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. 2022 Jul 27:3:942699.
doi: 10.3389/falgy.2022.942699. eCollection 2022.

Neutrophil and Eosinophil Responses Remain Abnormal for Several Months in Primary Care Patients With COVID-19 Disease

B N Jukema  1   2 , K Smit  3 , M T E Hopman  4 , C C W G Bongers  4 , T C Pelgrim  1   2 , M H Rijk  3 , T N Platteel  3 , R P Venekamp  3 , D L M Zwart  3 , F H Rutten  3 , L Koenderman  1   2
Affiliations

Neutrophil and Eosinophil Responses Remain Abnormal for Several Months in Primary Care Patients With COVID-19 Disease

B N Jukema et al. Front Allergy. .

Abstract

Introduction: Neutrophil and eosinophil activation and its relation to disease severity has been understudied in primary care patients with COVID-19. In this study, we investigated whether the neutrophil and eosinophil compartment were affected in primary care patients with COVID-19.

Methods: COVID-19 patients, aged ≥ 40 years with cardiovascular comorbidity presenting to the general practitioner with substantial symptoms, partaking in the COVIDSat@Home study between January and April 2021, were included. Blood was drawn during and 3 to 6 months after active COVID-19 disease and analyzed by automated flow cytometry, before and after stimulation with a formyl-peptide (fNLF). Mature neutrophil and eosinophil markers at both time points were compared to healthy controls. A questionnaire was conducted on disease symptoms during and 3 to 6 months after COVID-19 disease.

Results: The blood of 18 COVID-19 patients and 34 healthy controls was analyzed. During active COVID-19 disease, neutrophils showed reduced CD10 (p = 0.0360), increased CD11b (p = 0.0002) and decreased CD62L expression (p < 0.0001) compared to healthy controls. During active COVID-19 disease, fNLF stimulated neutrophils showed decreased CD10 levels (p < 0.0001). Three to six months after COVID-19 disease, unstimulated neutrophils showed lowered CD62L expression (p = 0.0003) and stimulated neutrophils had decreased CD10 expression (p = 0.0483) compared to healthy controls. Both (un)stimulated CD10 levels increased 3 to 6 months after active disease (p = 0.0120 and p < 0.0001, respectively) compared to during active disease. Eosinophil blood counts were reduced during active COVID-19 disease and increased 3 to 6 months after infection (p < 0.0001). During active COVID-19, eosinophils showed increased unstimulated CD11b (p = 0.0139) and decreased (un)stimulated CD62L expression (p = 0.0036 and p = 0.0156, respectively) compared to healthy controls. Three to six months after COVID-19 disease, (un)stimulated eosinophil CD62L expression was decreased (p = 0.0148 and p = 0.0063, respectively) and the percentage of CD11bbright cells was increased (p = 0.0083 and p = 0.0307, respectively) compared to healthy controls.

Conclusion: Automated flow cytometry analysis reveals specific mature neutrophil and eosinophil activation patterns in primary care patients with COVID-19 disease, during and 3 to 6 months after active disease. This suggests that the neutrophil and eosinophil compartment are long-term affected by COVID-19 in primary care patients. This indicates that these compartments may be involved in the pathogenesis of long COVID.

Keywords: SARS-CoV-2; activation; flow cytometry; granulocytes; long COVID; primary care.

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Conflict of interest statement

The AQUIOS CL® “Load & Go” flow cytometer is provided by the company Beckman Coulter Life Sciences, Miami, FL, USA. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Flowchart depicting the inclusion of patients for this study.
Figure 2
Figure 2
(A) Manual gating strategy for mature neutrophils. (B) Automated gating strategy for mature neutrophils (metacluster 3) and eosinophils (metacluster 5) by using the FlowSOM algorithm with 64 clusters and 6 metaclusters.
Figure 3
Figure 3
Median fluorescence intensity (MFI) in arbitrary units (AU) for markers on mature neutrophils during (t = 1) and 3 to 6 months after (t = 2) active COVID-19 disease in primary care patients. Healthy controls (HC) are displayed as reference. fNLF-samples are measured in the absence of fNLF, whereas fNLF+ samples are measured in the presence of the formylpeptide (10 μM). Statistical significance was tested using the Mann-Whitney U Test for continuous variables and the Wilcoxon matched-pairs signed rank test for paired analyses.
Figure 4
Figure 4
Median fluorescence intensity (MFI) in arbitrary units (AU) for markers on mature neutrophils in healthy controls (HC) and in primary care patients with active COVID-19 disease (t = 1). Paired analyses are shown for the samples measured in the absence (fNLF-) and presence (fNLF+) of a formylpeptide (10 μM). Statistical significance was tested using the Wilcoxon matched-pairs signed rank test.
Figure 5
Figure 5
Eosinophil blood counts and granulocyte/eosinophil blood count ratios during (t = 1) and 3 to 6 months after (t = 2) active COVID-19 disease in primary care patients. Statistical significance was tested using the Wilcoxon matched-pairs signed rank test.
Figure 6
Figure 6
Median fluorescence intensity (MFI) in arbitrary units (AU) for markers on eosinophils during (t = 1) and 3 to 6 months after (t = 2) active COVID-19 disease in primary care patients. Healthy controls (HC) are displayed as reference. fNLF-samples are measured in the absence of fNLF, whereas fNLF+ samples are measured in the presence of the formylpeptide (10 μM). Statistical significance was tested using the Mann-Whitney U Test for continuous variables and the Wilcoxon matched-pairs signed rank test for paired analyses.
Figure 7
Figure 7
Median fluorescence intensity (MFI) in arbitrary units (AU) in relation to disease severity for neutrophil CD11b 3 to 6 months after active COVID-19 disease. fNLF- samples are measured in the absence of fNLF, whereas fNLF+ samples are measured in the presence of the formylpeptide (10 μM). Trendlines were generated using a simple linear regression analysis.
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