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Neutrophil calprotectin identifies severe pulmonary disease in COVID-19

Hui Shi1,2*, Yu Zuo1*, Srilakshmi Yalavarthi1, Kelsey Gockman1, Melanie Zuo3, Jacqueline A.

Madison1, Christopher Blair4, Wrenn Woodward5, Sean P. Lezak5, Njira L. Lugogo6, Robert J.

Woods4, Christian Lood7, Jason S. Knight1‡, and Yogendra Kanthi8,9‡

* These authors contributed equally and share first authorship.

‡ Jason S. Knight and Yogendra Kanthi are co-corresponding authors.

Affiliations

1 Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor,

Michigan, USA

2 Division of Rheumatology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine,

Shanghai, China

3 Division of Geriatric and Palliative Medicine, Department of Internal Medicine, University of

Michigan, Ann Arbor, Michigan, USA

4 Division of Infectious Disease, Department of Internal Medicine, University of Michigan, Ann

Arbor, Michigan, USA

5 Michigan Clinical Research Unit, University of Michigan, Ann Arbor, Michigan, USA

6 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University

of Michigan, Ann Arbor, Michigan, USA

7 Division of Rheumatology, University of Washington, Department of Medicine, Seattle,

Washington, USA

8 Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan,

Ann Arbor, Michigan, USA

9 Division of Cardiology, Ann Arbor Veterans Administration Healthcare System, Ann Arbor,

Michigan, USA

Correspondence: Jason S. Knight, MD, PhD Yogendra Kanthi, MD

[email protected] [email protected]

Competing interests: The authors have no financial conflicts to report.

All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprintthis version posted May 17, 2020. ; https://doi.org/10.1101/2020.05.06.20093070doi: medRxiv preprint

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

mailto:[email protected]

mailto:[email protected]

https://doi.org/10.1101/2020.05.06.20093070

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ABSTRACT

Severe cases of coronavirus disease 2019 (COVID-19) are regularly complicated by respiratory

failure. While it has been suggested that elevated levels of blood neutrophils associate with

worsening oxygenation in COVID-19, it is unknown whether neutrophils are drivers of the

thrombo-inflammatory storm or simple bystanders. We now report that patients with COVID-19

(n=172) have markedly elevated levels of the neutrophil activation marker S100A8/A9

(calprotectin) in their blood. Calprotectin tracked with other acute phase reactants including C-

reactive protein, D-dimer, ferritin, and absolute neutrophil count, but was superior in identifying

patients requiring mechanical ventilation. In longitudinal samples, calprotectin rose as

oxygenation worsened. When tested on day 1 or 2 of hospitalization (n=94 patients),

calprotectin levels were significantly higher in patients who progressed to severe COVID-19

requiring mechanical ventilation (8039 ± 7031, n=32) as compared to those who remained free

of intubation (3365 ± 3146, p<0.0001). In summary, serum calprotectin levels track closely with

current and future COVID-19 severity, strongly implicating neutrophils as active perpetuators of

inflammation and respiratory compromise in COVID-19.



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INTRODUCTION

Since December 2019, the outbreak of coronavirus disease 2019 (COVID-19) caused by severe

acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to hundreds of countries

and has been declared a global pandemic. Severe COVID-19 results in death due to

progressive hypoxemia, acute respiratory distress syndrome (ARDS), and multi-organ failure

(1). The role of the host response in this progression remains to be fully defined.

S100A8 (MRP8) and S100A9 (MRP14) are calcium-binding proteins that belong to the S100

family. They exist mainly together as a biologically-functional heterodimer known as S100A8/A9

or calprotectin. Calprotectin is found in abundance in neutrophils, where it can account for

almost two-thirds of soluble protein in the cytosol. Calprotectin may also be detected at low

levels in monocytes, reactive macrophages, platelets, and squamous epithelial cells (2). Upon

neutrophil activation or death, calprotectin is released extracellularly where it has microbicidal

functions (via heavy-metal chelation) and also serves as a pro-inflammatory ligand for Toll-like

receptor 4 (3). Given its small size, easy diffusion between tissue and blood, and resistance to

enzymatic degradation, calprotectin is a sensitive and dynamic marker of neutrophil activation

anywhere in the body (4, 5). High levels of calprotectin have been found in many types of

infectious and inflammatory diseases—including sepsis, myocardial infarction, inflammatory

bowel disease, lupus, and adult-onset Still’s disease—where it tracks closely with disease

severity (6-10).

