key: cord-284576-nemh4wdo
authors: Sims, Jonathan T.; Krishnan, Venkatesh; Chang, Ching-Yun; Engle, Sarah M.; Casalini, Giacomo; Rodgers, George H.; Bivi, Nicoletta; Nickoloff, Brian J.; Konrad, Robert J.; de Bono, Stephanie; Higgs, Richard E.; Benschop, Robert J.; Ottaviani, Silvia; Cardoso, Anabela; Nirula, Ajay; Corbellino, Mario; Stebbing, Justin
title: Characterization of the Cytokine Storm Reflects Hyperinflammatory Endothelial Dysfunction in COVID-19
date: 2020-09-10
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2020.08.031
sha: 
doc_id: 284576
cord_uid: nemh4wdo

Background Physicians treating COVID-19 patients increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared to healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. Objective To identify and characterize the host inflammatory response to SARS-CoV-2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease, in patients stratified as mild, moderate, and severe, versus matched healthy controls. Methods Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers. Results Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within COVID-19 patients, and strong biomarker association with patient response as measured by Ordinal scale. As patients progress, we observe statistically significant dysregulation of IFNγ, IL-1RA, IL-6, IL-10, IL-19, MCP-1, -2, -3, CXCL9, CXCL10, CXCL5, ENRAGE and PARP-1. Furthermore, in a limited series of patients who were sampled frequently confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. Conclusion These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of SARS-CoV-2 pathogenesis and the host response.

Background: Physicians treating COVID-19 patients increasingly believe that the 54 hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating 55 biomarkers seen across the spectrum of COVID-19 have not been characterized compared to 56 healthy controls, but such analyses are likely to yield insights into the pursuit of interventions 57 that adequately reduce the burden of these cytokine storms. 58

Objective: To identify and characterize the host inflammatory response to SARS-CoV-2 59 infection, we assessed levels of proteins related to immune responses and cardiovascular 60 disease, in patients stratified as mild, moderate, and severe, versus matched healthy controls. 61

Methods: Blood samples from adult patients hospitalized with COVID-19 were analyzed using 62 high-throughput and ultrasensitive proteomic platforms and compared with age-and sex-63 matched healthy controls to provide insights into differential regulation of 185 markers. 64

Results: Results indicate a dominant hyperinflammatory milieu in the circulation and vascular 65 endothelial damage markers within COVID-19 patients, and strong biomarker association with 66 patient response as measured by Ordinal scale. As patients progress, we observe statistically 67 significant dysregulation of IFNγ, IL-1RA, IL-6, IL-10, IL-19, MCP-1, -2, -3, CXCL9, CXCL10, 68 CXCL5, ENRAGE and PARP-1. Furthermore, in a limited series of patients who were sampled 69 frequently confirming reliability and reproducibility of our assays, we demonstrate that 70 intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine 71 The COVID-19 pandemic created an overwhelming need to define host-derived molecular 96 mediators of disease severity evident in hospitalized patients. One approach to dissect 97 protective and pathological immune responses to COVID-19 infection is to measure plasma 98 biomarkers correlated to various stages of COVID-19 and determine which pro-inflammatory 99 mediators are modulated in response to therapies improving patient outcomes(1). 100

We utilized multiplex methodology from Olink Proteomics to assess 184 analytes, spanning a 104 broad range of cytokines and chemokines involved in inflammation (INF I) and cardiovascular-105 linked processes (CVD II), and, based on the inflammatory responses described in SARS-CoV-106 2-infected lung epithelium, we also measured IL-19 levels using a highly sensitive and specific 107 assay as previously described(4, 6, 7). Analysis of the differential protein regulation patterns 108 within severity classes of COVID-19 cases compared to HC revealed strong dysregulation of 109

IFNγ, IL-6, IL-10, MCP-3, CXCL9, CXCL10, CXCL11, ENRAGE, PARP-1, and IL-1RA (Fig. 1A , 110 B-bottom, C). These changes were significant and ranged from increases of 21-fold for IFNγ, 111

18-fold for IL-6, 12-fold for MCP-3, to 9-fold for CXCL10, to more than 3 fold for MCP-1 and -2, 

