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Expert Opinion on Drug Discovery
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iedc20
An overview of disease models for NLRP3
inflammasome over-activation
Hongliang Zhang, Ayesha Zahid, Hazrat Ismail, Yujie Tang, Tengchuan Jin &
Jinhui Tao
To cite this article: Hongliang Zhang, Ayesha Zahid, Hazrat Ismail, Yujie Tang, Tengchuan Jin &
Jinhui Tao (2021) An overview of disease models for NLRP3 inflammasome over-activation, Expert
Opinion on Drug Discovery, 16:4, 429-446, DOI: 10.1080/17460441.2021.1844179
To link to this article: https://doi.org/10.1080/17460441.2021.1844179
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REVIEW
An overview of disease models for NLRP3 inflammasome over-activation
Hongliang Zhang
a
,*
, Ayesha Zahid
b
,*
, Hazrat Ismail
c
, Yujie Tang
a
, Tengchuan Jin
a,b,d
and Jinhui Tao
a
a
Department of Rheumatology and Immunology, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science
and Technology of China, Hefei, China;
b
Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, CAS Key
Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei,
China;
c
MOE Key Laboratory for Cellular Dynamics & Anhui Key Laboratory for Chemical Biology, CAS Center for Excellence in Molecular Cell
Science. Hefei National Science Center for Physical Sciences at Microscale. University of Science and Technology of China, Hefei, China;
d
CAS Center
for Excellence in Molecular Cell Science, Shanghai, China
ABSTRACT
Introduction: Inflammatory reactions, including those mediated by the NLRP3 inflammasome, maintain
the body’s homeostasis by removing pathogens, repairing damaged tissues, and adapting to stressed
environments. However, uncontrolled activation of the NLRP3 inflammasome tends to cause various
diseases using different mechanisms. Recently, many inhibitors of the NLRP3 inflammasome have been
reported and many are being developed. In order to assess their efficacy, specificity, and mechanism of
action, the screening process of inhibitors requires various types of cell and animal models of NLRP3-
associated diseases.
Areas covered: In the following review, the authors give an overview of the cell and animal models
that have been used during the research and development of various inhibitors of the NLRP3
inflammasome.
Expert opinion: There are many NLRP3 inflammasome inhibitors, but most of the inhibitors have poor
specificity and often influence other inflammatory pathways. The potential risk for cross-reaction is
high; therefore, the development of highly specific inhibitors is essential. The selection of appropriate
cell and animal models, and combined use of different models for the evaluation of these inhibitors can
help to clarify the target specificity and therapeutic effects, which is beneficial for the development and
application of drugs targeting the NLRP3 inflammasome.
ARTICLE HISTORY
Received 4 July 2020
Accepted 27 October 2020
KEYWORDS
NLRP3 inflammasome;
disease models; inhibitor
screening; drug discovery;
inflammatory diseases
1. Introduction
To maintain the homeostasis of the body, an adaptive
response in the form of inflammation is made to tackle
the harmful stimuli within the body. Mainly tissue damage,
infections, and stress cause the inflammation [1].
Inflammasomes which are oligomeric complexes, are an
important component of innate immunity and are the
major deriving force for inducing the inflammation by
using pathogen-recognition receptors (PRRs) [2]. Three
major types of PRRs are known to regulate the function of
the inflammasomes which are named as NOD-like receptor
(NLR) containing family, the absent in melanoma 2 (AIM2),
and retinoic acid-inducible gene I (RIG-I) like receptors
(RLRs) [3]. Generally, in these inflammasomes, apoptosis-
associated speck-like protein containing a CARD (ASC) and
pro-caspase-1 form the core of the inflammasome wherein
ACS links pro-caspase-1 to pattern recognition proteins, and
the inflammasome activation is mediated by identifying
danger or pathogens-associated molecular patterns
(DAMPs/PAMPs) [4].
Taking microbial infection as an example, mast cells and
tissue-resident macrophages recognize PAMPs on the surface
of pathogenic microorganisms through PRRs such as toll-like
receptors (TLRs) and NLRs, and then release a series of inflam-
matory mediators such as vasoactive amines, chemokines,
cytokines, eicosanoids, and some proteolytic enzymes which
produce inflammation and exudation locally, so that plasma
proteins and neutrophils can be attracted from the capillary
wall to the infection site. The reactive oxygen species (ROS),
reactive nitrogen species, elastase, proteinase 3 and cathepsin
G help in eradicating the pathogens [5]. When the pathogen is
cleared, macrophages initiate the attenuation of inflammation,
prostaglandins turn into lipoxins to convert pro-inflammatory
state into anti-inflammatory state, impede the recruitment of
neutrophils, promote the recruitment of monocytes, and sin-
gle nuclear cells clear the dead cells to initiate tissue repair.
Resolvins and protectins transforming growth factor-β and
CONTACT Tengchuan Jin jint@ustc.edu.cn USTC, Department of Rheumatology and Immunology, The First Affiliated Hospital of USTC, Division of Life
Sciences and Medicine, Hefei 230026, China USTC, Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory
of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, Hefei, 230026, China;USTC, CAS Center for Excellence in Molecular Cell Science,
Shanghai, China, Hefei, 230026, China; Jinhui Tao taojinhui@ustc.edu.cn USTC, Department of Rheumatology and Immunology, The First Affiliated Hospital
of USTC, Division of Life Sciences and Medicine, Hefei, 230026, China;
*
Contributed equally to the manuscript
EXPERT OPINION ON DRUG DISCOVERY
2021, VOL. 16, NO. 4, 429–446
https://doi.org/10.1080/17460441.2021.1844179
© 2020 Informa UK Limited, trading as Taylor & Francis Group
growth factors produced by macrophages also participate in
inflammation relief [6]. If the pathogen remains unremoved,
the infiltrating neutrophils will be replaced by macrophages
and T cells. If this is still ineffective, the body will enter the
chronic inflammatory process [7].
1.1. NLRP3 inflammasome
At present, five members of PRRs are best known to form
inflammasome which are the nucleotide-binding oligomer-
ization domain (NOD), leucine-rich repeat (LRR)-containing
proteins (NLR) family members NLRP1, NLRP3, and NLRC4,
absent-in-melanoma 2 (AIM2) and pyrin [8,9]. Additionally,
some other members of PRRs including NLRP2, NLRP6,
NLRP7, NLRP12, and gamma-interferon-inducible protein
(IFI16) have also been reported to form inflammasome
[10–14].