While neutrophils are only beginning to be investigated in COVID-19, other pandemic viruses

such as influenza H1N1 and MERS-CoV elicit neutrophilic infiltration at sites of infection (11,

12). Indeed, the acute and exudative phase of ARDS is defined by an exuberant immune

response productive of pro-inflammatory cytokines and chemokines, as well as neutrophil-

mediated disruption of the alveolar epithelial-capillary barrier (13). Neutrophil-depleted mice

demonstrate milder lung pathology in response to influenza infection, with lower levels of

thrombomodulin, matrix metalloproteinases, and myeloperoxidase in bronchoalveolar lavage

fluid (14). Moreover, high levels of neutrophils portend a worse prognosis in patients with

influenza A infection (15).

Work to date exploring COVID-19 pathophysiology has focused especially on macrophages and

their products such as interleukin-6 (IL-6) and IL-1β. While it has been suggested that elevated

levels of blood neutrophils associate with worsening oxygenation in COVID-19 (16, 17), it is



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unknown whether neutrophils are drivers of the thrombo-inflammatory storm or simple

bystanders. In particular, there is a paucity of information about neutrophil catalysts,

checkpoints, and effector mechanisms—all of which could add actionable context to our

understanding of the COVID-19 inflammatory storm. Here, to better understand the potential

role of activated neutrophils in COVID-19, we measured serum calprotectin in hospitalized

patients and determined its relationship to severity of illness and respiratory status.

RESULTS

Detection of calprotectin in sera of COVID-19 patients. Serum samples were obtained from

172 patients hospitalized with COVID-19 at a large academic hospital (Table 1). As compared

with serum samples from 47 healthy controls, the COVID-19 samples showed markedly higher

levels of calprotectin (Figure 1A). For a subset of the patients, longitudinal serum samples

were available. Nine of those patients showed a clinically meaningful change in oxygenation

status during the period of collection (six worsening, three improving). Calprotectin levels

trended upward as oxygenation worsened and downward as oxygenation improved (Figure

1B). In summary, calprotectin is markedly elevated in the sera of patients with COVID-19 and

appears to rise as clinical status deteriorates.

Association between calprotectin and clinical lab studies. We next asked how calprotectin

compared to commonly available clinical tests. Specifically, we assessed potential correlations

with C-reactive protein, D-dimer, ferritin, lactate dehydrogenase, absolute neutrophil count,

lymphocyte count, hemoglobin level, and platelet count. Calprotectin demonstrated a positive

correlation with C-reactive protein, D-dimer, ferritin, lactate dehydrogenase, and absolute

neutrophil count (Figure 2A-E). There was no correlation with absolute lymphocyte count

(Figure 2F). There was a negative correlation with hemoglobin level (Figure 2G) and a positive

correlation with platelet count (Figure 2H). The strongest correlation was with absolute

neutrophil count (r=0.49, p<0.0001).

Calprotectin associates with severe disease including mechanical ventilation. We next

determined each patient’s clinical respiratory status at the time calprotectin was measured. As

compared with patients breathing room air, patients requiring mechanical ventilation had

significantly higher levels of calprotectin (Figure 3A). Interestingly, differences were also

appreciated between patients requiring nasal-cannula oxygen and mechanical ventilation

(Figure 3A). Absolute neutrophil count also differentiated between these same groups, as did



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neutrophil-to-lymphocyte ratio (Supplemental Figure 1A-B). In contrast, C-reactive protein did

not discriminate between patients requiring nasal-cannula oxygen and those requiring

mechanical ventilation (Figure 3B). Beyond clinical respiratory status, oxygenation efficiency

can also be measured by comparing pulse oximetry (SpO2) to the fraction of inspired oxygen

(FiO2). We tested the correlation between calprotectin and SpO2/FiO2 ratio, and found a strong

negative association (r=-0.42, p<0.0001, Figure 3C). Less robust associations were also

appreciated for C-reactive protein, absolute neutrophil count, and neutrophil-to-lymphocyte ratio

(Figure 3D and Supplementary Figure 1C-D).

Calprotectin levels early in hospitalization associate with a requirement for mechanical

ventilation at any point. Of the 172 patients evaluated here, 94 had sera available from the

first two days of their hospitalization. Calprotectin levels were significantly higher in those

individuals who required mechanical ventilation at any point during their hospitalization (n=32),

as compared with those who did not (p<0.0001, Figure 4A). C-reactive protein was also

analyzed (when available on the same day as the calprotectin measurement) and was found to

be predictive with a p-value of 0.0054 (Figure 4B). Taken together, these data suggest a

compelling relationship between neutrophil activation, as defined by serum calprotectin levels,

and severe respiratory disease in COVID-19.

