key: cord-0841916-tweqxttb
authors: Shi, H.; Zuo, Y.; Yalavarthi, S.; Gockman, K.; Zuo, M.; Madison, J. A.; Blair, C. N.; Woods, R. J.; Lood, C.; Knight, J. S.; Kanthi, Y.
title: Neutrophil calprotectin identifies severe pulmonary disease in COVID-19
date: 2020-05-10
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2020.05.06.20093070
sha: bd1e9dea98f7bfcaedc793659820a0a678792676
doc_id: 841916
cord_uid: tweqxttb

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=96) 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=52 patients), calprotectin levels were significantly higher in patients who progressed to severe COVID-19 requiring mechanical ventilation (7805 +/- 7585, n=23) as compared to those who remained free of intubation (3123 +/- 2990, p=0.0059). 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.

Severe cases of coronavirus disease 2019 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=96) have markedly elevated levels of the neutrophil activation marker S100A8/A9 (calprotectin) in their blood. Calprotectin tracked with other acute phase reactants including Creactive 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=52 patients), calprotectin levels were significantly higher in patients who progressed to severe COVID-19 requiring mechanical ventilation (7805 ± 7585, n=23) as compared to those who remained free of intubation (3123 ± 2990, p=0.0059). 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.

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 preprint this version posted May 10, 2020. . https://doi.org/10.1101/2020.05.06.20093070 doi: medRxiv preprint

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] [7] [8] [9] [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 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 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 preprint this version posted May 10, 2020. . https://doi.org/10.1101/2020.05.06.20093070 doi: medRxiv preprint 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.

Detection of calprotectin in sera of COVID-19 patients. Serum samples were obtained from 96 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. Eight of those patients showed a clinically meaningful change in oxygenation status during the period of collection (five 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, neutrophil-to-lymphocyte ratio, hemoglobin level, and platelet count. Calprotectin demonstrated a positive correlation with C-reactive protein, D-dimer, ferritin, lactate dehydrogenase, absolute neutrophil count, and neutrophil-to-lymphocyte ratio (Figure 2A-F) . There was a negative correlation with hemoglobin level ( Figure 2F ) and a positive correlation with platelet count ( Figure 2G ). The strongest correlations were with C-reactive protein (r=0.51, p<0.0001) and absolute neutrophil count (r=0.50, 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 ). Differences were also appreciated between patients requiring nasal-cannula oxygen and mechanical ventilation ( Figure 3A) .

Absolute neutrophil count also differentiated between these same groups, albeit with lower p values ( Figure 3B ). In contrast, neither neutrophil-to-lymphocyte ratio nor C-reactive protein discriminated between patients requiring nasal-cannula oxygen and mechanical ventilation 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 preprint this version posted May 10, 2020. . https://doi.org/10.1101/2020.05.06.20093070 doi: medRxiv preprint (Figure 3C-D) . 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 ( Figure 3E) . Of the 96 patients evaluated here, 52 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=23), as compared with those who did not (p=0.0059, Figure 3F ). Taken together, these data suggest a compelling relationship between neutrophil activation, as defined by serum calprotectin levels, and severe respiratory disease in COVID-19.

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 selfamplifying thrombo-inflammatory storm that afflicts many COVID-19 patients. Calprotectin engages innate immune sensors such as TLR4 28 to activate the transcription factor NFκB 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 . 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 preprint this version posted May 10, 2020. 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.

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 96 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 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 preprint this version posted May 10, 2020. 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 preprint this version posted May 10, 2020. (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 preprint this version posted May 10, 2020. 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 preprint this version posted May 10, 2020. were compared to clinical laboratory results (when available on the same day as the research 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 preprint this version posted May 10, 2020. were grouped by clinical status (room air vs. nasal cannula vs. mechanical ventilation) and analyzed for calprotectin (A), absolute neutrophil count (B), neutrophil-to-lymphocyte ratio (C), and C-reactive protein (D). Groups were compared by Kruskal-Wallis test corrected by Dunn's test for multiple comparisons; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. E, Calprotectin was compared to SpO2/FiO2 ratio for each patient. Correlation was determined by Pearson's test. F, For 52 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=23) during their hospitalization. Groups were compared by Mann-Whitney test; **p<0.01. 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 preprint this version posted May 10, 2020. . https://doi.org/10.1101/2020.05.06.20093070 doi: medRxiv preprint

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China

Distribution of a new myelomonocytic antigen (L1) in human peripheral blood leukocytes. Immunofluorescence and immunoperoxidase staining features in comparison with lysozyme and lactoferrin

Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock

Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study

The leukocyte protein L1 in plasma and synovial fluid from patients with rheumatoid arthritis and osteoarthritis

Calprotectin and calgranulin C serum levels in bacterial sepsis

Fecal calprotectin in diagnosis and clinical assessment of inflammatory bowel disease

Pro-inflammatory S100 proteins are associated with glomerulonephritis and anti-dsDNA antibodies in systemic lupus erythematosus

Serum calprotectin--a promising diagnostic marker for adultonset Still's disease

Myeloid-related protein 8/14 and the risk of cardiovascular death or myocardial infarction after an acute coronary syndrome in the Pravastatin or Atorvastatin Evaluation and Infection Therapy: Thrombolysis in Myocardial Infarction (PROVE IT-TIMI 22) trial

Immunopathogenesis of coronavirus infections: implications for SARS

A Pathophysiologic Approach to Biomarkers in Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome

Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis

High Level of Neutrophil Extracellular Traps Correlates With Poor Prognosis of Severe Influenza A Infection

Immune phenotyping based on neutrophil-to-lymphocyte ratio and IgG predicts disease severity and outcome for patients with

COVID-19 early warning score: a multi-parameter screening tool to identify highly suspected patients

Neutrophil extracellular traps cause airway obstruction during respiratory syncytial virus disease

Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death

Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood

Targeting potential drivers of COVID-19: Neutrophil extracellular traps

Neutrophil extracellular traps are associated with disease severity and microbiota diversity in patients with chronic obstructive pulmonary disease

Cystic fibrosis sputum DNA has NETosis characteristics and neutrophil extracellular trap release is regulated by macrophage migration-inhibitory factor

Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury

Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury

Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans

Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent

Extracellular DNA, Neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma

Neutrophil Extracellular Traps (NETs) promote macrophage inflammation and impair atherosclerosis resolution in mice with diabetes

Biological basis and pathological relevance of microvascular thrombosis

Platelets, neutrophils, and neutrophil extracellular traps (NETs) in sepsis

Adenosine receptor agonism protects against NETosis and thrombosis in antiphospholipid syndrome

Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19

Neutrophil Extracellular Traps: Villains and Targets in Arterial, Venous, and Cancer-Associated Thrombosis

At the Bedside: Neutrophil extracellular traps (NETs) as targets for biomarkers and therapies in autoimmune diseases

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. 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)

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 preprint this version posted