NLRP3 inflammasome is the best-characterized inflam-
masome known to help in the maturation and secretion of
interleukin 1-beta (IL-1β) and IL-18. Emerging studies have
linked NLRP3 inflammasome in the pathogenesis of many
human diseases, such as auto-inflammatory disorders, dia-
betes, and neurodegenerative disorders [15–17]
The NLRP3 protein consists of three parts, the N-terminal
PYD domain, the NACHT domain with nucleic acid binding
and oligomerization in the middle, and the C-terminal leucine-
rich repeat (LRR) domain. The PYD domain of NLRP3 and the
PYD domain of ASC interact to initiate the assembly of inflam-
masome. The NACHT domain has ATPase activity and provides
energy for oligomerization activation. NLRP3 inflammasome
can be activated by a plethora of stimuli having different
chemical natures. These stimuli can be divided into three
groups: endogenous stimuli such as glucose, MSU, ATP, amy-
loid β, environment-related stimuli such as silica, asbestos, and
pathogenic-related stimuli. Several cellular and molecular
events are used as a trigger for the activation of NLRP3
inflammasome such as reactive oxygen species (ROS), Ca2
+
signaling, K
+
efflux, lysosomal rupture, and mitochondrial dys-
function [18]. A unifying model for the activation of NLRP3
inflammasome remains controversial and multiple pathways
are associated with it which include classical or canonical
activation pathway, non-canonical activation pathway, and
alternative activation pathway [19,20]. A two-signal model is
proposed for classical activation which is mainly divided into
two steps: 1) A first signal called priming signal is provided by
the microbial components or endogenous cytokines, which
leads to the activation of nuclear factor kappa-light-chain-
enhancer of activated B cells (NF-κB), which induces the
expression of downstream NLRP3 and pro-interleukin 1-beta
(pro-IL-1β) genes to be up-regulated and prepared for activa-
tion [21]. Recent studies have provided evidence that FAS-
associated death domain protein (FADD) and caspase-8 are
also involved in the priming step [22]. 2) A second signal
provided by extracellular ATP [23], pore-forming toxins and
particles, etc., activates the assembly of NLRP3 inflammasome
leading to the activation of caspase-1 which subsequently
results in the processing and release of IL-18 and IL-1β [24].
In addition to the maturation of proinflammatory cytokines,
caspase-1 activation also promotes pyroptosis through clea-
vage of an acid cytoplasmic protein gasdermin D (GSDMD).
Pyroptosis, a form of programmed cell death which is a key
defense mechanism against microbial infections further facil-
itates the release of pro-inflammatory mediators such as high
mobility group box 1 (HMGB1) [25]. Non-canonical activation
pathway depends upon caspase-11 in mice and caspase4-/5 in
humans rather than caspase-1 [20] and is stimulated mainly by
Gram-negative bacteria which activate toll/IL-1 receptor
homology-domain-containing adapter-inducing interferon-β
(TRIF) and TLR4–MyD88 pathways leading to the translocation
of NF-κB which promotes the transcription of interferon reg-
ulatory factor (IRF)-3 and IRF7 genes as well as IL-1β, IL-18, and
NLRP3 [26]. IRF3 and IRF7 form a complex to induce the
expression of interferon (IFN)-α/β. The resulting IFN-α and
IFN-β bind to their receptors IFN-α/β receptor 1 (IFNAR) and
IFNAR2 ultimately inducing the activation of JAK/STAT path-
way and subsequent caspase-11 transcription [25]. Activated
caspase-11 promotes the activation of NLRP3-ASC-caspase-1
pathway to release 1 L-1β, induces the release of HMGB1 and
IL-1α, and induces pyroptosis via cleavage of GSDMD [27]. The
Priming step, which is indispensable for canonical activation
due to low basal levels of caspase-1, seems unnecessary for
non-canonical pathway activation in human cells in which
caspase-4 is present abundantly [28]. More recently, alterna-
tive activation of NLRP3 inflammasome was reported in
human monocytes which are proposed to be triggered with-
out activation signals [19]. This pathway is species-specific and
does not require K
+
efflux. Experiments have shown that this
activation is dependent on TLR4-TRIF-RIPK1-FADD-caspase-8
signaling leading to low IL-1β secretion in a P2X7 independent
manner and does not result in pyroptosis or ASC speck for-
mation [19,29]. After the activation of inflammasome, secreted
IL-1β and IL-18 induce the expression of some other down-
stream cytokines such as IL-6, IL-12, tumor necrosis factor
alpha (TNF-α), and interferon gamma (IFN-γ). These cytokines
contribute to innate immune responses to stress or infection
resulting in a generalized pro-inflammatory environment [30].
Caspase-1, IL-1β, and IL-18 are commonly utilized in research
as indicators of NLRP3 activation [18].
1.2. NLRP3-associated diseases
Regulated activation of NLRP3 inflammasome is vital for the
removal of pathogens, repair of damage, and maintenance of
homeostasis. However, over-activated inflammasome can
cause cytokine storm leading to edema, internal bleeding,
respiratory distress syndrome, and even death. Table 1
Article Highlights
●The most comprehensive and up-to-date overview of disease models
for NLRP3 inflammasome over-activation.
●Both cellular and animal models were included and their use in NLRP3
targeted drug discovery.
●3.Strategies for high specific NLRP3 inhibitor development were
provided.
430 H. ZHANG ET AL.
describes the various diseases which have been associated
with NLRP3 inflammasome dysregulation.
1.3. NLRP3 inflammasome-targeted drug discovery
In recent years, many molecules were reported to modulate
inflammasome signaling by interacting with different compo-
nents of inflammasome complexes. However, the difficult
reconstitution of the inflammasome in in vitro and in vivo
settings has limited the development of specific on-target
biochemical assays for compound activity confirmation and
drug discovery in high throughput screening setups. The func-
tions of NLRP3 inflammasome are diverse and regulating these
processes by inhibiting NLRP3 is a very effective treatment.
The current mainstream target drug sources mainly include
computational synthesis based on target structure, chemical
structure improvement based on existing drugs, and screen-
ing based on molecular libraries [40]. The efficiency of compu-
tational synthesis based on target structure is higher, but for
a long time due to the lack of structures of NLRP3 and other
component proteins, there were obstacles in structure-based
design. In 2019, Nature reported the electron microscope
structures of NLRP3 and NEK7 for the first time [41], which is
expected to promote structure-based targeted drug design.
Natural products are another important source of NLRP3 inhi-
bitors and are widely used in Chinese medicine and can be
used for clinical treatment once their effects are tested. There
have been many articles summarizing the existing inhibitors of
NLRP3 inflammasome [42–44].
2. In vitro models for NLRP3 inflammasome
inhibitors screening
The in vitro screening model for NLRP3 inflammasome is
mainly a cell-based model. The cells that can be activated
by inflammasomes include macrophages, monocytes, dendri-
tic cells, epithelial cells, keratinocytes, and splenic centro-
blasts [45–47]. The use of such cell lines has significantly
enabled the dissection of various aspects of inflammasome
biology. These cell lines can be primed and induced to form
NLRP3 inflammasome and can be utilized for testing the
effects and efficacy of various drugs or inhibitors.
A frequently used cell line in inflammasome study is human
monocytic cell line THP-1, which stems from leukemic mono-
cytes [48].
2.1. Inflammasome priming in in vitro models
The widely accepted model in research settings has two-
steps: utilizing priming and activating signals for the for-
mation of NLRP3 inflammasome. The purpose of using
a priming signal is to activate the NF-κB pathway which
ultimately increases the expression of NLRP3 and pro-IL-1β
and stimulates the NLRP3 post-translational modifications
(PTMs) [49]. Table 2 outlines the commonly used priming
and inducing agents for NLRP3 inflammasome.
2.2. Activating/inducing the inflammasome
By using activation signals, important cellular and molecular
events necessary for NLRP3 inflammasome activation are
fueled leading to maturation of caspase-1 and secretion of
Table 1. NLRP3-associated diseases and various inhibitors tested for them.
