COVID-19 Severity Shifts the Cytokine Milieu Toward a Proinflammatory State in Egyptian Patients: A Cross-Sectional Study

Abstract

Despite extensive research to decipher the immunological basis of coronavirus disease (COVID-19), limited evidence on immunological correlates of COVID-19 severity from MENA region and Egypt was reported. In a single-center cross-sectional study, we have analyzed 25 cytokines that are related to immunopathologic lung injury, cytokine storm, and coagulopathy in plasma samples from 78 hospitalized Egyptian COVID-19 patients in Tanta University Quarantine Hospital and 21 healthy control volunteers between April 2020 and September 2020. The enrolled patients were divided into 4 categories based on disease severity, namely mild, moderate, severe, and critically ill. Interestingly, interleukin (IL)-1-α, IL-2Rα, IL-6, IL-8, IL-18, tumor necrosis factor-alpha (TNF-α), FGF1, CCL2, and CXC10 levels were significantly altered in severe and/or critically ill patients. Moreover, principal component analysis (PCA) demonstrated that severe and critically ill COVID-19 patients cluster based on specific cytokine signatures that distinguish them from mild and moderate COVID-19 patients. Specifically, levels of IL-2Rα, IL-6, IL-10, IL-18, TNF-α, FGF1, and CXCL10 largely contribute to the observed differences between early and late stages of COVID-19 disease. Our PCA showed that the described immunological markers positively correlate with high D-dimer and C-reactive protein levels and inversely correlate with lymphocyte counts in severe and critically ill patients. These data suggest a disordered immune regulation, particularly in severe and critically ill Egyptian COVID-19 patients, manifested as overactivated innate immune and dysregulated T-helper1 responses. Additionally, our study emphasizes the importance of cytokine profiling to identify potentially predictive immunological signatures of COVID-19 disease severity.

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COVID-19 Severity Shifts the Cytokine Milieu Toward
a Proinflammatory State in Egyptian Patients:
A Cross-Sectional Study
Mohamed L. Salem,
1,2
Madonna M. Eltoukhy,
3
Rasha E. Shalaby,
4,5
Kamal M. Okasha,
6
Mohamed R. El-Shanshoury,
7
Mohamed A. Attia,
3
Mohamed S. Hantera,
8
Asmaa Hilal,
9
and Mohammed A. Eid
9
Despite extensive research to decipher the immunological basis of coronavirus disease (COVID-19), limited
evidence on immunological correlates of COVID-19 severity from MENA region and Egypt was reported. In
a single-center cross-sectional study, we have analyzed 25 cytokines that are related to immunopathologic
lung injury, cytokine storm, and coagulopathy in plasma samples from 78 hospitalized Egyptian COVID-19
patients in Tanta University Quarantine Hospital and 21 healthy control volunteers between April 2020 and
September 2020. The enrolled patients were divided into 4 categories based on disease severity, namely mild,
moderate, severe, and critically ill. Interestingly, interleukin (IL)-1-a,IL-2Ra, IL-6, IL-8, IL-18, tumor
necrosis factor-alpha (TNF-a), FGF1, CCL2, and CXC10 levels were significantly altered in severe and/or
critically ill patients. Moreover, principal component analysis (PCA) demonstrated that severe and critically
ill COVID-19 patients cluster based on specific cytokine signatures that distinguish them from mild and
moderate COVID-19 patients. Specifically, levels of IL-2Ra, IL-6, IL-10, IL-18, TNF-a, FGF1, and CXCL10
largely contribute to the observed differences between early and late stages of COVID-19 disease. Our PCA
showed that the described immunological markers positively correlate with high D-dimer and C-reactive
protein levels and inversely correlate with lymphocyte counts in severe and critically ill patients. These data
suggest a disordered immune regulation, particularly in severe and critically ill Egyptian COVID-19 patients,
manifested as overactivated innate immune and dysregulated T-helper1 responses. Additionally, our study
emphasizes the importance of cytokine profiling to identify potentially predictive immunological signatures
of COVID-19 disease severity.
Keywords: COVID-19, Egyptian COVID-19 patients, proinflammatory cytokines, cytokine profile, coagulo-
pathy, Luminex assay
Introduction
Coronavirus disease (COVID-19), a contagious disease
caused by severe acute respiratory syndrome cor-
onavirus 2 (SARS-CoV-2), represents the third coronavirus-
related zoonotic disease of global public health importance
after severe acute respiratory syndrome (SARS) and the
Middle East respiratory syndrome (MERS) that primarily
emerged in 2003 and 2012, respectively (Azhar et al., 2019;
Hui and Zumla, 2019; Richardson et al., 2020). Severe
COVID-19 cases usually suffer from pneumonia that cor-
relates with both localized and systemic proinflammatory
Departments of
1
Zoology and
9
Botany and Microbiology, Faculty of Science, Tanta University, Tanta, Egypt.
2
Center of Excellence in Cancer Research, Teaching Hospital, Tanta University, Tanta, Egypt.
Departments of
3
Clinical Pathology,
5
Microbiology, and Immunology,
6
Internal Medicine,
7
Pediatrics, and
8
Chest, Faculty of Medicine,
Tanta University, Tanta, Egypt.
4
Department of Basic Medical Sciences, Faculty of Medicine, Galala University, Suez, Egypt.
