Received: 24 October 2020 | Revised: 23 December 2020 | Accepted: 22 January 2021

DOI: 10.1002/jmv.26826

REVIEW

Role of Toll‐like receptors in the pathogenesis of COVID‐19

Shaghayegh Khanmohammadi MD Candidate1,2 | Nima Rezaei MD, PhD1,2,3

1
Research Center for Immunodeficiencies,
Children's Medical Center, Tehran University Abstract
of Medical Sciences, Tehran, Iran
Coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syn-
2
Network of Immunity in Infection,
Malignancy and Autoimmunity (NIIMA),
drome coronavirus 2 (SARS‐CoV‐2), has led to a pandemic since March 2020. The
Universal Scientific Education and Research exact pathogenesis of SARS‐CoV‐2 and the role of each component of the innate
Network (USERN), Tehran, Iran
and adaptive immune system is still unknown. However, available data from other
3
Department of Immunology, School of
Medicine, Tehran University of Medical coronavirus families, such as SARS‐CoV and the Middle East respiratory syn-
Sciences, Tehran, Iran drome and also new findings could be useful for a better understanding of
SARS‐CoV‐2. Toll‐like receptors (TLR) play an important role in recognition of viral
Correspondence
Nima Rezaei, MD, PhD, Research Center for particles and activation of the innate immune system. Activation of TLR pathways
Immunodeficiencies, Children's Medical
leads to secretion of pro‐inflammatory cytokines, such as interleukin‐1 (IL‐1), IL‐6,
Center Hospital, Dr. Qarib St, Keshavarz Blvd,
Tehran 14194, Iran. and tumor necrosis factor‐α, as well as type 1 interferon. Different TLRs, like TLR2,
Email: rezaei_nima@tums.ac.ir
TLR3, TLR4, TLR6, TLR7, TLR8, and TLR9 are potentially important in COVID‐19
infection. It is also worth mentioning that we should bear in mind both the beneficial
and harmful effects of TLR in confronting COVID‐19 infection. TLRs could be a
potential target in controlling the infection in the early stages of disease and pro-
duction of vaccine against SARS‐CoV‐2.

KEYWORDS
COVID‐19, cytokines, SARS‐CoV‐2, Toll‐like receptors

1 | INTRODUCTION important role in the pathogenesis of SARS‐CoV and the Middle East
respiratory syndrome (MERS).1 The aim of this article is to discuss
Toll‐like receptors (TLRs) belong to the family of innate immune the possible role of different TLRs in COVID‐19.
receptors, which play an important role in the activation of innate
immunity, regulation of cytokine expression, indirect activation of
the adaptive immune system, and the recognition of pathogen‐ 2 | TO LL‐ LIKE RECEPTORS
associated molecular patterns (PAMPs).1–3 Severe acute respiratory
syndrome coronavirus 2 (SARS‐CoV‐2), also known as novel cor- TLRs have ten family members in humans. Some of the TLRs are
onavirus disease 2019 (COVID‐19), has appeared in Wuhan, China, located in the cell membrane, and the others are situated in endo-
spread rapidly and led to a pandemic in 2020.4–6 The innate immune somes, such as TLR3, TLR7, TLR8, and TLR9.8 TLRs are expressed on
system protects the body against pathogens. Interestingly, new different immune cells, such as dendritic cells (DCs), macrophages,
studies on mesenchymal stem cells (MSCs) have revealed that MSCs natural killer cells, and cells of the adaptive immunity – T cells and B
can promote an anti‐inflammatory condition in affected tissues by cells.9 TLR3 recognizes double‐strand RNA (ds RNA), TLR4 re-
increasing anti‐inflammatory cytokines and M2 macrophages (anti‐ cognizes lipopolysaccharide (LPS), TLR7/8 recognizes single‐strand
inflammatory phenotype) in COVID‐19 patients, especially in the RNA (ssRNA), and TLR9 recognizes unmethylated CpG DNA.8
7
severe form of the disease. TLRs' pathways, as a component of Myeloid differentiation primary response 88 (MyD88) and
innate immunity, could be involved in the pathogenesis of SARS‐ TIR‐domain containing adaptor, inducing interferon‐β (IFN‐β) (TRIF,
CoV‐2 because several studies have shown that TLRs play an also known as TICAM1) are two main pathways for the transduction