Our analysis is consistent with a more prominent increase in IFNγ and IL-10 levels in COVID-19 118 patients as seen in the moderate/severe and critical patients who are more likely to suffer from 119 adult respiratory distress syndrome(9). While interferon signaling is certainly implicated with the 120 J o u r n a l P r e -p r o o f dramatic increase in IFNγ, additional insights into type I interferon family members was out of 121 scope of this manuscript due to the lack of reliable ultrasensitive assays for these analytes. 122

Furthermore, we observed PTX3, Gal9, and CD8A upregulation in all patients regardless of 123 severity (Fig. 1A clinical inflammatory markers, such as LDH, CRP, and Ferritin, but we specifically observed that 148 MCP-3, CTSL1, and PTX3 were more meaningfully correlated to changes in the Ordinal Scale 149 than these widely used readouts (Supplementary Table 4 ). We observed several analytes 150 inversely correlated to the same magnitude (Cor<-0.7) with both LDH, CRP, and Ferritin and the 151

Ordinal Scale: MMP12, GIF, GDF-2, BOC, SCF, and CD6 (Fig. 1A, B- In addition to these identified immunological changes, there is a growing consensus on the Changes in PARP-1 were most pronounced in the severe, and critical patients and this marker 171 have been implicated in cardiovascular disease(21). It is also well known that PARP-1 activity 172 correlates with viral infection and, conceivably, this marker reflects the added tissue viral burden 173 in such patients; however, in this limited set, PARP-1 levels did not correlate with change in the 174

Ordinal Scale or viral load (Fig. 1B) . Furthermore, it has been reported that GDF-2 (BMP-9) 175 plays a protective role against inflammation-mediated damage on endothelial and cardiac 176 tissues. Likewise, we observed downregulation of GDF-2 in the plasma of COVID-19 patients 177 (Fig. 1B) . In GDF-2 -/mice subjected to transverse aortic constriction, loss of GDF-2 activity 178 promoted cardiac fibrosis and impairs heart function(22). Therefore, reductions in GDF-2 179 potentially may result in endothelial and cardiac damage in an inflammatory condition, and 180 therapeutic restoration of factors such as GDF-2, such as observed with Baricitinib-treated 181 patients, can help stabilize cardiac function in these at-risk patients (Fig. 2D) . 182

Changes in cytokines and other biomarkers in a series of 4 patients treated with Baricitinib 184 offers insights in the pleiotropic nature of this JAK1/JAK2 inhibitor. The biomarkers that were 185 most robustly decreased longitudinally in the Baricitinib-treated cohort compared to baseline 186 include IL-6, MCP-3, IL-10, CXCL10, and IFNγ ( Fig. 1-middle; Fig. 2 The authors would like to thank healthcare workers across the world for their response to 213 COVID-19. We thank Jochen Schmitz for scientific input and support. We thank Cynthia Abbott 214 and Diane Stothard for manuscript preparation support. moderate/severe (middle), and critical (right) patients compared to HC. The y-axis is the -log10 317 of the p-value with higher numbers reflecting greater significance and the x-axis represents the 318 log2 fold change, wherein color designation of blue or red represents decreased or increased 319 presence of these markers relative to HC, respectively. 

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World Health Organization

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CoV-2 infection

Remdesivir in adults with severe 262

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Thromboembolic risk and anticoagulant therapy in COVID-19 patients: emerging evidence and 275 call for action

Soluble 277 CD40 ligand, interleukin (IL)-6, and hemostatic parameters in metabolic syndrome patients with 278 and without overt ischemic heart disease

Mapping of 79 loci for 83 plasma protein biomarkers in cardiovascular disease

Role of poly(ADP-ribose) polymerase 1 (PARP-1) in cardiovascular 283 diseases: the therapeutic potential of PARP inhibitors

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PTX3 in small-vessel 289 vasculitides: An independent indicator of disease activity produced at sites of inflammation

Circulating levels of the 292 long pentraxin PTX3 correlate with severity of infection in critically ill patients

A 295 single-cell atlas of the peripheral immune response in patients with severe COVID-19