Bio-Plex Pro Cytokine Assays

Aberrant Cytokine Activity in the
Host Immune Response to COVID-19
Leads to Cytokine Release Syndrome
By Kenneth Oh

Emerging medical reports from the COVID-19 pandemic include a broad spectrum of patient symptoms
due to the host immune response. Host cells utilize cytokines and chemokines to coordinate nearly all
aspects of an immunogenic response to pathogens: acute inflammation, innate response, and adaptive
immunity. Evidence increasingly suggests that cytokine release syndrome (CRS, or cytokine storm), a typeof exuberant immune response, may be responsible for severe complications, including acute respiratory
distress syndrome (ARDS) and death, as well as many of the milder symptoms observed.
As of May 7, 2020, there were more than 3.7 million confirmed
cases of, and over 264,000 deaths from, COVID-19 disease
worldwide (Johns Hopkins University COVID-19 Dashboard,coronavirus.jhu.edu/map.html). While many scientists race toperform virological assessments of the SARS-CoV-2 virion,
others are focusing on the host immune response, given
the range of host responses in the context of comorbidities,complications, and causes of death from COVID-19. In ameta-analysis of 5,700 hospital-admitted patients from theNew York area, the most common comorbidities werehypertension (n = 3,026; 56.6%), obesity (n = 1,737; 41.7%), anddiabetes (n = 1,808; 33.8%) (Richardson et al. 2020). All threeof the most prevalent comorbidities in patients with COVID-19
are associated with chronic high- or low-grade inflammation(Bartoloni et al. 2017; Ellulu et al. 2017; Tsalamandris et al.
2019). Chronic inflammation is a physiological state of elevatedcirculating proinflammatory cytokines due to one or multiple
preexisting conditions (comorbidities), a prior or current
infection, or both.
A serious complication from COVID-19, which results in severe
illness or death, is ARDS. ARDS is the condition of hypoxemia(low blood oxygen) and results in a rapid onset of respiratory
failure. ARDS can be caused by a variety of mechanisms.
Beginning with an acute inflammatory phase, a macrophageor infected-cell–driven proinflammatory cytokine mediation(IL-1, IL-1β, IL-6, IL-8, and TNF-α) acts locally to activate
neutrophils and trigger perforation at the alveoli, resulting ina buildup of mucosal and endemic fluids and cellular debris.
This strong proinflammatory response is controlled by antiinflammatory cytokines, such as macrophage inhibitory factor(MIF), IL-10, and IL-11. However, if immunological homeostasis
is not achieved, counterproductive fibrotic lung repair takes
place, and the alveolar space becomes filled with stem cells
and new blood vessels (the “ground-glass” effect), leading
to poor oxygen uptake and often resulting in death. A recentevaluation of 150 patients from Wuhan, China lists respiratory
failure as a result of ARDS as the leading cause of death fromCOVID-19 (Ruan et al. 2020).
As our understanding of the pathophysiology of COVID-19
has evolved, a host immune hyperinflammatory response has
been implicated as a major contributor to severe illness, pooroutcomes, and death. CRS is the aberrant overproduction ofproinflammatory cytokines, such as IFN-γ, IL-1α, IL-1β, IL-6,IL-8, GM-CSF, M-CSF, and TNF-α (Tisoncik et al. 2012),
resulting in high concentrations of systemic circulatingcytokines. At the onset of viral infection, an acute inflammatory
event is triggered by foreign pattern recognition receptors,
which recruit other immune cells to destroy the foreign
pathogen while simultaneously inflicting localized collateral
damage. In an ideally functioning immune response,
a reparative process is initiated shortly thereafter, andhomeostasis is achieved through anti-inflammatory cytokines
and host tissue repair (primarily by endothelial cells).
However, excess proinflammatory cytokines may be present
due to a hyperinflammatory response to a virulent pathogen;preexisting circulating cytokines, chemokines, and theircognate soluble receptors (chronic, low-grade, or high-grade
inflammation); or both. When coupled with inflammationinduced vascular leakage, an influx of proinflammatory

Aberrant Cytokine Activity in the Host Immune Response to COVID-19 Leads to Cytokine Release Syndrome

Bio-Plex Pro Human Cytokine 27-Plex Assay (see Table 1 forassay composition). The researchers reported higher plasma
concentrations for IL-1β, IL-1Rα, IL-7, IL-8, IL-9, IL-10, FGF basic,G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β,PDGF-BB, TNF-α, and VEGF in both ICU patients and non-ICU
patients than in healthy adults. Higher plasma concentrationsof IL-6 were found in patients in ICU only. Notably, further
comparisons indicated higher levels of IL-2, IL-7, IL-10, G-CSF,IP-10, MCP-1, MIP-1α, and TNF-α in ICU patients than innon-ICU patients (Huang et al. 2020).