J Med Virol. 2021;1–5. wileyonlinelibrary.com/journal/jmv © 2021 Wiley Periodicals LLC | 1

2 | KHANMOHAMMADI AND REZAEI

of TLR's signals. TRAF and IRAK proteins in signaling pathways cause 4 | TLRs IN SARS ‐C o V A N D M E R S‐ Co V
activation of nuclear factor‐kB (NF‐kB) and Interferon regulatory
factor (IRF), which in turn leads to the production of type 1 IFN and Unfortunately, the exact pathway of the COVID‐19 pathogenesis is still
pro‐inflammatory cytokines such as interleukin‐1 (IL‐1), IL‐6, tumor unknown. Therefore, most of the information regarding the COVID‐19
necrosis factor‐α (TNF‐α), and IL‐12. Besides this, TLRs indirectly pathogenesis arises from the available data about SARS‐CoV and
play role in the adaptive immune system by monitoring the expres- MERS‐CoV.9 Results of studies suggest that type 1 IFN plays an im-
sion of costimulatory molecules (Figure 1). portant role in SARS‐CoV and MERS‐CoV. They interfere with signaling
pathways of the host cell and decrease the expression of IFN receptors,
which in turn lead to a systemic inflammatory response.9 As the pro-
3 | T LR s A N D V I R U S E S duction of type 1 IFN is mediated by TLRs, they could play a vital role in
the pathogenesis of CoVs. Moreover, a cytokine storm exists in MERS‐
Many viruses activate the innate immune system with TLRs, CoV, SARS‐CoV, and SARS‐CoV‐2 that plays a crucial role in dete-
which contributes to the elimination of viruses, although it can rioration of these infections.17 Several studies have shown that TLR3
also harm the host due to persistent inflammation and tissue via the TIRF pathway leads to a protective response in SARS‐CoV and
destruction. For instance, severity of COVID‐19 is associated MERS‐CoV infections. Although TLR3 in mouse models leads to acti-
with production of IL‐6 in patients which could be produced by vation of IRF3 and NF‐kB pathways and production of type 1 IFN and
induction of TLR pathways. Activation of TLRs by SARS‐CoV‐2 pro‐inflammatory cytokines, in knock‐out mice for TLR3, no reduction
leads to activation of inflammasome and production of IL‐1β, in the secretion of these cytokines is seen in coronavirus infection.
which induces IL‐6. Excessive activation of the inflammasome is Therefore, other pathways are also involved in the production of pro‐
associated with poor outcome in COVID‐19 patients.10 In addi- inflammatory cytokines and type 1 IFN.1 Induction of the TLR3 path-
tion, activation of Janus kinase transducers (JAK/STAT), which is way, but not TLR2/4/7, in murine coronavirus infection in mouse
induced by TLRs, could lead to macrophage activation syndrome. models, stimulates the production of IFN‐β in macrophages and hinders
8,11–15
Therefore, TLRs have a dual role in viral infections. Besides infection.18 Besides this, MERS‐CoV 4a protein can bind to dsRNA and
this, TLRs also contribute to the activation of the adaptive im- suppress the production of type 1 IFN.19 TLR4 also activates the same
mune system via the upregulation of major histocompatibility pathway. Although TLR4 plays a vital role in bacterial infections, it can
complex on dendritic cells.16 be activated by oxidized phospholipids, which also appears in viral lung