This article has been updated on June 13, 2023 after first online publication of May 26, 2023 to reflect the edit made in the co-author’s
name Mohamed R. Shanshoury which has been changed to Mohamed R. El-Shanshoury.
JOURNAL OF INTERFERON & CYTOKINE RESEARCH
Volume 43, Number 6, 2023
ªMary Ann Liebert, Inc.
DOI: 10.1089/jir.2023.0029
257
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immune responses, SARS-CoV-2 RNAemia, and predispose
to a life-threatening acute respiratory distress syndrome
(ARDS) (Huang et al., 2020; Lei et al., 2020).
COVID-19 mortality rates at the beginning of the pandemic
ranged from 2.84% to 5.5%, which decreased approximately to
0.08% worldwide. Mortality rates peaked among elderly pa-
tients whose age was 75 years or older (Depalo, 2021; Velavan
and Meyer, 2020). Unlike patients younger than 70 years, the
average duration between the onset of symptoms and mortality
was shorter than 14 days (11.5 days) in patients older than 70
years (Wang et al., 2020b). This indicates that elderly COVID-
19 patients are highly susceptible to complications caused by
SARS-CoV-2 variants of concern. Therefore, envisaging pre-
cision immunological biomarkers for elderly patients seems
crucial for early therapeutic intervention.
Most COVID-19 patients demonstrate lymphocytopenia
and significantly high neutrophils, D-dimer, and C-reactive
protein (CRP) levels in the blood; however, the predictive
power of these parameters as precision biomarkers of disease
outcome is still not fully established (Barrett et al., 2021;
Gonc¸ alves et al., 2021; Wang et al., 2020b). COVID-19 may
also cause multi-organ dysfunction, presumably due to en-
dotheliitis, loss of vascular integrity, and coagulation pathway
activation (Perico et al., 2021). Additionally, a multi-organ
hyperinflammatory state, known as cytokine storm or macro-
phage activation syndrome (MAC), usually ensues in severe
and critically ill cases and inflicts tissue damage and fibrosis,
particularly in the lungs (Que et al., 2022; Satyam et al., 2021).
Varying levels of inflammatory cytokines, including interleu-
kin (IL)-6 and tumor necrosis factor-alpha (TNF-a), have been
detected in the plasma of COVID-19 patients, with the highest
levels detected in severe cases (Tufa et al., 2022).
Some studies have proposed redefining COVID-19 as
multiple organ dysfunction syndrome (MODS-CoV-2) be-
cause of its complex and systemic pathological sequelae. An
essential dimension of MODS-CoV-2 in COVID-19 patients
is the connection between deregulated inflammatory response
and hypercoagulability state because of inflammatory injury
to the microvasculature that activates the coagulation system
leading to extensive microthrombosis (Chen et al., 2020a;
Dennis et al., 2023; Tian et al., 2020; Wang et al., 2020a;
Zhao et al., 2022). According to previous reports, 40% of
COVID-19 patients are at risk of venous thromboembolism.
Additionally, disseminated intravascular coagulation has been
involved in 46% of COVID-19 deaths demonstrating that
coagulation dysfunction is one of the primary causes of death
in severe COVID-19 patients (Wang et al., 2020a; Wu and
McGoogan, 2020).
The regulation of particular cytokines in COVID-19 pa-
tients and their potential relationship to immunopathology
and coagulopathy have been the subject of numerous reports
(Al-Ani et al., 2022; Lax et al., 2020; Pla
´s
ˇek et al., 2022;
Que et al., 2022; Savla et al., 2021). However, the ability to
distinguish a protective COVID-19 immune response from
an immunopathologic response across the severity spectrum
of COVID-19 in individuals with various ethnic back-
grounds has not been thoroughly analyzed (Kopel et al.,
2020; Vadgama et al., 2022). Using a highly standardized
Luminex xMAP multiplex assay, we comprehensively ex-
amined the levels of 25 cytokines previously shown to
regulate antiviral immune response and anti-inflammatory
and proinflammatory states in Egyptian COVID-19 patients
of varying disease severity. Additionally, we have mapped
the relatedness between immunological and clinical
258 SALEM ET AL.
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laboratory parameters using principal component analysis
(PCA) for identifying predictive biomarkers of COVID-19
disease progression.
Materials and Methods
Study design and ethical approval statement
In a cross-sectional study, 78 adult COVID-19 patients
were enrolled from the inpatient wards and the ICU Depart-
ment of Tanta University Quarantine Hospital, Tanta, Egypt,
between April 2020 and September 2020. Cases were se-
lected to be older than 18 years and meet the COVID-19
virological case definition [positive detection of viral genome
in nasopharyngeal swabs by reverse transcription-polymerase
chain reaction (RT-PCR)]. Patients with more than one of the
following chronic diseases, namely diabetes mellitus, hyper-
tension, cardiac disease, and grade 2 or 3 liver disease, were
excluded. Demographic and clinical data were collected as
described in Table 1. The enrolled patients were classified
into 4 groups according to the World Health Organization
(WHO) clinical stage definition for COVID-19 as follows: 10
cases with mild infection, 18 cases with moderate illness, 30
cases with severe illness, and 20 critically ill cases. Twenty-
one healthy control volunteers were also enrolled.