cytokines into the circulatory system results, inducing systemic
inflammation and tissue damage. Complications, such ascardiac injury or arrest, sepsis, ARDS, lung injury, organ failure,
encephalopathy, and death — all of which have been observedin hospitalized patients with COVID-19 (Jiang et al. 2020; Zenget al. 2020; Poyiadji et al. 2020; Filatov et al. 2020) — may result.
CRS is often not listed as a clinical cause of death, in part
because an exact definition does not yet exist, and partly
because the formal clinical classification schemes rely
mainly on physiological qualitative characterization and thepresence of general inflammation markers, such as C-reactive
protein. However, studies of COVID-19 pathophysiology haveincreasingly implicated CRS as a root cause or underlying factor
in COVID-19–related deaths and a serious result of immune
system dysregulation that warrants significantly more research.
CRS is also a major concern and is considered a high-risk factor
in treatment with advanced forms of T-cell–recruiting therapies,
such as chimeric antigen receptor T-cell therapy (Garcia
Borrega et al. 2019). Therefore, the pathophysiologies of CRS,
ARDS, and chronic inflammation merit much more investigation.

In another report, researchers performed a longitudinal
study (three time points over 15+ days) measuring theplasma cytokine and chemokine levels of twelve patientswith COVID-19, eight patients with bacterial pneumonia,
eight patients with influenza virus A H7N9, and eight healthy
subjects in Shenzhen, China using the Bio-Plex Pro Human
Cytokine Screening Panel, 48-Plex. The composition ofthis panel and two other Bio-Rad human cytokine andchemokine panels is described in Table 1. Their analysis
revealed significant elevations in 38 of the 48 cytokines andchemokines measured in patients with COVID-19. Further
analysis revealed a constellation of 17 cytokines linked toSARS-CoV-2 virion load and 15 cytokines strongly associatedwith Murray lung injury score, which could predict disease
severity according to receiver operating characteristic (ROC)
and ROC/area under the ROC curve (ROC/AUC) curves for
COVID-19 (Liu et al. 2020).

Proinflammatory cytokines, such as IFN-γ, IL-1β, IL-6, IL-8,
GM-CSF, M-CSF, and TNF-α, have received significant
attention in CRS and ARDS research (Figure 1). Furthermore,proinflammatory cytokines are often investigated in relation
to both CRS and ARDS, as these two diseases share similar
pathologies, although the exact causal relationship is still
under investigation. Many noncanonical cytokines warrant
more research as well, as their role in immune response andregulation is critical.

As the need to emerge from this global pandemic grows,
performing more research on host immune responses byevaluating multiple cytokines (ideally in longitudinal studies)
is essential and urgently needed in order to understand thepathophysiology of COVID-19 and the underlying mechanismsof CRS.

One of the first reports emerging from the epicenter of theSARS-CoV-2 outbreak in Wuhan, China measured cytokine
levels in hospitalized patients with COVID-19 using the Bio-Rad

Number of journal articles

ARDS

CRS

1,000

May 2020

800
600
400200

VEGF

TRAIL

TNF-α

TNF-β

SDF-1αSCF

SCGF-βPDGF

RANTES

β-NGF

MIP-1αMIP-1β

MIF

MIG

MCP-3

M-CSF

LIF

MCP-1

IL-18

IP-10

IL-16

IL-17A

IL-13

IL-15

IL-12 (p70)

IL-12 (p40)

IL-9

IL-10

IL-7

IL-8

IL-5

IL-6

IL-3

IL-4

IL-2IL-2RαIL-1α

IL-1βIFN-γIL1-ra

HGF

IFN-α2GRO-αG-CSF

GM-CSFFGF basicCTACKEotaxin

0Biomarker

Fig. 1. Number of publications associating cytokine and chemokine biomarkers with CRS and ARDS between 1990 and April 2020, according to a
PubMed search. Blue bars, publications referencing CRS. Orange bars, publications referencing ARDS.

© 2020 Bio-Rad Laboratories, Inc.

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Aberrant Cytokine Activity in the Host Immune Response to COVID-19 Leads to Cytokine Release Syndrome

May 2020

Table 1. The composition of three human cytokine and chemokine panels.