DISCUSSION

Here, we report markedly elevated levels of serum calprotectin indicative of neutrophil activation

in the majority of patients hospitalized with COVID-19. Furthermore, we found that high levels

of serum calprotectin on day 1 or 2 of hospitalization tracked with a requirement for mechanical

ventilation at any point during the admission, a finding that should be urgently assessed in

larger prospective cohorts. These data provide strong evidence in support of activated

neutrophils as indispensable players in moderate-to-severe cases of COVID-19. Triggers of

neutrophil activation are potentially numerous and might include virus-damaged epithelial cells

(14, 18); activated endothelial cells (19); activated platelets (20, 21); cytokines such as IL-1β

(22, 23), granulocyte colony stimulating factor (24, 25), and IL-8 (26, 27); and potentially even

direct interaction with the virus itself, in particular if opsonized with IgG antibodies.

In addition to being an inflammatory marker, calprotectin may also have a direct role in the self-

amplifying thrombo-inflammatory storm that afflicts many COVID-19 patients. Calprotectin

engages innate immune sensors such as TLR4 (28) to activate the transcription factor NFκB



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(29) and ultimately trigger the production of pro-inflammatory cytokines (30). Depending on the

system, calprotectin has been detected both upstream (31) and downstream (32) of IL-6, which

has emerged as a possible therapeutic target in COVID-19. Calprotectin is also a potent

stimulator of neutrophils themselves, promoting degranulation and phagocytosis (33, 34), as

well as other more recently described effector functions such as neutrophil extracellular trap

(NET) release (35). Intriguingly, crosstalk between neutrophils, platelets, and calprotectin

appears to play a role in both arterial and venous thrombosis (35, 36), which are being

increasingly identified as complications of COVID-19 (37, 38).

Not considered in this study is the specific form of neutrophil activation and cell death that floods

COVID-19 patients with excess calprotectin. One consideration is NET release (39, 40). NETs

are extracellular webs of DNA, histones, and microbicidal proteins that appear to perpetuate

many types of lung disease including smoking-related disease (41), cystic fibrosis (42), and

ARDS (14, 43, 44). NETs leverage calprotectin as an antimicrobial strategy against Candida

(45) and Aspergillus (46), but, when left unchecked, NETs are also an important source of

macrophage activation (47, 48) and microvascular occlusion (49, 50). This is another topic that

in our opinion requires further and urgent investigation.

Dipyridamole is an FDA-approved antiplatelet drug with a favorable side-effect profile that our

group recently discovered to inhibit neutrophil activation via agonism of adenosine A2A receptors

(51). In a small clinical trial performed in China, dipyridamole-treated patients demonstrated

improvements in platelet counts and D-dimer levels (52), along with possible stabilization of

clinical status. Given the urgent need for effective treatments of COVID-19, a randomized study

to characterize the impact of dipyridamole on COVID-19-related neutrophil activation, thrombo-

inflammatory storm, and clinical outcomes seems warranted. Other approaches to combatting

neutrophil effector functions include dismantling already-formed NETs (deoxyribonucleases)

and strategies that might prevent neutrophil activation and NET release such as inhibitors of

neutrophil elastase and peptidylarginine deiminase 4 (53, 54).

As we await definitive antiviral and immunologic solutions to the current pandemic, we posit that

anti-neutrophil therapies (53, 54) may be part of a personalized strategy for some individuals

affected by COVID-19 who are at risk for progression to respiratory failure. In this context,

calprotectin is well-positioned to be an early indicator of patients with COVID-19 likely to

progress to respiratory failure and who therefore require immunomodulatory treatment.



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METHODS

Human samples. Serum samples from 172 hospitalized COVID-19 patients were used in this

study. Blood was collected into serum separator tubes by a trained hospital phlebotomist. After

completion of biochemical testing ordered by the clinician, the remaining serum was stored at

4°C for up to 48 hours before it was released to the research laboratory. Serum samples were

immediately divided into small aliquots and stored at -80°C until the time of testing. All 172

patients had a confirmed COVID-19 diagnosis based on FDA-approved RNA testing. This study

complied with all relevant ethical regulations, and was approved by the University of Michigan

Institutional Review Board (HUM00179409), which waived the requirement for informed consent

given the discarded nature of the samples. Healthy volunteers were recruited through a posted

flyer; exclusion criteria for these controls included history of a systemic autoimmune disease,

active infection, and pregnancy. For preparation of control serum, blood was collected into

serum separator tubes by a trained hospital phlebotomist. These serum samples were divided

into small aliquots and stored at -80°C until the time of testing.

Quantification of S100A8/A9 (calprotectin). Calprotectin levels were measured with the

Human S100A8/S100A9 Heterodimer DuoSet ELISA (DY8226-05, R&D Systems) according to

the manufacturer’s instructions.

Statistical analysis. When two groups were present, normally-distributed data were analyzed

by two-sided t test and skewed data were analyzed by Mann-Whitney test. For three or more

groups, analysis was by one-way ANOVA or Kruskal-Wallis test with correction for multiple

comparisons. Correlations were tested by Pearson’s correlation coefficient. Data analysis was

with GraphPad Prism software version 8. Statistical significance was defined as p<0.05.