F I G U R E 1 Toll‐like receptor signaling pathways. TLR2/6 and 4 localize in the cell membrane, and TLR3, TLR7/8, and 9 localize in the
endosome surface. Activation of two major adaptive downstream proteins, MYS88 and TRIF, leads to production of pro‐inflammatory cytokines
and IFN I. Activation of TRIF induces activation of TRAF3, which activates IRF3 and leads to production of IFN I. MYD88 induces activation of
TRAF6 in TLR2/6 as well as TLR4 pathway but leads to activation of IRAK4 and indirectly TRAF6 and TRAF3 in TLR7/9 and TLR9 pathway.
Activation of NF‐kB signaling through TRAF6 increases production of pro‐inflammatory cytokines and activation of IRF7 leads to production of
IFN I. dsRNA, double strand RNA; IFN I, interferon type 1; LPS, lipopolysaccharides; MYD88, myeloid differentiation primary response 88;
NF‐kB, nuclear factor k‐light‐chain‐enhancer; ssRNA, single strand RNA; TRAF, tumor necrosis factor receptor‐associated factor;
TRIF, TIR‐domain‐containing adapter‐inducing interferon‐β
KHANMOHAMMADI AND REZAEI | 3

infections.1 In addition, neutrophil extracellular traps (NETs) are acti- prophylactic administration of TLR2/6 agonist reduces SARS‐CoV‐2
vated by the TLR4 signaling pathway. Studies have shown that NET transmission and provides protection against COVID‐19. Stimulation
formation in COVID‐19 patients could lead to sustained inflammation of TLR2 leads to activation of the innate immune response, sup-
which could deteriorate the condition of patients. Moreover, the higher pression of excessive inflammation and tissue damage, as well as
production of TLR4 in men is due to a higher testosterone level; this promotion of the integrity of local epithelial barrier function.
fact can explain the higher level of pro‐inflammatory cytokines in men
compared to women.20 Interestingly, neutrophil myeloperoxidase,
which produces oxidized phospholipids, has a higher level in COVID‐19 6 | TL R7 /8 IN C O VID‐ 19
patients and also TLR4 pathway activation in the pulmonary phase of
infection causes oxidative injury. Therefore, TLR4 could also be in- TLR7/8 are tandem duplicated genes on the X‐chromosome, which
21,22
volved in the pathogenesis of COVID‐19. An in silico study has also are located in the endosome membrane and recognize ssRNA and
revealed a close relationship between bat SARS‐CoV and SARS‐CoV‐2 synthetic oligoribonucleotides, such as imidazoquinoline, imiquimod,
(based on the spike protein and ACE2) and found that SARS‐CoV‐2 and R‐848. Therefore, they could be involved in the recognition of
spike protein binds with TLR1, 4, and 6, with a higher affinity for TLR4. the SARS‐CoV‐2 genome.24 Binding of the superficial S glycoprotein
TLR4 antagonist could be administered for treating COVID‐19 pa- on the envelope of the virus to ACE2 could be sensed by TLR7. TLR7
tients.23 According to another study, TLR8 is seen in the lung, and TLR7 is expressed on monocyte‐macrophages and DC and its activation
and TLR8 could lead to a cytokine storm in SARS‐CoV‐1 and cause leads to the production of IL‐1, IL‐6, monocyte chemoattractant
24
different side effects. protein‐1, MIP‐1A, TNF‐α, and type 1 IFN.30 Also, a gender‐
dependent response could be seen in ssRNA viruses such as SARS‐
CoV‐2 as the TLR7/8 gene is on the X chromosome. Higher ex-
5 | T LR S I N SA R S‐C O V ‐2 pression of TLR7 could lead to a better prognosis in ssRNA viral
infections, inducing a higher immune response. Interestingly, the le-
+
SARS‐CoV‐2 has ssRNA like other CoVs. Spike S glycoproteins on vel of IL‐6 after viral infection is lower in women compared with
the SARS‐CoV‐2 envelope bind to angiotensin‐converting enzyme 2 men.24,31 Moreover, van der Made et al. found loss of function var-
(ACE2) and the virus enters inside the cell by receptor‐mediated iants of X‐chromosomal TLR7 in 4 male patients with severe