Disease NLRP3 Inhibitors tested References
Alzheimer disease CP-456, 773, Fenamate [17,24,25]
Atherosclerosis CP-456, 773 [24,26]
Asthma and allergic airway inflammation CP-456, 773 [27–29]
Amyotrophic lateral sclerosis MCC950 [30]
Cryopyrin-associated periodic syndromes OLT177, CY-09, BHB
CP-456, 773, Glyburide
[40–44]
Gout BHB, Quercetin [45,46]
Glomerulonephritis Antroquinonol [47]
Inflammatory bowel disease and related models CP-456, 773
INF39, FC11A-2
[31,32,48,49]
Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis CP-456, 773 [33,34]
Hypertension CP-456, 773 [35]
Myocardial infarction CP-456, 773
16,673–34-0
[36–38]
Multiple sclerosis or experimental autoimmune encephalitis CP-456, 773
JC-171, IFNβ
MCC950, OLT1177
[39,41,51–53]
Oxalate-induced nephropathy CP-456, 773 [54,55]
Hyperinflammation following influenza infection CP-456, 773 [56,57]
Graft-versus-host disease None [58]
Stroke Ibrutinib, CP-456, 773 [59–61]
Silicosis None [62]
Type 1 diabetes None [63]
Traumatic brain injury CP-456, 773 [64,65]
Obesity-induced inflammation or insulin resistance CY-09 [40,66]
Rheumatoid arthritis
osteoarthritis (OA)
VX-740 and VX-765 [67,68]
Myelodysplastic syndrome CP-456, 773 [69]
Contact hypersensitivity CP-456, 773 [29,70]
Joint inflammation triggered by chikungunya virus CP-456, 773 [71]
Parkinson’s Disease Kaempferol (Ka) [72]
EXPERT OPINION ON DRUG DISCOVERY 431
IL-1β. The inducers which can be used for canonical activation
(via caspase-1) or non-canonical activation (via caspase 4/5 in
humans and caspase-11 in mice) of NLRP3 inflammasome are
mentioned in Table 2. The result of both activation models is
cell lysis and secretion of inflammatory cytokines [51–53]. For
in vitro experiments, it is very crucial to monitor the NLRP3
inflammasome formation, secretion of cytokines, cell lysis, and
the inhibition efficiency of inhibitors. Different assays are used
to check all these parameters.Inflammasome components
detection
To detect the various components of inflammasome such
as ASC, NLRP3, pro-caspase-1, pro-IL-1β, and pro-IL-18, immu-
noblotting with specific antibodies is routinely used. These
antibodies can be obtained from various commercial compa-
nies, moreover, different easy to use detection kits for inflam-
masome components are also available.
2.3. ASC speck formation detection
A direct measure for the formation of inflammasome is the assess-
ment of ASC specks which are considered the hallmarks of NLRP3
inflammasome activation. Usually, light microscope or advanced
flow cytometry are used to assess the formation of ASC specks. For
visualization of ASC specks through light microscopy, ASC protein
is fused with a fluorescent protein and the presence of distinct
bright points upon stimulation indicates the inflammasome acti-
vation [54,55]. The flow cytometry method is more sensitive, offers
quantitative assessment of ASC speck formation, and reduces the
risk of false-positive readouts [56]. Furthermore, a transgenic
mouse containing ASC fluorescent protein (ASC-citrine) has been
developed whose primary cells and tissue exhibit ASC speck for-
mation upon treatment with NLRP3 inflammasome activators [57].
2.4. Detection of pro-caspase-1 to caspase-1 conversion
Another gold-standard to detect the activation of inflamma-
some is to check the presence of active caspase-1. Routinely
used assays for the detection of pro-caspase-1 to caspase-1
conversion include Western blot, ELISA, and fluorescently
labeled inhibitors of caspase (FLICA), however, they lack high
specificity and sensitivity. Besides, genetic constructs that
upon transfection into cells act as biosensors for caspase-1
activity are also available such as iGLuc reporter system [58].
A single-step plate-based, highly through-put bioluminescent
method for caspase-1 activity measurement was reported
recently, which has been tested successfully in human primary
monocytes, J774A.1 mouse macrophage, THP-1 cells, and
bone marrow-derived macrophages [59].
2.5. Caspase-1 activity on pro-IL-1β and pro-IL-18
Western blots of processed IL-1β and IL-18 or ELISA of released
cytokines are often used as indicators for the activation of
inflammasome and caspase-1 activation [60]. ELISA assay to
detect IL-1β and IL-18 as a measure of caspase-1 activity is also
used, however, cross-reactivity with pro-IL-1β and pro-IL-18
can yield inaccurate readouts [61], therefore, it is important
that immunoblot assessment of caspase-1 cleavage is also
done alongside to ensure the accuracy of results.
2.6. Cell lysis and pyroptotic cell death measurement
Inflammasome activation results in pyropototic cell death,
thus, it can be used as a measure of inflammasome responses.
One method of detecting pyroptotic cell death is based on
measuring the release of cytosolic lactate dehydrogenase
(LDH) into the medium, which is released upon pyroptosis
following caspase-1 activation [62]. Since LDH release also
occurs during other forms of cell death, therefore, lack of
specificity can be a problem and this assay must be done in
the presence of caspase 1, 11, 4, or 5 inhibition to ensure if
pyroptosis still occurs [62]. Another approach is the utilization
of a microscope to determine the uptake of cell impermeant
dyes corresponding to the loss of cell integrity. This approach
can be combined with live-cell imaging to get a temporal
assessment of pyroptosis [63].
Recently, a whole blood-based assay for the evaluation of
both canonical and non-canonical NLRP3 inflammasome activa-
tion was reported. In this method, following LPS priming, the
addition of ATP resulted in the stimulation of unprocessed
human blood cells [64]. The advantage of this method of using
whole unprocessed blood cells is that separation of peripheral
blood mononuclear cells (PBMCs) or other types of cells from the
blood can result in undesirable side effects such as apoptosis
[65]. The effects of handling-related cell activation will also be
minimal in such settings resulting in the most accurate estima-
tion of cytokine production [66]. While using in vitro models of
NLRP3 inflammasome activation for drug screening, it should be
kept in mind that different cell types have specific requirements,
different stimuli, and differences in the dosing of LPS. Moreover,
some inhibitors target NLRP3 inflammasome as well as another
inflammasome, therefore, while testing an inhibitor its specificity
should be determined.
3. In vivo models for NLRP3 inflammasome
inhibitors screening
Based on the drugs screened in in vitro experiments, further
application in clinical settings first requires in vivo evaluation
in animal models. There are many types of inflammatory
Table 2. Commonly used priming agents and inducers for canonical and non-canonical activation of NLRP3 inflammasome for in vitro models.
Priming Agents Lipopolysaccharide (LPS), Synthetic triacylated lipopeptide, Polyinosine-polycytidylic acid (poly(I:C)) [18,21,31]
Canonical
Inducers
Alum, Adenosine triphosphate (ATP), Synthetic heme crystal (Hemozoin), Monosodium urate crystals (MSU crystals),
Nanoparticles of silica dioxide (Nano-SiO2), Nigericin, Synthetic analog of the cord factor (TBD)
[18,20,31–34]
Non-canonical
inducers
Outer Membrane Vesicles of Gram-negative bacteria (OMVs), Beta-1,3-glucan from A. faecalis (Curdlan), Heat-killed C. albicans
(HKCA), Beta-D-glucan from lichen Lasallia pustulata (Pustulan), Zymosan Depleted (Hot alkali treated zymosan)
[19,35–39]
432 H. ZHANG ET AL.
disease models available involving the activation of NLRP3
inflammasome [67–73]. To facilitate the researchers, we have
summarized common disease models and classified them
according to the type of diseases.