The detailed grouping criteria of enrolled participants are
listed as follows: (1) healthy controls (CO) including individ-
uals with no COVID-19 infection, no known contact with a
confirmed or suspected COVID-19 case in the previous 14
days, no upper or lower respiratory tract infection, and no
history of a serious chronic disease; (2) mild illness (MI) in-
cluding individuals with laboratory-confirmed positive RT-
PCR for SARS-CoV-2 who have any of the various signs and
symptoms of COVID-19 (eg, fever, cough, sore throat, ma-
laise, headache, muscle pain, nausea, vomiting, diarrhea, loss
of taste and smell) but do not have complicated symptoms such
as shortness of breath, dyspnea, or abnormal chest imaging;
(3) moderate illness (MO) including individuals with
laboratory-confirmed SARS-CoV-2 infection who show evi-
dence of lower respiratory disease during clinical assessment
or imaging and have an O
2
saturation (SpO
2
)‡94% on room
air; (4) severe illness (SE) including individuals with
laboratory-confirmed SARS-CoV-2 infection and hospitalized
with COVID-19 as a primary reason for admission with re-
spiratory support [intubation, continuous positive airway
pressure (CPAP), bilevel positive airway pressure (BiPAP)]
who have SpO
2
<94% on room air, respiratory frequency >30
breaths/min, or lung infiltrates >50% on CT chest; (5) critical
illness (CL) including individuals with laboratory-confirmed
SARS-CoV-2 infection and hospitalized with COVID-19 as a
primary reason for admission and on respiratory support (in-
tubation, CPAP, BiPAP) who have respiratory failure, septic
shock, and multiple organ dysfunction.
The study protocol has been approved by the Ethical
Committee Review Board, Faculty of Medicine, Tanta
University (approval code ‘‘34112/9/20’’). The sample size
for healthy controls and COVID-19 cases was determined
based on sample collection feasibility at the time of con-
ducting the study.
Routine hematological parameters
Laboratory tests such as hemoglobin (Hb) level, total and
differential white blood cells count, platelet counts (PLT),
Table 1. Demographic and Clinical Characteristics of COVID-19 Patients and Healthy Controls
Demographic
and clinical
characteristic
Groups of enrolled study participants
Healthy controls Mild COVID-19 cases Moderate COVID-19 cases Severe COVID-19 cases Critically ill COVID-19 cases
nM–SEM nM–SEM pn M–SEM pn M–SEM pn M–SEM p
Gender 21 8 males, 3 females 9 6 males, 3 females 18 10 males, 8 females 29 16 males, 13 females 17 10 males, 7 females
Age (years) 21 34.1818 –2.00825 9 49.6667* –3.50397 0.014 18 48.2778* –3.73187 0.009 29 55.6897* –2.84713 0.000 17 61.0588* –3.18599 0.000
Fever (C) 21 36.6909 –0.05633 9 37.8889* –0.03514 0.0001 18 38.0944* –0.06882 0.0001 29 38.4552* –0.08590 0.0001 17 38.4765* –0.10450 0.0001
O
2
% 21 98.0000 –0.42640 9 92.0000* –0.64550 0.031 18 89.3889* –0.73270 0.0001 29 85.6207* –1.23816 0.0001 17 80.5882* –2.30418 0.0001
Hb concentration
(gm/dL)
21 12.6364 –0.33771 10 12.7900 –0.49832 0.867 11 13.0636 –0.66295 0.634 27 12.2259 –0.48612 0.585 12 11.7917 –0.54889 0.337
PLT (10
3
/mL) 21 299.0909 –27.38462 10 293.9000 –35.03441 0.900 11 238.1818 –16.01693 0.134 27 247.7778 –20.28903 0.132 12 261.7500 –23.59928 0.345
WBC (10
9
/L) 21 7.7000 –0.60347 10 6.7500 –0.51516 0.660 11 6.6000 –0.83350 0.601 27 10.5333 –1.19321 0.112 12 11.2750 –1.85289 0.086
Lymphocyte
percentage
(%)
21 31.5455 –2.06866 10 15.4000* –2.16641 0.0001 11 19.0909* –2.70170 0.0001 27 11.7185* –1.00268 0.0001 12 11.6833* –1.33948 0.0001
D-dimer
(ng/mL)
21 191.8182 –7.11186 6 459.1667* –76.66395 0.001 4 437.5000* –87.50000 0.007 17 686.9412* –35.82602 0.0001 8 808.2500* –68.89452 0.0001
CRP (mg/L) 21 6.9091 –0.47586 10 37.6200 –4.15558 0.164 9 50.9111 –19.66659 0.054 25 89.0800* –12.07683 0.0001 11 86.0000* –17.82134 0.0001
*Asterisks representa significant difference between the selected group with the healthy controls using 1-way ANOVA at P<0.01.
ANOVA, analysis of variance; CRP, C-reactive protein; Hb, hemoglobin; M, mean; n, number of human subjects; PLT, platelet counts; SEM, standard error mean; WBC, white blood cells.
CYTOKINE STORM IN COVID-19 PATIENTS: A CROSS-SECTIONAL STUDY 259
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CRP, and the coagulation parameter, D-dimer, were as-
sessed using standard clinical laboratory methods for all
recruited participants.