endocytosis.9 The results of several studies have shown that most of COVID‐19 infection that leads to impairment of type 1 and 2 IFN
the patients suffer from lymphopenia and have elevated serum levels response. Whole‐genome sequencing of SARS‐CoV, MERS‐CoV, and
of pro‐inflammatory cytokines, such as TNF‐α, IL‐6, and interleukin‐2 SARS‐CoV‐2 revealed that TLR7 could be more involved in the pa-
receptor (IL‐2R). It is also worth mentioning that higher serum levels thogenesis of SARS‐CoV‐2 in comparison to SARS‐CoV and MERS‐
of TNF‐α, IL‐1, IL‐6, and IL‐8, a higher number of neutrophils, as well CoV as it has more ssRNA motifs that could bind to TLR7.32 Agonists
as the decrease of lymphocytes are associated with the severity of of TLRs could lead to a strong immune response in COVID‐19.16 For
9 −/−
the disease. Besides this, IL6 mice are characterized by less lung instance, an innate immunity stimulator, Imiquimod, could be an
damage in viral lung infections.1 Based on the studies on other CoVs option to control COVID‐19. Imiquimod binds to TLR7/8 and induces
and COVID‐19, TLRs could play a crucial role in COVID‐19 disease. the production of pro‐inflammatory cytokines, such as TNF‐α, IL‐1,
Conti et al.25 suggested that activation of the TLRs in COVID‐19 IL‐2, IL‐6, IL‐8, IL‐12, as well as IFN‐α. However, it should be noticed
infection could lead to the production of pro‐inflammatory cytokines, that Imiquimod in the late stage of the infection could lead to cy-
such as IL‐1β. Moreover, immunopathological consequences causing tokine storm and side effects of consistent inflammation. Therefore,
death in COVID‐19 patients are due to the interaction of TLRs with Imiquimod is appropriate in the early stages of COVID‐19 infection.9
26
virus particles. A study by Totura et al. revealed that both TRIF‐ It should be mentioned that another candidate for triggering NET
driven and MyD88‐driven pathways, induced by TLRs, provide the formation in COVID‐19 patients is TLR7 and activation of TLR7/8
most effective antiviral defense against SARS‐CoV and lethal can induce a strong pro‐inflammatory response in patients, leading to
SARS‐CoV disease is seen in removal of TLR signaling pathways. For acute lung injury. Therefore, it may have a dual role in progression of
−/− −/−
instance, TLR3 , TLR4 mice are more susceptible to SARS‐CoV the disease.17,33 Another clinical trial in China (ChiCTR2000029776)
without an increase in mortality, but TLR3/TLR4 adaptor TRIF de- evaluates the therapeutic effects of the induction of TLR pathways in
ficient mice are highly susceptible to SARS‐CoV with high risk of COVID‐19 patients. Moreover, the TLR2/6/9 agonist is also ad-
mortality. Also, irregular signaling pathways and pro‐inflammatory ministered to activate innate immune cells and lung epithelial cells to
−/−
cytokine production following infection of TRIF mice, were similar produce various factors against infection. Interestingly, several clin-
to patients with severe outcomes in SARS‐CoV or MERS‐CoV in- ical trials evaluate the effect of anti‐inflammatory factors to reduce
fection.27 However, other studies on SARS‐CoV‐2 have revealed the death caused by persistent inflammation of the lung in COVID‐19
pathologic role of TLR4 in excessive inflammation in COVID‐19 pa- patients. For instance, CD24Fc conjugate is used to block TLR
tients as it leads to NETosis and activation of inflammasome, as activation.16 Also, using TLR4 antagonists such as glycyrrhetinic acid
20,22,28
mentioned before. TLR agonists could also be used as has anti‐inflammatory effects in the lung of mice with acute re-
prophylactic drugs for SARS‐CoV‐2. Proud et al.29 revealed that spiratory distress syndrome, and protects the tissue from
4 | KHANMOHAMMADI AND REZAEI

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