Peripheral blood was collected with 4 mL Vacutainer ® (BD Biosciences, San Diego, CA)

with EDTA as anti-coagulant. Samples were immediately stored on ice, processed within 30 minutes and plasma was isolated by centrifugation at 2000g for 20 minutes at 4°C to obtain platelet-poor plasma. Plasma were immediately stored at -80 0 C in several aliquots until analysis. Samples were thawed only once.

Plasma levels of sCD14, sCD163, sCD25, sTIM-3 and MPO were measured in duplicate by 

Cardiac markers and hsCRP were measured at department of Medical Biochemistry at the two centers: OUH: NTproBNP and hsCRP were analyzed on COBAS 8000 (Roche Diagnostics, Basel, Switzerland). Drammen Hospital: hsCRP was analyzed on Alinity/Architect ci8200 (Abbott, Abbott Park, IL) and NTproBNP on a Cobas E411 (Roche). Reference values for NT-proBNP: women: <50 years (y) ≥170 ng/L; 50-69 y ≥300 ng/L; ≥70 y ≥760 ng/L, men:

<50 y ≥85 ng/L; 50-69 y ≥250 ng/L; ≥70 y ≥500 ng/L.

Patient characteristics were compared using student's t-test or chi-square for continuous and categorical variables, respectively (Table 1 and (Table 3) . Correlation analysis between selected markers was also performed at individual time-points (Pearson, Figure 2 ).

To limit multiple comparisons, post-hoc testing was only performed on variables where outcome or outcome*time interaction was significant using multivariate regression at each time-point with eGFR and age as covariate ( Figure 1 ). In addition, we multiplied the pvalues with the number of leukocyte markers assessed, i.e. with five in the overall univariate and partial correlation analysis ( Figure 1 and Table 3 ) and with three in the post-hoc and individual correlation analysis (Figure 1 and 2).

P-values are two-sided and considered significant when <0.05. SPSS release 26.0.0.1 was used for statistical analysis.

The sponsor of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and takes final responsibility for the decision to submit for publication. Table 2 . Of the 39 COVID-19 patients, 9 were admitted to ICU (death n=3, 2 due to respiratory failure, 1 due to multi-organ failure related to . The ICU patients were characterized by low P/F ratio and lymphocyte counts and high NT-proBNP and hsCRP levels. No significant differences in age, time from symptoms to admission, major comorbidities or eGFR were detected in relation to ICU ( Table 2) .

The temporal course of leukocyte markers in relation to ICU admission (ICU n=9; non-ICU n=30) is shown in Figure 1 . Patients admitted to ICU were characterized by high levels of the T cell activation marker sCD25 and sTIM-3 in à priori analysis, and for sTIM-3 also in posthoc testing. Moreover, whereas sCD25 displayed stable levels between samples obtained at 3-5 and 7-10 days, sTIM-3 levels increased until 7-10 days in the ICU group. Finally, sCD25 and sTIM-3 levels were markedly higher than control levels at all time-points.

The neutrophil marker MPO was significantly higher in the ICU group compared with the non-ICU group at all time points (Figure 1 ). MPO levels in the non-ICU patients were comparable to control levels. However, in contrast to the T cell markers, MPO in the ICU patients decreased during follow-up.

In contrast to the T cell markers and MPO, plasma levels of sCD14 and sCD163, reflecting activation of monocytes/macrophages, were similar in ICU and non-ICU patients ( Figure 1 ).

For the above analysis of outcomes, age and eGFR were included as covariates when comparing time-points, but excluding these covariates gave similar results. Table 3 shows correlations between leukocyte activation markers and P/F ratio as a marker of respiratory function and NT-proBNP as a marker of cardiac involvement during the course of the study. sCD25, sTIM-3 and MPO were negatively correlated with the P/F ratio and positively correlated with NT-proBNP. Notably, these correlations were consistent with a similar pattern at all time-points, in particular for sTIM-3 ( Figure 2 

Whereas MPO levels were positively correlated with neutrophil counts (Table 3) , none of the T cell markers correlated with lymphocyte counts, of which T cells is the dominating cell subset, suggesting that the increased levels of sCD25 and sTIM-3 do not merely reflect altered numbers of T cells. All markers were correlated with kidney function, but importantly, the associations of sCD25, sTIM-3 and MPO with ICU admission was seen also after adjustment for eGFR. Finally, the two markers of T cell activation, sCD25 and sTIM-3, were strongly correlated (r=0.71, p<0.001).

with severe clinical outcome, characterized by high levels of inflammatory cytokines and low lymphocyte counts [10] [11] [12] . Several studies demonstrate that lymphopenia, and in particular low number of T cells, is an important feature of COVID-19 infection 8, 10, 11, 13, 14 . We show that despite signs of T cell depletion, severe COVID-19 infection as reflected by ICU admission is also characterized by raised levels of sTIM-3 suggesting activated and potentially exhausted T cells.

There are a few reports of elevated sTIM-3 levels in infectious disorders such as HIV 15 , hepatitis B 16 and C 17 , malaria falciparum 18 and pulmonary tuberculosis 19 . This is, however, to the best of our knowledge, the first report on sTIM- finding may suggest that excessive T cell and neutrophil activation also could be involved.

Increasing evidence indicate that COVID-19 infected patients are at higher risk of developing cardiac involvement or CV related death 3, 27 and accordingly, a majority of ICU patients had elevated NT-proBNP levels in our study. The spectrum of cardiac involvement in COVID-19 infection is ranging from type 1 and type 2 myocardial infarction to myocarditis, and in more general terms, T cells are known to participate in these conditions 28, 29 

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Patients (n=39) Women, n (%) 7 (44) 10 (26) Age, years 66±7 61±15Time from symptoms, days -9.6±3.7Caucasian, n (%) 16 (100) 27 (69) Current smoker, n (%) 3 (19) 8 (21) P/F ratio (kPa) -41±15Need for oxygen therapy, n (%) -28 (72) Comorbidities Cardiovascular, n (%) 0 (0) 9 (23) Pulmonary, n (%) 0 (0) 9 (23) Renal, n (%) 0 (0) 4 (10)Liver, n (%)Obesity, n (%) 0 (0) 5 (13) Diabetes, n (%) 0 (0) 3 (8) Rheumatic, n (%) 0 (0) 4 (10) J o u r n a l P r e -p r o o f