3.1. Traumatic brain injury (TBI) models
TBI refers to a group of brain injuries that have complex etiology,
prognosis, and severity. TBI can result in various neurodegenera-
tive diseases such as Parkinson’s disease and Alzheimer’s disease.
Various alarmins such as ATP, DNA/RNA, uric acid, and interleu-
kin-1α (IL-1α), etc., are released at the site of injury in TBI which
triggers the inflammation and adaptive immune response [74].
These alarmins prime NF-κB and also bind with TXNIP to activate
NLRP3 [75]. Several studies have reported the activation of
NLRP3 inflammasome in TBI [76–78]. NLRP3 is expressed in
neurons, astrocytes, and microglia in the pericontusional cortex.
Once an injury happens, NEK7, NLRP3, ASC, and caspase-1 are
upregulated resulting in enhanced secretion of IL-1β and IL-18
[79]. Many inhibitors targeting NLRP3 have been validated in
relevant animal models, including rats and mice which are men-
tioned in Table 3. NLRP3 targeted inhibitors such as ω-3 fatty
acids alleviated inflammation and behavioral deficits, BAY
11–7082 revealed more preserved brain structure and reduced
edema, MCC950 protected mice from edema, and resulted in
improved neurological function, Treatment with mangiferin
(1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside), a component
of traditional Chinese medicine, alleviated brain damage by
inhibition of ROS-TXNIP-NLRP3 inflammasome pathway. Some
inhibitors like Omega 3 fatty acids and Propofol have already
been used in many clinical studies. Rodents are the most com-
mon model of TBI due to their small size, reasonable cost, and
ease of handling. Four models that are frequently used in
research settings are discussed here.
3.2. Fluid Percussion Injury (FPI) Models
These models can have midline FPI in which injury is applied
at sagittal suture or lateral FPI in which injury is applied over
the parietal cortex. A fluid pulse is targeted directly onto the
surface of the dura which causes deformation of the brain
tissue [80]. In FPI models, the location and severity of the
injury can be changed to reproduce different neurological
impairments. Due to its extensive use, the results of FPI can
be compared between different laboratories, however, the
device needs to be adjusted according to the model and
size of the animal [81].
3.3. Controlled cortical impact (CCI) injury model
In this model, an electromagnetic impactor device is used to
carry out mechanical deformation in the cortex [82] which can
be altered by varying the velocity, depth, and dwell time of
the impactor [83,84]. In CCI, the impact on the cortex results in
cortical tissue loss, blood-brain barrier (BBB) dysfunction, con-
cussion, axonal injury, acute subdural hematoma, and even
coma [83,85].
3.4. Weight drop–impact acceleration injury
In this model, a free-falling, guided weight impacts the skull
resulting in injury whose severity can be changed by adjusting
the height and mass of the impactor [86]. It is the most
common model of TBI at present, which has been tested in
many NLRP3 inflammasome inhibitor studies [87–92].
3.5. Blast-related traumatic brain injury model
Various test methods such as open-field blasts, shock tubes,
and blast tubes have been developed to replicate the explo-
sive blast injuries suffered by humans. In laboratories, blast-
tubes are most commonly used [93].
It should be noted that due to the complexity of TBI, NLRP3
is not the only activated inflammatory pathway, and the NF-
κB, MAPK, and NLRP1 pathways are also involved. Some stu-
dies have reported that inhibitors target all these in TBI,
although it is beneficial for the treatment of diseases, it is
not suitable for evaluating the effect of inhibitors targeting
individual pathways. When using such models for NLRP3 inhi-
bitor research, it is best to simultaneously detect the activity of
these pathways to evaluate the efficacy of the inhibitors
accurately.
3.6. Neurodegenerative disease models
Neuroinflammation plays a pivotal role in several neurodegen-
erative disorders including brain injury, Parkinson’s disease,
Alzheimer’s disease, and depression, etc. The activation of
Table 3. Different models for TBI and NLRP3 inflammasome inhibitors tested in them.
Model of TBI Species Inhibitor NLRP3 specificity Clinical study
CCI Mouse MCC950 Yes (NLRP3 NACHT domain) NA
CCI Rat JC124 Yes NA
CCI, weight drop, and fluid Percussion Rat and mouse Bay 11–7082 No NA
Blast Rat Mangiferin No NA
CCI Mouse Omega-3 fatty acids No NCT03345550;
NCT03032302;
NCT02990091;
NCT02762539;
NCT01515917
Blast Rat Propofol No NCT04034771;
NCT03285165;
NCT03285165;
NCT01007773;
NCT00336882
EXPERT OPINION ON DRUG DISCOVERY 433
NLRP3 inflammasome is frequently detected in neuroinflam-
mation, and activated inflammation promotes or even leads
the disease process. Neuroinflammation and neurodegenera-
tion often result from the aberrant accumulation of aggre-
gated host proteins such as α-synuclein, amyloid-β, and
prions which contribute to the activation of inflamma-
some [94].
3.6.1. Alzheimer’s disease (AD) models
AD is a progressive neurodegenerative disorder characterized
by memory loss and dementia [95]. The intracellular aggrega-
tion of neurofibrillary tangles (NFTs) and extracellular accumu-
lates of amyloid plaques are the pathological hallmarks of AD
[96]. Helleet al. reported the involvement of NLRP3 inflamma-
somes in AD for the first time [97].
The models of AD which have been tested for NLRP3
inflammasome inhibitors include APP/PS1 mouse;
TgCRND8 AD mouse; triple transgenic Alzheimer’s disease
(3xTgAD) mouse and mice injected with stereotaxic injection
of β-amyloid [98]. Due to the unknown etiology of
idiopathic AD, most animal models are based upon the
genetic mutations associated with familial AD (fAD). The ratio-
nale used is that after the initial stimulus, downstream events
are reasonably similar. None of these models exhibits all the
aspects of the AD, however, they are still invaluable tools for
studying the disease process and drug designing.
Yin et al. reported the formation and activation of NLRP3
inflammasome in TgCRND8 mice, which was inhibited by the
treatment with inhibitor JC-124 [99]. APPswe/PS1dE9 mice over-
express the Swedish mutation of APP K670N/M671L together
with Presenilin-1 (PS1) deletion in exon 9 and have parenchymal
Aβ load with Aβ plaques [100]. The study by Henekaet al.
reported that knockdown of NLRP3 gene reduces the accumula-
tion of Aβ and averts the cognitive and behavioral dysfunction in
the aged APP/PS1 mice. APP/PS1/NLRP3
−
/
−
mice also displayed
decreased buildup of Aβ plaque in the hippocampus [17]. Shi
et al. showed that in APPswe/PS1dE9 mice treatment with arte-
misinin, an antimalarial drug, considerably blocked the activation
of NALP3 inflammasome and NF-κB, and lowered the amyloid
plaque aggregates in the hippocampus and cortex [101].
3.6.2. Depression
Depression is a neurological disorder, which affects the mental
and physical health of patients. Mild depression patients com-
monly display low mood, sadness, weakness, and insomnia,
while severe patients can have repeated suicidal thoughts or
may even attempt suicide. The current pathogenesis of
depression is ambiguous, shows strong heterogeneity, and
has limited clinical treatment. The NLRP3 inflammasome has
been linked with the pathogenesis of depression [102].