ACKNOWLEDGEMENTS

The work was supported by a COVID-19 Cardiovascular Impact Research Ignitor Grant from the

Michigan Medicine Frankel Cardiovascular Center as well as by the A. Alfred Taubman Medical

Research Institute. YZ was supported by career development grants from the Rheumatology

Research Foundation and APS ACTION. JAM was partially supported by the VA Healthcare

System. JSK was supported by grants from the NIH (R01HL115138), Lupus Research Alliance,

and Burroughs Wellcome Fund. YK was supported by the NIH (K08HL131993, R01HL150392),

Falk Medical Research Trust Catalyst Award, and the JOBST-American Venous Forum Award.



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AUTHORSHIP

HS, YZ, SY, KG, MZ, JM, CNB, WW, and SPL conducted experiments and analyzed data. HS,

YZ, NLL, RJW, CL, JSK, and YK conceived the study and analyzed data. All authors

participated in writing the manuscript and gave approval before submission.

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Table 1: COVID-19 patient characteristics

Demographics

Number 172

Age (years)* 61.48 ± 17.7 (26-96)

Hospital day** ‡ 4.6 ± 5.2 (1-25)

Female 75 (44%)

White/Caucasian 67 (39%)

Black/African-American 75 (44%)

Comorbidities

Diabetes 69 (40%)

Ischemic heart disease 52 (30%)

Renal disease 51 (30%)

Lung disease 46 (27%)

Autoimmune 9 (5%)

Cancer 23 (13%)

History of stroke 16 (9%)

Obesity 83 (48%)

Hypertension 116 (67%)

Immune deficiency 12 (7%)

History of smoking 45 (26%)

Medications‡

Hydroxychloroquine 31 (18%)

Anti-IL6 receptor 21 (12%)

ACE inhibitor 8 (5%)

Angiotensin receptor blocker 1 (0.6%)

Antibiotic 64 (37%)

Antiviral 6 (3%)

Respiratory status‡

Mechanical ventilation 61 (39%)

High-flow oxygen 12 (7%)

Nasal-cannula oxygen 59 (34%)

Room air 40 (23%)

* Mean ± standard deviation ** Median = 3 ‡ At time of first sample available for testing



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Figure 1: Detection of calprotectin in sera of COVID-19 patients. A, Sera from COVID-19

patients (n=172) and healthy controls (n=47) were assessed for calprotectin (note log scale).

COVID-19 samples were compared to controls by Mann-Whitney test; ****p<0.0001. B, Nine of

the COVID-19 patients had longitudinal samples available during a time in which they had a

significant change in oxygenation status.



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Figure 2: Association between calprotectin and clinical studies. Calprotectin levels

(n=172) were compared to clinical laboratory results (when available on the same day as the



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research sample). Correlation coefficients were calculated for C-reactive protein (A), D-dimer

(B), ferritin (C), lactate dehydrogenase (D), absolute neutrophil count (E), neutrophil-to-

lymphocyte ratio (F), hemoglobin (G), and platelets (H).



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Figure 3: Levels of calprotectin track closely with oxygenation status. Patients (n=172)

were grouped by clinical status (room air vs. nasal cannula vs. mechanical ventilation) and

analyzed for calprotectin (A) and C-reactive protein (B). Groups were compared by Kruskal-

Wallis test corrected by Dunn's test for multiple comparisons; *p<0.05 and ****p<0.0001.

Calprotectin (C) or C-reactive protein (D) were compared to SpO2/FiO2 ratio for each patient.

Correlation was determined by Pearson’s test.



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Figure 4: Levels of calprotectin early in hospitalization associate with requirement for

mechanical ventilation at any point. A, For 94 patients, a calprotectin level was available

from hospital day 1 or 2. Here, patients are divided by whether they at any point required

mechanical ventilation (vent ever, n=32) during their hospitalization. Groups were compared by

Mann-Whitney test; ****p<0.0001. B, A similar analysis was performed for C-reactive protein;

**p<0.01.



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Supplemental Figure 1: Levels of calprotectin track closely with oxygenation status.

Patients (n=172) were grouped by clinical status (room air vs. nasal cannula vs. mechanical

ventilation) and analyzed for calprotectin absolute neutrophil count (A) or neutrophil-to-

lymphocyte ratio (B). Groups were compared by Kruskal-Wallis test corrected by Dunn's test for

multiple comparisons; **p<0.01, ***p<0.001, and ****p<0.0001. Absolute neutrophil count (C) or

neutrophil-to-lymphocyte ratio (D) was compared to SpO2/FiO2 ratio for each patient.

Correlation was determined by Pearson’s test.



https://doi.org/10.1101/2020.05.06.20093070

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