Cytokine profiling using Luminex xMAP
multiplex assay
A volume of 2 mL of peripheral blood samples was drawn
from the enrolled participants and centrifuged directly at
13,000 rpm for 10 min to separate plasma (500mL). Plasma
samples were then blindly coded and used for analysis. Lu-
minex 200 xMAP Technology (Luminex Corp, Austin, TX)
was used to measure plasma levels of a panel of 25 cytokines
(kit catalog no. LXSAHM-25, and kit lot no. L137457).
Briefly, 50 mL of plasma was diluted 2-folds and then incu-
bated with magnetic beads (Molecular Probes; Life Tech-
nologies, Carlsbad, CA) coated with cytokine-specific capture
antibodies. After several washes and incubation periods, the
beads were detected with the Luminex system (Luminex
Corp, Austin, TX). Data were obtained and calculated using a
5-parametric curve fit using xPONENT

, version 4.03, in a
blinded manner with measurement performed with the Flex-
MAP3D system (Luminex Corp) (Datta et al., 2008).
Statistical analysis
Statistical analysis was performed using SPSS 26.0 for
Windows (SPSS, Inc., Chicago, IL). The figures and graphs
were prepared with GraphPad Prism 9.0. (La Jolla, CA).
Pearson’s correlation or Fisher’s exact test was used as
appropriate to compare the frequency of categorical vari-
ables. Means and standard error values for each continuous
variable were calculated. The comparison of continuous
variables between 2 groups was performed using the 1-way
analysis of variance (ANOVA), and the correlation analysis
was performed using Spearman’s correlation analysis. A P-
value <0.05 was considered statistically significant.
Results
Demographic and clinical criteria of enrolled
study participants
The basic information of enrolled participants is listed in
Table 1. We have enrolled 78 COVID-19 cases (59.5%
males; mean age 53 years) and 21 healthy controls (58%
male; mean age 34 years). No significant differences in
gender, age, Hb level, white blood cells, and PLT were
found among all patient groups (P>0.05). The mean age of
severe and critically ill patients was 1.2-fold higher than that
of mild and moderate cases. Fever was the most prevalent
symptom among patients (78.78%); however, higher tem-
peratures were observed in severe and critically ill patients.
D-dimer and CRP levels were significantly elevated in se-
vere and critically ill patients (P£0.0001) compared with
other groups, which may reflect active inflammation and
hypercoagulable state in severe and critically ill patients.
Furthermore, severe and critically ill patients had significant
lymphopenia compared with other groups (P£0.0001). Si-
milar results have been observed for oxygen saturation in
severe and critically ill patients requiring assisted ventila-
tion, indicating pulmonary dysfunction.
Upregulation of both proinflammatory
and anti-inflammatory cytokines in severe
and critically ill COVID-19 patients
The primary focus of our analysis was to measure the
levels of proinflammatory cytokines potentially related to
immunopathologic lung injury to monitor disease progres-
sion in COVID-19 patients, including IL-1a, soluble IL-
2Ra, IL-6, IL-8, IL-18, CCL4, and TNF-a. These cytokines
were only significantly upregulated in severe and/or criti-
cally ill cases who had very low oxygen saturation and re-
quired assisted ventilation compared (Fig. 1 and Table 1).
However, no significant difference was detected for the
proinflammatory IFN-gand CCL3 between patient groups,
and the proinflammatory IL-1b, IL-2, IL-17, and TNF-b
were not detected in the plasma of all enrolled patients (data
not shown). Interestingly, the anti-inflammatory cytokine
IL-10 was markedly upregulated in severe and critically ill
patients compared with healthy controls and mild and
moderate cases (Fig. 1).
Additionally, the proangiogenic FGF1 was significantly
upregulated in severe and critically ill patients compared
with healthy controls and/or patients with a milder form of
the disease (Fig. 2).
Of note that the following immunomodulatory markers
IL-3, IL-4, IL-5, IL-7, IL-13, IL-15, and Granulocyte-
Macrophage Colony-Stimulating Factor showed no signifi-
cant difference between patient groups (Fig. 3 and data not
shown).
Upregulation of CCL2 and CXCL10 as potential
hypercoagulability markers in critically ill cases
CCL2 and CXCL10 levels were significantly increased in
the critically ill group compared with mild, moderate, and
severe cases with extremely high levels of D-dimer (Fig. 2
and Table 1). Additionally, CXCL10 levels showed a strong
positive correlation with D-dimer levels in COVID-19 pa-
tients (Fig. 4).
PCA identifies potentially predictive biomarkers
of COVID-19 severity
To map the relationship between plasma cytokine levels,
clinical parameters, and COVID-19 severity, we performed
PCA for COVID-19 patients of various severity levels
(Fig. 4). Proinflammatory markers, including CRP, soluble
IL-2Ra, IL-6, IL-18, CXCL10, and hypercoagulability mar-
ker D-dimer mainly contributed to the first principal com-
ponent and exhibited differential association with severe and
critically ill patients. Likewise, the proangiogenic FGF1
clustered with severe and critically ill patients (Fig. 4).