Elevated protein expression of NLRP3, ASC, caspase-1, and
increased mRNA levels of proinflammatory cytokines were
observed in the prefrontal cortex of the rat depression
model. NLRP3 inflammasome-dependent pyroptosis and
apoptosis in hippocampus neurons mediate depression-like
behavior in diabetic mice. NLRP3 inflammasome-targeted
drugs can improve depression behavior, such as Metformin
improves depression-like symptoms in mice via inhibition of
peripheral and central NF-κB-NLRP3 inflammation activation.
Astragalin exerted antidepressant-like action through SIRT1
signaling which modulated NLRP3 inflammasome deactiva-
tion. Trans-cinnamaldehyde reverses depression-like behaviors
in chronic unpredictable mild stress rats by inhibiting NF-κB/
NLRP3 inflammasome pathway. At present, the methods used
to construct depression models are mechanistically divided
into two major categories, one of which is the use of neuro-
transmitters, including monoamine, butyric acid, glutamic
acid, prohormone, and arginine vasopressin, etc.; the other
category is the use of oxidative stress and disturbance of
inflammatory pathways [103,104]. The depression modeling
includes the following methods:
3.7. Reserpine-induced depression
Reserpine can block the recovery of vesicles and block synap-
tic signal transmission in the brain to cause depression. The
main advantage of this model is that it saves time and is
effective to distinguish the pharmacological and antidepres-
sant effects of psychoactive drugs, but this model has a high
lethal rate and is different from human depression because of
long-term stress and mental burden [105].
3.8. The learned helpless model
In this model, animals show a lowered intention to escape in
an unpredictable and uncontrollable injury stimulation envir-
onment. This model bears a close resemblance to the char-
acteristics of human depression, but the duration is shorter,
and different experimental animals respond differently. It is
appropriate for investigating the etiology of depression and
used in the antidepressant manufacturing process [106].
3.9. Chronic mild stress model
It uses a long-term inescapable stimulation method composed
of a series of trials, such as fixation, electric shock, day and
night reversal, ice walking, food or water deprivation, and tail
clipping. This method has reliable predictive validity, however,
the mortality of model animals is higher than other methods
[105]. Yamanashi et al. tested beta-hydroxybutyrate (BHB), an
inhibitor of NLRP3 inflammasome using this model. Potential
antidepressant and anti-inflammatory effects on rats were
observed upon treatment with BHB [107].
3.10. Social failure stress model
In this method using frequent stimulation, model animals
develop long-term psychosocial and behavioral changes,
including a reduction in sexual behavior and reduced commu-
nication with other members. The model construction method
is simple and shorter than other chronic models. The disad-
vantage of this model is that experimental animals will experi-
ence anxiety which can confuse the evaluation [104].
3.10.1. Multiple sclerosis (MS)
MS is an autoimmune disease which is caused by the
progressive loss of myelin sheaths of neurons. Several
434 H. ZHANG ET AL.
studies have shown the contribution of NLRP3 inflamma-
some in the development of MS. In PMBCs obtained from
MS patients, the expression of caspase-1 was considerably
augmented [108]. In another study, the mRNA expression
of NLRP3 inflammasome-associated molecules such as
NLRP3, caspase-1, and IL-1β was reported to be up-
regulated in the PBMC of MS patients [109].
The extensively used model for MS is known as experi-
mental autoimmune encephalomyelitis (EAE) [110], which
can be induced in many animals such as rats, mice, guinea
pigs, and primates. These models of EAE have a varying
degree of efficacy with different specific features that allow
a wide spectrum of choice for experiments [111]. In mice
EAE model having Nlrp3
−
/
−,
the severity of EAE reduced
along with a substantial decrease in inflammatory cells
infiltration such as CD4
+
, CD8
+
T cells, dendritic cells, and
macrophages [112]. Treatment with MCC950, an inhibitor
of the NLRP3 inflammasome, reduced the severity of EAE
and alleviated the severe memory deficits which were
manifested in the late phase of EAE [51,113].
3.10.2. Parkinson’s disease (PD)
The second most common neurodegenerative disorder is
PD, which is characterized by progressive loss of dopami-
nergic (DA) neurons in the midbrain [114]. The aggregates
of misfolded α-synuclein (α-Syn) are the pathological hall-
mark of PD [115]. The genes which are linked to PD
include α-synuclein, leucine-rich repeat kinase 2 (LRRK2),
Parkin, DJ-1, phosphatase and tensin homolog (PTEN)-
induced novel kinase 1 (PINK1) and glucocerebrosidase
(GBA) [116]. The up-regulation of NLRP3 and activation of
caspase-1 results in IL-1β secretion upon exposure of
monocytes to fibrillar α-Syn [117]. The alleviated levels of
IL-1β have been linked to NLRP3 inflammasome activation
in the microglia and midbrain of the mouse model of PD
[118]. Currently, many animal models of PD such as mice,
Drosophila melanogaster, and Caenorhabditis elegans are
available. Additionally, many intoxication models are also
used such as reserpine- or haloperidol-treated rats which
manifest one or more symptoms of PD; classical 6-hydro-
xydopamine (6-OHDA) rat which shows deteriorated dopa-
minergic nigro-striatal pathway and 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) mouse models, in which
neurotoxin MPTP is used to induce a syndrome which
virtually mimics PD. In a study, MPTP mouse model was
used to evaluate the effect of inhibition of NLRP3 on
systemic inflammation of PD. A recent study reported
that kaempferol (Ka), a small molecule inhibitor of NLRP3
inflammasome lowered the severity of PD in mice mod-
els [119].
3.10.3. Amyotrophic lateral sclerosis (ALS)
ALS is a fatal neurodegenerative disease associated with pro-
gressive degeneration of the upper and lower motor neurons.
The progression of the ALS is mediated by astrocytes and
microglia. The pathological hallmark of familial ALS (fALS) is
the aggregation of a mutant form of superoxide dismutase 1
(SOD1) enzyme [120]. At present, the mouse model available
of ALS carries SOD1
G93A
mutated human SOD1 gene [121]. An
increase in the expression of components of NLRP3 inflamma-
some and IL-1β has been observed in the spinal cord tissues
from the patients and in SOD1
G93A
mouse model [122].
Furthermore, anterodorsal thalamic nucleus of SOD1
G93A
mice also depicted an alleviated expression of ASC and
NLRP3 [121]. A recent 2020 study has tested MCC950 in
SOD1
G93A
mice and concluded that pretreatment with the
inhibitor abrogated the secretion of IL-1β from microglia [123].
3.11. Rheumatic diseases
Rheumatic diseases are chronic autoimmune degenerative dis-
eases that mainly affect connective tissues, such as cartilages in
joints, bones, ligaments, tendons, and muscles leading to con-
siderable health issues and mortality. More than 100 rheumatic
diseases have been reported accounting for the largest group of
inflammatory/autoimmune diseases. The patients of these dis-
eases are present throughout the world, hence, there is a huge
interest in therapeutic interventions for such diseases. The most
common of these diseases are rheumatoid arthritis (RA), gout,
ankylosing spondylitis (AS), systemic lupus erythematosus (SLE),
and Sjögren’s syndrome (SS) [124].
3.11.1. Gout animal models
Gout is one of the few rheumatic diseases with relatively clear
pathogenesis. It is an inflammatory type of disease closely
related to hyperuricemia. The high concentration of uric acid
causes the precipitation and deposition of urate crystals.