Cytokine or Chemokine

Alternate Cytokine orChemokine Designation

Bio-Plex Pro
Human Cytokine Screening
48-Plex Panel

Bio-Plex Pro
Human Cytokine
27-Plex Assay

Bio-Plex Pro
Human Immunotherapy
20-Plex Panel——CTACK

CCL27

Eotaxin

CCL11—

FGF basic

FGF-β

—

G-CSF

GCSF; CSF3—

GM-CSF

CSF2

GRO-α

CXCL1

—

—

HGF

Hepatocyte growth factor;
scatter factor (SF)——IFN-α2

IFN-a2

—

—

IFN-γ

—
—

—

IL-1RαIL-1Ra

IL-1α

IL-1a; hematopoietin 1

IL-1β

IL-1b; leukocytic pyrogen

IL-2———

IL-2Rα

CD25—

—

IL-3

Colony-stimulating factor

——

IL-4—

IL-5

—

IL-6—

IL-7

—

IL-8

CXCL8

IL-9—

IL-10

CSIF

IL-12 (p40)

NKSF

IL-12 (p70)

NKSF1

IL-13

—

IL-15

—

IL-16

—

IL-17A—

IL-18

IFN-γ inducing factor

IP-10

CXCL10—
—————

—

LIF

Leukemia inhibitory factor

—

—

M-CSF

Macrophage colonystimulating factor 1

—

—

MCP-1

CCL2; MCAF

MCP-3

CCL7—

—MIF

MMIF; GIF——MIG

CXCL9—

MIP-1αCCL3

MIP-1βCCL4β-NGF

Nerve growth factor, beta

PDGF-BB

Platelet derived growth factor

RANTES

CCL5

—

—
—

SCF

Stem cell factor; steel factor——SCGF-βStem cell growth factor, beta——

SDF-1αCXCL12; stromal cell–derivedfactor 1

—

—

TNF-α

Cachexin; cachectin; DIF;
TNFSF2

TNF-β

Lymphotoxin-α

—

—

TRAIL

TNFSF11

—

—

VEGF

—

© 2020 Bio-Rad Laboratories, Inc.

—

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Aberrant Cytokine Activity in the Host Immune Response to COVID-19 Leads to Cytokine Release Syndrome

May 2020References
Bartoloni E et al. (2017). Role of inflammatory diseases in hypertension.
High Blood Press Cardiovasc Prev 24, 353–361.
Ellulu MS et al. (2017). Obesity and inflammation: The linking mechanism andthe complications. Arch Med Sci 13, 851–863.
Filatov A et al. (2020). Neurological complications of coronavirus disease(COVID-19): Encephalopathy. Cureus 12, e7352.
Garcia Borrega J et al. (2019). In the eye of the storm: Immune-mediatedtoxicities associated with CAR-T cell therapy. Hemasphere 3, e191.
Huang C et al. (2020). Clinical features of patients infected with 2019 novel
coronavirus in Wuhan, China. Lancet 395, 497–506.
Jiang F et al. (2020). Review of the clinical characteristics of coronavirus
disease 2019 (COVID-19). J Gen Intern Med 35, 1,545–1,549.
Johns Hopkins University & Medicine. Coronavirus Resource Center.
COVID-19 Dashboard by the Center for Systems Science and Engineering(CSSE) at Johns Hopkins University. coronavirus.jhu.edu/map.html,
accessed May 8, 2020.
Liu Y et al. (2020). Elevated plasma level of selective cytokines in COVID-19
patients reflect viral load and lung injury. National Science Review [publishedonline ahead of print March 9, 2020]. Accessed May 7, 2020.
Poyiadji N et al. (2020). COVID-19–associated acute hemorrhagic necrotizing
encephalopathy: CT and MRI features. Radiology [published online ahead ofprint March 31, 2020]. Accessed May 7, 2020.

Richardson S et al. (2020). Presenting characteristics, comorbidities, andoutcomes among 5,700 patients hospitalized with COVID-19 in the New York
City area. JAMA [published online ahead of print April 22, 2020].
Accessed May 7, 2020.
Ruan Q et al. (2020). Clinical predictors of mortality due to COVID-19 based on
an analysis of data of 150 patients from Wuhan, China. Intensive Care Med 46,846–848.
Tsalamandris S et al. (2019). The role of inflammation in diabetes: Current
concepts and future perspectives. Eur Cardiol 14, 50–59.
Tisoncik JR et al. (2012). Into the eye of the cytokine storm. Microbiol Mol Biol
Rev 76, 16–32.
Zeng JH et al. (2020). First case of COVID-19 complicated with fulminant
myocarditis: A case report and insights. Infection [published online ahead ofprint April 10, 2020]. Accessed May 7, 2020.

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