Correlation analysis for the detected cytokines
and clinical laboratory parameters showed
variable relationships
To examine the potential interplay between the detected
cytokines and clinical laboratory parameters, we have per-
formed a correlation analysis between the levels of cyto-
kines and clinical laboratory parameters throughout the
COVD-19 clinical continuum (Fig. 5). The data showed a
significant positive correlation between CCL2 and IL-6,
260 SALEM ET AL.
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CO MI MO SE CL
0
2
4
6
8
10
IL-1α
****
****
****
CO MI MO SE CL
0
500
1000
1500
soluble IL-2Rα
****
****
CO MI MO SE CL
0
50
100
150
IL-6
****
****
****
****
****
****
****
CO MI MO SE CL
0
20
40
60
80
IL-8
****
****
**** ****
CO MI MO SE CL
0
5
10
15
20
25
IL-10
****
****
****
****
****
****
****
CO MI MO SE CL
0
1000
2000
3000
IL-18
***
****
**
**
CO MI MO SE CL
0
100
200
300
CCL4
****
****
****
CO MI MO SE CL
0
5
10
15
TNF-α
****
****
****
****
****
****
pg/ml
Patient groups
FIG. 1. Levels of proinflammatory cytokines potentially related to lung injury and anti-inflammatory cytokines. Concentrations of IL-1a, soluble IL-2Ra, IL-6, IL-8, IL-10,
IL-18, CCL4, and TNF-awere measured by Luminex assay 100/200 in plasma of 21 healthy controls (CO), 10 mild COVID-19 (MI), 18 moderate COVID-19 (MO), 30
severe COVID-19 (SE), and 20 critically ill COVID-19 (CL) cases. The mean cytokine levels plus standard errors from 2 independent analyses are shown. Asterisks represent
a significant difference between the selected groups using 1-way ANOVA at P<0.01. ANOVA, analysis of variance; IL, interleukin; TNF-a, tumor necrosis factor-alpha.
261
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CO MI MO SE CL
0
10
20
30
IL-4
****
****
CO MI MO SE CL
0
5
10
15
20
IL-7
****
****
****
**** ****
CO MI MO SE CL
0
50
100
150
IL-13
CO MI MO SE CL
0
2
4
6
IL-15
pg/ml
Patient groups
FIG. 3. Levels of immunomodulatory cytokines in COVID-19 patients. Concentrations of IL-4, IL-7, IL-13, and IL-15
were measured by Luminex assay 100/200 in plasma of 21 healthy controls (CO), 10 mild COVID-19 (MI), 18 moderate
COVID-19 (MO), 30 severe COVID-19 (SE), and 20 critically ill COVID-19 (CL) cases. The mean cytokine levels plus
standard errors from 2 independent analyses are shown. Asterisks represent a significant difference between the selected
groups using 1-way ANOVA at P<0.01.
CO MI MO SE CL
0
5
10
15
20
FGF1
****
****
****
****
****
CO MI MO SE CL
0
100
200
300
400
500
CCL2
****
****
****
****
CO MI MO SE CL
0
50
100
150
200
250
CXCL10
****
****
****
****
****
****
****
Patient groups
pg/ml
FIG. 2. Levels of proangiogenic cytokines and potentially associated cytokines with a hypercoagulable state in COVID
patients. Concentrations of FGF1, CCL2, and CXCL10 were measured by Luminex assay 100/200 in plasma of 21 healthy
controls (CO), 10 mild COVID-19 (MI), 18 moderate COVID-19 ( MO), 30 severe COVID-19 (SE), and 20 critically ill
COVID-19 (CL) cases. The mean cytokine levels plus standard errors from 2 independent analyses are shown. Asterisks
represent a significant difference between the selected groups using 1-way ANOVA at P<0.01.
262
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FIG. 4. Distinct cytokine profiles related to COVID-19 severity. PCA of tested cytokines and different clinical parameters. The analysis identified 8 cytokines and 3 clinical
parameters (CRP, D-dimer, and lymphocyte counts) that were important in explaining the observed variations between patient groups. PC1 and PC2 scores for the tested
variables in the table rank the variables based on their influence on the observed variations between patient groups and determine the clustering of patient groups along PC1
and PC2. CRP, C-reactive protein; PCA, principal component analysis.
263
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CCL4, TNFa, CXCL10, D-dimer and CRP, IL-10, IL-15,
IL-18, and soluble IL-2Ra, respectively (P£0.001). How-
ever, a significant negative correlation was observed be-
tween CCL2, TNF-aand Hb concentration, IL-2Ra, TNF-a,
IL-10 and PLT, IL-2Raand lymphocyte percentage, re-
spectively (P£0.001). These findings reveal a potential in-
terplay between hematological and inflammatory parameters
in COVID-19 patients.
Discussion
Cytokine-induced modulation of host immune response
has been shown to play a critical role in governing the
outcome of many potentially serious viral infections, in-
cluding influenza, SARS-CoV, and MERS (Alhetheel et al.,
2020; Cheung et al., 2005; Page et al., 2012; Zhou et al.,
2014). A growing body of evidence suggests that the se-
verity of COVID-19 is driven by disordered immunoregu-
lation, which often results in ARDS, a leading cause of
death in affected patients (Quan et al., 2021). However,
there is a limited consensus on specific cytokine signatures
that predict the clinical outcome and guide precision therapy
of COVID-19 (Pereira et al., 2021; Wang et al., 2021).
Moreover, high-throughput cytokine profiling is particularly
understudied among Egyptian COVID-19 patients (Gharib
et al., 2021; Shafiek et al., 2021). The present study aims to
address the current gap by clustering hospitalized Egyptian
COVID-19 patients of varying disease severity based on
their cytokine profile.