NLRP3 inflammasome is activated by urate crystals and the
secretion of IL-β initiates the inflammatory cascade [125]. The
models of gout are available in various animal species, but rats
and mice are used most frequently to study the progression
and pathogenesis of the disease due to ease of handling, cost,
and homogeneity of the genetic background [126].
3.12. Intra-articular injection of urate crystal suspension
This method is the classic and most established in which
uric acid crystals along with auxiliary reagents such as
palmitic acid and bovine serum albumin, etc., are injected
into the animal to activate the inflammation. The model
preparation cost is low and the degree of recognition is
high, but it is different from the gout in the human
body [126].
3.13. Uricase knockout
Knocking out the uricase enzyme gene perturbs the uric acid
metabolism pathway leading to high levels of uric acid in the
blood which cause gout. The cost of this model is high, takes
a longer time to develop and the knockout mice are easy to
die due to metabolic defects [127].
3.14. Using urease inhibitors to inhibit urease activity
To produce hyperuricemia, urease inhibitors are used, and
then urate crystal suspension is injected into the joint cavity.
This method overcomes the shortcomings of the above two
EXPERT OPINION ON DRUG DISCOVERY 435
methods and the only problem with this method is the easy
inactivation of the inhibitor [128].
3.14.1. Rheumatoid arthritis (RA) models
RA is a serious lifelong autoimmune disease with high preva-
lence worldwide. NLRP3 inflammasome activation has been
reported in RA [129]. Many animal models for RA are available,
among them, collagen-induced arthritis (CIA) and adjuvant-
induced arthritis (AIA) models are the most common and
easy to use. CIA, streptococcal cell-wall-induced arthritis
(SCW) and K/BxN mice models in which mice express both
the T cell receptor (TCR) transgene KRN and the MHC class II
molecule A(g7) are frequently used for the mechanistic under-
standing of the disease [126,130]. NLRP3 inhibitor MCC950 has
been verified in the CIA model of RA [129].
3.14.2. Lupus nephritis (LN)
Lupus is an autoimmune disease caused by immune abnorm-
alities. LN acceleration and deterioration have been linked
with the activation of NLRP3 inflammasome activation, reac-
tive oxygen species (ROS), and mononuclear leukocyte infiltra-
tion in the kidney [131]. The commonly used animal models of
LN include MRL
lpr
and gld models, the BXSB model, and the
NZB/NZW F1 model [132]. Using NZB/Wf1 mice, citral
(3,7-dimethyl-2,6-octadienal), and epigallocatechin-3-gallate
were tested which lowered the disease burden by inhibiting
NLRP3 inflammasome activation [133,134]. Anesthetic isoflur-
ane (ISO) was tested in MRL
lpr
mice for LN and it significantly
enhanced the survival rate and offset the renal damage in
mice by abrogating NLRP3 inflammasome formation and acti-
vation in the kidney [135]. Furthermore, two other inhibitors of
NLRP3 inflammasome, Bay11-7082, and Sophocarpine have
also been tested in MRL
lpr
murine models [136,137].
3.15. Fibrotic diseases
Fibrosis is an excessive scarring process that happens in many
tissues during the aberrant wound healing process. During
fibrosis, normal parenchymal tissues are replaced by connec-
tive tissues which result in significant tissue remodeling lead-
ing to permanent scar tissues. Various organs such as the lung,
liver, heart, kidney, brain, and skin can be affected by fibrosis
with lung and liver fibrosis occurring most frequently [138].
3.15.1. Liver fibrosis
Liver fibrosis is a serious health problem that could develop
into liver cirrhosis and hepatocellular carcinoma. Factors like
alcohol consumption, nonalcoholic steatohepatitis (NASH),
viral hepatitis [hepatitis B (HBV) and hepatitis C (HCV)], auto-
immune hepatitis, nonalcoholic fatty liver disease (NAFLD),
and cholestatic liver diseases could induce liver fibrosis.
NLRP3 inflammasome plays a key role during liver fibrosis
and, many factors can activate NLRP3 and contribute to the
disease progress [139]. For example, chenodeoxycholic acid
activates NLRP3 inflammasome and contributes to cholestatic
liver fibrosis, androgen aggravates liver fibrosis by activation
of NLRP3 inflammasome in CCl4-induced liver injury mouse
model, mitochondrial reactive oxygen species were induced
by liver injury to activate NLRP3. Once activated NLRP3 could
result in IL-1β and IL-18 secretion, which promotes the trans-
formation of hematopoietic stem cells (HSCs) into mechano-
cytes. Besides, pyroptosis can be induced by caspase-1
followed by the activation of NLRP3 inflammasome. NLRP3
inflammasome activation is required for fibrosis development
in NAFLD, moreover, NLRP3 inflammasome blockade reduces
liver inflammation and fibrosis in experimental NASH in mice.
There are mainly five animal models for liver fibrosis: chemi-
cal-based, diet-based, surgery-based, genetically modified,
and infection-based. One common model is the bile duct
ligation (BDL) model based on surgery which consists of
a doubly ligated bile duct transection between 2 ligatures
leading to cholestatic injury and periportal biliary fibrosis.
Both MCC950 and Calcipotriol had been tested in this model
[140]. Methionine and choline-deficient diet (MCD)-induced
NASH model, which mimics the hepatic stress caused by the
fatty acid flux from adipose tissue to the liver as well as
increased production of triglycerides, resulting in liver steato-
sis and lipotoxicity was used to test MCC950 and Sweroside
[141]. Schistosoma japonicum-infected mice models having
a high resemblance to human infection were used to test
MCC950. Calcipotriol was tested in 3,5-Diethoxycarbonyl-
1,4-dihydrocollidine (DDC)-induced cholestasis model.
Triiodothyronine was tested in alcoholic liver disease mouse
model and BAY 11–7082 was tested in hepatic fibrosis mouse
model [139].
3.15.2. Pulmonary fibrosis
Pulmonary fibrosis is a kind of lung disease caused by aberrant
wound healing. Microbial and environmental elements such as
mechanical stretch, airborne fine particulate matter (PM2.5),
asbestos, carbon black nanoparticle (CBNP), LPS, and crystal-
line silica could lead to repeated alveoli injury, chronic inflam-
mation, excess deposition of the extracellular matrix
components, mainly collagen, and scarring of lung tissue
which can cause respiratory failure. NLRP3 inflammasome
plays a key role in pulmonary fibrosis development [142].
Many inducers of pulmonary fibrosis can activate NLRP3. For
example, PM2.5 could activate NLRP3 by cathepsin B release,
ROS production, and K
+
efflux once internalized by cells, lead-
ing to pulmonary fibrosis. NLRP3 knockout could prevent
mechanical stretch-induced pulmonary fibrosis in mice there-
fore, it is a potential target for pulmonary fibrosis treatment.