Luminex xMAP technology was utilized to analyze 25
cytokines in plasma samples isolated from patients with
varying degrees of COVID-19 severity, including proin-
flammatory cytokines potentially associated with lung
damage in COVID-19 patients, such as IL-1a, IL-1b, IL-2,
1.00
0.02
0.64
0.76
0.81
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0.97
0.78
0.89
-0.31
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0.70
0.47
0.20
-0.78
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0.62
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0.60
0.40
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0.77
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0.62
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0.91
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1.00
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-0.43
-0.91
-0.70
-0.65
-0.60
0.20
0.72
-0.32
0.94
-0.79
-0.84
-0.21
-0.08
-0.36
-0.33
-0.35
0.85
1.00
-0.25
-0.37
-0.27
-0.84
0.09
-0.59
-0.50
-0.28
-0.93
-0.05
0.48
0.04
0.77
-0.57
-0.52
0.97
0.19
0.76
0.86
0.91
-0.23
-0.25
1.00
0.87
0.96
-0.22
-0.22
0.33
0.81
0.63
0.18
-0.82
-0.15
0.72
-0.44
0.70
0.55
0.78
0.62
0.98
1.00
0.99
-0.57
-0.37
0.87
1.00
0.97
-0.03
0.25
0.71
0.98
0.91
0.13
-0.89
-0.42
0.89
-0.75
0.95
0.87
0.89
0.45
0.90
0.96
0.99
-0.39
-0.27
0.96
0.97
1.00
-0.14
0.04
0.54
0.92
0.82
0.12
-0.88
-0.34
0.85
-0.59
0.84
0.74
-0.31
0.08
0.05
-0.05
-0.02
-0.75
-0.84
-0.22
-0.03
-0.14
1.00
0.08
0.51
0.15
0.08
0.79
0.46
-0.64
-0.35
-0.51
0.24
0.33
-0.35
0.91
0.44
0.29
0.18
-0.43
0.09
-0.22
0.25
0.04
0.08
1.00
0.66
0.29
0.59
-0.43
-0.28
-0.47
0.49
-0.42
0.40
0.58
0.16
0.85
0.83
0.72
0.68
-0.91
-0.59
0.33
0.71
0.54
0.51
0.66
1.00
0.81
0.88
0.26
-0.44
-0.82
0.60
-0.89
0.87
0.96
0.70
0.65
0.98
0.98
0.97
-0.70
-0.50
0.81
0.98
0.92
0.15
0.29
0.81
1.00
0.93
0.26
-0.80
-0.55
0.83
-0.84
0.98
0.93
0.47
0.87
0.98
0.93
0.89
-0.65
-0.28
0.63
0.91
0.82
0.08
0.59
0.88
0.93
1.00
-0.03
-0.79
-0.66
0.90
-0.75
0.92
0.96
0.20
-0.29
0.07
0.09
0.15
-0.60
-0.93
0.18
0.13
0.12
0.79
-0.43
0.26
0.26
-0.03
1.00
0.27
-0.28
-0.32
-0.49
0.29
0.20
-0.78
-0.58
-0.86
-0.90
-0.87
0.20
-0.05
-0.82
-0.89
-0.88
0.46
-0.28
-0.44
-0.80
-0.79
0.27
1.00
0.09
-0.96
0.48
-0.75
-0.66
0.07
-0.63
-0.54
-0.44
-0.46
0.72
0.48
-0.15
-0.42
-0.34
-0.64
-0.47
-0.82
-0.55
-0.66
-0.28
0.09
1.00
-0.30
0.54
-0.56
-0.72
0.62
0.76
0.91
0.91
0.87
-0.32
0.04
0.72
0.89
0.85
-0.35
0.49
0.60
0.83
0.90
-0.32
-0.96
-0.30
1.00
-0.54
0.80
0.77
-0.36
-0.62
-0.80
-0.74
-0.69
0.94
0.77
-0.44
-0.75
-0.59
-0.51
-0.42
-0.89
-0.84
-0.75
-0.49
0.48
0.54
-0.54
1.00
-0.92
-0.90
0.60
0.70
0.97
0.94
0.91
-0.79
-0.57
0.70
0.95
0.84
0.24
0.40
0.87
0.98
0.92
0.29
-0.75
-0.56
0.80
-0.92
1.00
0.96
0.40
0.84
0.95
0.88
0.84
-0.84
-0.52
0.55
0.87
0.74
0.33
0.58
0.96
0.93
0.96
0.20
-0.66
-0.72
0.77
-0.90
0.96
1.00
CCl2
CCL4
solubleIL2Rα
CXCL10
FGF1
GCSF
IFNγ
IL1α
IL4
IL6
IL7
IL8
IL15
IL18
TNFα
IL13
Hb
Platelets
WBC
Lymphocytes
Ddimer
CRP
CCl2
CCL4
solubleIL2Rα
CXCL10
FGF1
GCSF
IFNγ
IL1α
IL4
IL6
IL7
IL8
IL15
IL18
TNFα
IL13
Hb
Platelets
WBC
Lymphocytes
Ddimer
CRP
-1.0
-0.5
0
0.5
1.0
FIG. 5. Correlation heat map for the measured cytokines and clinical parameters in various COVID-19 severity groups.
Positive numbers represent a positive correlation, and negative numbers represent a negative correlation between the tested
parameters. Color intensity determines the strength of the correlation.
264 SALEM ET AL.
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soluble IL-2Ra, IL-6, IL-17, IL-8, CCL3, CCL4, IFN-g,
TNF-a, and TNF-b. Additionally, we have examined cyto-
kines potentially related to a hypercoagulable state such as
CCL2, CCL3, and CXCL10. Finally, we have examined
cytokines known to be essential for immune modulation,
namely IL-1a, IL-3, IL-4, IL-5, IL-10, IL-13, IL-15, IL-17,
IL-18, and TNF-b(Chen et al., 2021; Chen et al., 2020b;
Chen et al., 2018; Huang et al., 2005; Jakobs et al., 2022;
Julian et al., 2021; Robba et al., 2020; Zhang and An, 2007).
Our comprehensive analysis identified distinct cytokine
profiles throughout the COVID-19 severity spectrum, sug-
gesting dysregulated adaptive immune responses with a
proinflammatory cytokine shift that is exacerbated in higher
COVID-19 severity levels.
Our profiling of cytokines demonstrated that the proin-
flammatory cytokines IL-2Ra, IL-8, IL-18, TNF-a,and
CXCL10 were significantly upregulated in severe and/or
critically ill COVID-19 patients compared with healthy
controls or cases with milder forms of the disease (Figs. 1–3).
Likewise, IL-4 and IL-15 levels demonstrated a similar, al-
though nonsignificant, surging trend toward severe forms of
COVID-19. Notably, IL-1a, IL-6, and CCL2 were exclu-
sively upregulated in critically ill patients who had respiratory
failure and required mechanical ventilation (Figs. 1 and 2).
However, IL-13 was downregulated in severe and criti-
cally ill COVID-19 patients, whereas no change was
observed in IFN-glevel across all severity levels of COVID-
19, suggesting compromised T-helper (Th)1 responses
(Fig. 3 and data not shown). This profile is consistent with
the development of hyperinflammatory status and MAC in
severe and critical COVID-19 cases implying a potentially
significant role of the host’s innate immune responses in the
immunopathology of COVID-19 (Cabaro et al., 2021;
Dorgham et al., 2021; Go
´mez-Escobar et al., 2021; Gustine
and Jones, 2021; Russick et al., 2021; Zhou et al., 2020).
The concurrent rise in the levels of macrophage inflam-
matory cytokines, including IL-1a, IL-18, IL-6, IL-8, TNF-
a, CXCL10, and CCL2 in severe or critically ill patients
(Figs. 1–3), has also been reported by other research groups
(Abers et al., 2021; Cabaro et al., 2021; Dorgham et al.,
2021; Go
´mez-Escobar et al., 2021; Russick et al., 2021;
Satısxet al., 2021; Zhou et al., 2020). Multiple studies have
suggested that high levels of the proinflammatory cytokines
IL-1 and IL-6 in COVID-19 patients may predict poor dis-
ease outcomes, including MAC and ARDS (Han et al.,
2020; Russick et al., 2021; Shafiek et al., 2021).
However, clinical trials incorporating IL-1 and IL-6 in-
hibitors in the treatment protocols of COVID-19 patients
demonstrated variable results without a significant im-
provement in disease outcomes (Mariette et al., 2021; Stone
et al., 2020). These data imply that the concerted action of
macrophage cytokines potentially governs the outcome of
COVID-19. Therefore, it has also been proposed that a
combination of these cytokines may be used as prognostic
markers to predict COVID-19 severity (Bhaskar et al.,
2020). In agreement with our results, other research groups
have demonstrated that CCL2 and CXCL10 levels were
significantly elevated in severe COVID-19 cases. Specifi-
cally, a high CXCL10 level has been associated with ARDS
development and respiratory failure (Cabaro et al., 2021).
The immune modulator IL-18, a vital member of the IL-1
cytokine family, has been demonstrated to play a crucial role in
promoting the development of MAC and cytokine release
syndrome in various autoinflammatory conditions (Weiss et al.,
2018). Our data show a significant increase in IL-18 levels in
severe and critically ill cases compared with healthy controls
and patients with milder forms of the disease (Fig. 3). In con-
gruence with our findings, other studies have detected high
levels of IL-18 among COVID-19 patients with severe pneu-
monia, which also correlated with mortality and poor disease
outcomes (Go
´mez-Escobar et al., 2021; Satısxet al., 2021).
However, contradictory evidence from a different study
showed that mortality in critically ill COVID-19 patients
who required extracorporeal membrane oxygenation corre-
lated with low levels of IL-18 (Dorgham et al., 2021). These
data suggest that unraveling the ultimate role of this cyto-
kine in COVID-19 pathogenesis requires further elucidation
of the molecular mechanisms and the immunological con-
texts regulating its function.
The observation that the proinflammatory TNF-a, IL-8, and
IL-2Rawere significantly upregulated in severe COVID-19
patients has been corroborated by other studies (Fig. 1)
(Dorgham et al., 2021; Kaya et al., 2021; Ma et al., 2021; Zhang
et al., 2020). Notably, one study has shown that high levels of
soluble IL-2Rain blood from severe COVID-19 patients are
inversely correlated with T cell numbers. The same study has
also shown that the in vitro addition of recombinant IL-2Ra
could inhibit the proliferation and function of activated T cells
derived from peripheral blood mononuclear cells. These data
signify that high IL-2Ralevels may play a role in compro-
mising adaptive immune responses and COVID-19 prognosis
(Zhang et al., 2020).