Many inhibitors had been tested in various kinds of related
animal models, such as the acute lung injury (ALI) model,
which is generated by intratracheal injection of LPS into
male C57BL/6 J mice has been used to testPirfenidone which
attenuated ALI by blocking NLRP3 inflammasome activation
[143]. Another widely used model is the bleomycin model
which is generated by transoral instillation or direct endotra-
cheal injection of bleomycin. This model shows good repro-
ducibility and ease of induction. Lycorine, Fluorofenidone, and
Raltegravir were tested in this model since it can reproduce
many aspects of idiopathic pulmonary fibrosis and other fibro-
tic interstitial lung diseases [142]. Recently, the role of NLRP3-
induced inflammation was reported in the pathogenesis of
cystic fibrosis. Pretreatment of monocytes containing cystic
fibrosis-associated mutations with NLRP3 inhibitors
436 H. ZHANG ET AL.
significantly lowered the high levels of L-18, IL-1β, caspase-1
activity, and ASC-speck release [144]. Besides, MCC950 was
tested in a murine cystic fibrosis model generated by aero-
solized LPS [145] and Resveratrol was tested in an ambient PM
exposed model [146].
3.16. Metabolic diseases
Metabolic diseases represent a wide range of disorders such as
cardiovascular diseases, obesity-induced inflammation, Type 2
diabetes, and gout. NLRP3 inflammasome plays a dual role in
metabolism; it helps to control metabolic balance and on the
other hand, it can promote metabolic disorder if unregulated.
Many metabolic products can activate NLRP3 inflammasome
such as protein crystals, protein aggregates, or cholesterol
crystals, etc. [147].
3.16.1. Type 2 diabetes (T2D) and obesity
Obesity is a major risk factor for T2D and insulin resistance, in
which metabolic balance is interrupted and leads to increased
glucose and fatty acids in tissues and blood. These fatty acids
especially saturated fatty acids can activate NLRP3 inflamma-
some in a ROS-MAPK dependent manner, meanwhile also
inducing TLR2/4. Ceramides are also upregulated in fatty tis-
sue to activate NLRP3 inflammasome and hyperglycemia
could promote IL-1β production in fatty tissue and diomyo-
cyte cell line. High glucose induces TLR2 and TLR4 in THP-1
cell line and chronic hyperglycemia stimulates peptide hor-
mone islet amyloid polypeptide (IAPP) production, which
tends to be misfolded to induce NLRP3 inflammasome and
damage β cell. Besides, diabetic patients manifest high levels
of uric acid and ROS, which are NLRP3 inducers [148]. Diabetic
animal models can be divided into two groups: genetic mod-
els and chemically induced models. One genetic model is
zucker diabetic fatty (ZDF) rat model, which possesses
mutated leptin receptor that induces hyperphagia, obesity,
and eventually β cell failure resulting in T2D in male rats
when fed a high-energy rodent diet. MCC950 is tested in this
model for NLRP3 inhibition [149]. Another genetic model is
goto-kakizaki (GK) rat model, which is generated by repeated
inbreeding of Wistar rats. This model is spontaneous and non-
obese. GK rats may help study the pathogenic mechanisms
and therapeutic approaches of human non-obese T2D
patients [150]. Alloxan and streptozotocin (STZ) are cytotoxic
glucose analogs that tend to accumulate in pancreatic β cells
through glucose transporter 2 (GLUT2), both are potent dia-
betogenic chemicals which are used to induce diabetes mod-
els [151].
3.16.2. Cardiovascular disease
Another metabolic disease where NLRP3 inflammasome
involvement is known as atherosclerosis, which is the
main cause of cardiovascular disease. The hallmark of
atherosclerosis is inflammatory plaques formed in blood
vessels. Cholesterol levels increase during atherosclerosis
and form crystals in the plaques to activate NLRP3 inflam-
masome, meanwhile, cholesterol is transformed into low-
density lipoproteins (LDL) in plaques and oxidized to
activate TLRs, or introduced into cells by CD36 to form
crystal to activate NLRP3. The plaques always show a low
pH and this extracellular acidosis can also induce NLRP3
[152]. NLRP3 knockout is reported to efficiently protect LDL
receptor defects in mice. The most used animal model for
atherosclerosis is apolipoprotein E knockout (ApoE
−/-
) mice
fed with a high-fat diet (HFD). ApoE has been widely
recognized as a risk factor for cardiovascular diseases,
ApoE
−/-
alone proved to be sufficient for developing aortic
atherosclerotic plaques in mice. HFD markedly accelerates
plaque development in these mice and MCC950 has been
tested in this model [153]. Another atherosclerosis model
is the nicotine model, which uses oral administration of
nicotine dissolved in peanut oil to rats. Rosmarinic acid,
a polyphenol with anti-inflammatory activity, shows high
efficiency in this model [154]. APOE*3Leiden.CETP mouse
model is a double-transgenic mouse and represents
a valuable model for the preclinical evaluation of interven-
tions because of its humanized lipoprotein metabolism.
The diet-induced development of atherosclerosis in these
mice has a pro-inflammatory plaque phenotype and shows
responsiveness to all lipid-modulating interventions that
are being used in the clinic [155].
3.16.3. Models for infectious diseases involving NLRP3
inflammasome
Many pathogenic microorganisms including bacteria, viruses,
and fungi cause inflammation during the process of infection.
3.16.3.1. Bacterial infections.
Some structural components of bacteria such as LPS and
some metabolites such as nigericin can activate NLRP3
inflammasome, but NLRP3 is not the only activated inflam-
masome, bacterial DNA often activates AIM2, NLRP1,
NLRC4, and NLRP6 inflammasome as well [156]. Inhibitors
that target NLRP3 inflammasome have been tested in var-
ious bacterial disease models such as streptococcal toxic
shock-like syndrome (STSLS) [157], polymicrobial sepsis
[158], and intra-amniotic inflammation-induced preterm
birth [159].
3.16.3.2. Viral infections
.Many viral infections can activate NLRP3 inflammasome,
such as Zika virus [160–162], Mayaro virus [163], Dengue
virus [164,165], Chikungunya virus [166], flu virus [167] and
Coronaviruses, etc. Some viruses pass their own proteins to
the host for inflammasome activation, for example, in SARS
coronavirus, ORF8 [168], ORF3 [169], E protein [170], RNA,
or DNA are used to activate NLRP3 inflammasome. Viruses
and bacteria can also cause superinfections such as influ-
enza/Staphylococcus aureus superinfection in which NLRP3
inflammasome plays a key role [171]. Inhibitors targeting
NLRP3 can greatly improve the survival rate of patients in
the absence of specific drugs and vaccines in such infec-
tions. Inhibitor MCC950 has been tested in mice infected
with Chikungunya virus and influenza A virus to target
NLRP3 inflammasome [166,172].
The disease model preparation for these infections is
relatively simple. Injecting pathogens, pathogen analogs,
EXPERT OPINION ON DRUG DISCOVERY 437
or their pathogenic components into the target animal is
carried out to manifest the disease symptoms. It should be
noted that the infection dose of different pathogens is
different, and the inflammation-inducing time is also
slightly different.
3.17. Models for other diseases
In addition to the above-mentioned diseases, NLRP3
inflammasome has been implicated in a plethora of other
diseases and various models of these diseases have been
reported to test NLRP3 inhibitors or other drugs [173].
3.17.1. Glomerulonephritis model
Primary glomerulonephritis (PGN) is the most common
reason inducing end-stage renal disease in China and the
activation of NLRP3 inflammasome has been linked with
the pathogenesis of PGN [174]. AcP-IgAN, which is a mouse
model of renal fibrosis was used to test the promising
effects of compound antroquinonol on the inhibition of
NLRP3 inflammasome [175].