Additional studies have associated high levels of IL-8 and
IL-2Rain severe COVID-19 cases with prolonged illness,
respiratory failure, and higher mortality rates (Dorgham
et al., 2021; Kaya et al., 2021; Ma et al., 2021). These data
suggest a complex interplay between distinct cytokines in
governing COVID-19 outcomes. The data may also suggest
a potential protective role of IL-2 signaling in restoring T
cell responses or delaying the onset of compromised T cell
responses (Zhang et al., 2020).
T-helper (Th) polarization is well known as a critical
determinant of the outcome of various viral infections
(Calarota and Weiner, 2004; Kamperschroer and Quinn,
2002; Neidleman et al., 2020; Roncati et al., 2020). By
profiling Th2-related cytokines, namely IL-4, IL-6, and IL-
10, their levels were modestly upregulated among patients
with more severe forms of COVID-19 (Figs. 1 and 3),
whereas IL-15 displayed a modest increase across all
COVID-19 severity levels (Fig. 3), and these data are con-
sistent with other similar studies (Gil-Etayo et al., 2021;
Kandikattu et al., 2020; Petrey et al., 2021; Vaz de Paula
et al., 2020). Conversely, the anti-inflammatory cytokine IL-
13, secreted by activated Th2 cells, was differentially
downregulated in severe COVID-19 cases (Fig. 3).
Contradictory evidence from various studies demon-
strated high IL-13 levels in severe COVID-19 patients who
developed ARDS, lung damage, and acute renal failure
(Donlan et al., 2021; Petrey et al., 2021; Vaz de Paula et al.,
2020). On the contrary, other studies suggested that higher
expression levels of IL-4 and IL-13 in asthmatic patients
may lower the risk of developing severe COVID-19 due to
the downregulation of angiotensin-converting enzyme-2
receptors (Song et al., 2021).
Interestingly, our PCA revealed that severe and critically ill
patients with high mortality rates exhibited distinguishing
CYTOKINE STORM IN COVID-19 PATIENTS: A CROSS-SECTIONAL STUDY 265
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cytokine signatures from mild and moderate COVID-19 pa-
tients. Specifically, the observed variations between early and
late stages of COVID-19 disease can be primarily attributed to
the levels of IL-2Ra, IL-6, IL-10, IL-18, TNF-, FGF1, and
CXCL10. Our PCA also demonstrated that the described im-
munological markers positively correlate with high D-dimer
and CRP levels and inversely correlate with lymphocyte
counts in severe and critically ill patients (Fig. 4). In addition, a
significant positive correlation was detected between IL-2Ra
and IL-10, IL-15, IL-18, and TNF-a(Fig. 5). These data raise
the hypothesis that SARS-CoV2 might dysregulate the adap-
tive immune response by disturbing the balance between the
Th1 and Th2 responses. Further characterization of individual
cytokine functions and cytokine cross talk in future studies
may define stage-specific roles of cytokine signaling in gov-
erning COVID-19 disease outcomes.
To the best of our knowledge, this is the first study to per-
form a high-throughput cytokine analysis spanning the whole
COVID-19 severity spectrum and show an apparent proin-
flammatory cytokine surge among severe and critically ill
COVID-19 patients from Egypt. Despite that, the reported
variability for some of the data points could be attributed to the
small sample size and to receiving dexamethasone therapy by
COVID-19 patients. Further molecular characterization of
cell-type-specific responses to individual cytokines using
systems biology approaches may explain the reported inter-
patient variability and allow envisaging novel precision diag-
nostic, prognostic, and therapeutic modalities for COVID-19.
Authors’ Contributions
Conceptualization: M.A.E. Methodology: M.L.S.,
M.M.E., R.E.S., K.M.O., M.R.S., M.A.A., M.S.H., A.H.,
and M.A.E. Formal analysis: M.L.S., R.E.S., and M.A.E.
Software: M.A.E. Investigation: M.L.S., R.E.S., and M.A.E.
Data curation: M.L.S., R.E.S., and M.A.E. Supervision:
M.L.S., R.E.S., and M.A.E. Writing—original draft prepa-
ration: R.E.S. and M.A.E. Writing—review and editing: all
authors read and approved the final article.
Ethical Approval and Consent to Participate
This study complies with all relevant ethical regulations.
The experimental protocol was established according to the
International Ethical Guidelines for Biomedical Research
Involving Human Subjects and were approved by the Ethi-
cal Committee Review Board, Faculty of Medicine, Tanta
University, Tanta, Egypt, granted this cross-sectional study
(approval code ‘‘34112/9/20’’).
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This work was funded entirely by Tanta University,
Tanta, Egypt, through a project (TU:20-03-04).
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Address correspondence to:
Dr. Mohammed A. Eid
Department of Botany and Microbiology
Faculty of Science
Tanta University
Tanta 31527
Egypt
E-mail: mohamed.eid@science.tanta.edu.eg
Received 25 February 2023/Accepted 16 April 2023
268 SALEM ET AL.
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