3.17.2. Cryopyrin-associated periodic syndrome (CAPS)
CAPS is used to define a group of auto-inflammatory dis-
orders which result due to activation mutations in NLRP3
gene. CAPS include Muckle–Wells syndrome (MWS), familial
cold auto-inflammatory syndrome (FCAS), neonatal-onset
multisystem inflammatory disorder (NOMID) also known
as chronic infantile neurological cutaneous articular
(CINCA) syndrome [176]. To date, nearly 200 CAPS-
associated mutations have been reported in NLRP3 gene
[177]. The mouse line for MWS, FCAS, and NOMID has been
developed to have a better understanding of CAPS. The
lethal, systemic inflammation displayed by these mice is
due to ASC, caspase 1, IL-1β, and IL-18 [178]. An example
of inhibitor testing in such models is MCC950 whose treat-
ment lowered neonatal lethality in a mouse model of CAPS
[51]. Furthermore, inhibition of NLRP3 inflammasome with
various agents has been tested using mouse models of
non-reperfused and reperfused acute myocardial infarction
[179], mouse model of dextran sulfate sodium (DSS)-
induced experimental colitis [180], mouse model of skin
and pulmonary inflammation [181], mouse model of peri-
tonitis [182] and many other disease models.
4. Inhibitor specificity evaluation
Target specificity is the bottleneck faced by all inhibitors in
clinical application. Most inhibitors will target molecules other
than the target, causing known or unexpected side effects,
leading to the failure of clinical trials and huge economic
losses. Specific inhibitors undoubtedly have better clinical
prospects, so early evaluation of specificity in the screening
process of inhibitors can fully clarify potential side effects.
Figure 1. Signaling pathways that may interfere with NLRP3 inhibitor specificity. A. Targeting other inflammasomes; the stimulus signal that activates NLRP3
can often activate other inflammatory bodies, which have the same downstream products as NLRP3 B. Some signaling pathways that can activate NLRP3, which
need to be considered during the evaluation of inhibitors. ATP, adenosine triphosphate; AD, Alzheimer’s disease; BAK/BAX, Bcl-2 homologous antagonist/killer/Bcl-
2-associated X protein; CASR/GPRC6A, Ca
2+
-sensing receptor/G protein-coupled receptor C6A; DAMP, death-associated molecular patterns, CLIC, chloride intracel-
lular channel; FADD, caspase-8 and FAS-mediated death domain protein; IL, interleukin; LN, lupus nephritis; MAVS, mitochondrial antiviral-signaling protein; MyD88,
myeloid differentiation primary response protein; MS, multiple sclerosis, NF- κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PAMPs, pathogen-
associated molecular pattern; P38 MAPK, P38 mitogen-activated protein kinase, P2X7R, P2X purinergic receptor 7; TLR, toll-like receptor; TNFR, tumor necrosis factor
receptor; TRAF6, TNF receptor-associated factor 6.
438 H. ZHANG ET AL.
4.1. Inhibitors targeting other inflammasomes
The stimulus that activates NLRP3 inflammasome can often
activate other inflammasomes, which share the same
downstream products as NLRP3 inflammasome. NLRP3,
NLRP1, NLRP6, NLRC4, and AIM2 can be activated by bac-
terial infections, NLRP3, AIM2, and NLRP9 can be activated
by viral infection. NLRP3, NLRC4, and NLRP1 can be acti-
vated by sterile inflammation. NLRP3 and NLRP1 are simul-
taneously activated in AD and TBI [78,183], NLRP3, and
NLRC4 are simultaneously activated in MS and SSc [184],
while NLRP1, NLRP3, and AIM2 are simultaneously acti-
vated in LN [170] as depicted in Figure 1a. Therefore, it is
important that during the evaluation of inhibitors, the
state of the corresponding activated inflammasome must
be tested in a specific model. Although inhibitors targeting
other inflammasomes may benefit the disease treatment,
side effects may also exist since diseases are complicated.
4.2. Inhibitors targeting other unwanted signaling
pathways
Some signaling pathways are directly involved in the activa-
tion of NLRP3 inflammasome, inhibitors targeting these path-
ways may achieve similar effects as NLRP3 inhibition, however,
since these pathways may also be involved in other cellular
responses, the regulation of these pathways will also produce
side effects, which need to be avoided during the evaluation
of inhibitors. For example, NF-κB pathway is involved in the
priming process of NLRP3 activation, NF-κB inhibition can also
produce inflammation inhibitory phenomenon which is similar
to NLRP3 inhibition, however, NF-κB is involved in a wide
range of functions, such as inflammation, immune responses,
apoptosis, and stress. Inhibitors that could target NF-κB will
undoubtedly produce side effects. Other pathways that can be
targeted by NLRP3 inhibitors include toll-like receptor (TLR),
P2X purinergic receptor 7 (P2X7R), interleukin-1 receptor (IL-
1 R), and tumor necrosis factor receptor (TNFR), etc. [185] as
shown in Figure 1b. NLRP3 targeted inhibitors that interfere
with these pathways should be avoided.
Furthermore, the activity of NLRP3 is indirectly regu-
lated by many pathways and molecules, inhibitors aimed
at these molecules can affect the activity of NLRP3 [173],
however, these molecules often play a key role in other
signaling pathways, are indispensable for normal physiolo-
gical function, and can have erratic side effects. Therefore,
considering the effects of these pathways during inhibitor
development using animal models is necessary. These
molecules or pathways mainly include: 1. NLRP3 ubiquiti-
nation involving membrane-associated RING finger protein
7 (MARCH7), F-box/LRR-repeat protein 2 (FBXL2), Pellino2,
E3 ubiquitin-protein ligase ARIH2 and tripartite motif-
Figure 2. Potential off-target pathways for NLRP3 inhibitor development. 1. NLRP3 ubiquitination 2. NLRP3 phosphorylation 3. NLRP3 sumolyzation 4. ASC
ubiquitination. In addition, other molecules such as BTK, Hsp90, etc. can also regulate NLRP3 activity. ABRO1, abraxas brother 1; ARIH2, E3 ubiquitin-protein ligase
ARIH2; BRCC3, BRCA1/BRCA2-containing complex subunit 3; BTK, Bruton’s tyrosine kinase; FBXL2, F-box/LRR-repeat protein 2; Hsp90, heat shock protein 90; IL,
interleukin; JNK1, JUN N-terminal kinase 1; LRR, leucine-rich repeat; LUBAC, linear ubiquitin chain assembly complex; MAPL, mitochondrial-anchored protein ligase;
MARCH7, membrane-associated RING finger protein 7; NACHT, central nucleotide-binding and oligomerization; Pellino, RING E3 ubiquitin ligases, mediating Lys-63-
type polyubiquitination of IRAK; PKA, protein kinase A; PKD, protein kinase D; PP2A, protein phosphatase 2A; PTPN22, protein tyrosine phosphatase non-receptor
type 22; PYD, pyrin domain; TRIM31, tripartite motif-containing protein 31; SENP6/7, sentrin-specific protease 6/7; STG1, stargazin-like protein; Syk, spleen associated
tyrosine kinase.
EXPERT OPINION ON DRUG DISCOVERY 439
containing protein 31 (TRIM31) ubiquitination enzymes and
Abraxas brother 1/BRCA1/BRCA2-containing complex sub-
unit 3 (ABRO1/BRCC3) and other deubiquitination enzymes
which can regulate the activation of NLRP3 ubiquitination
[185]. 2. Phosphorylation of NLRP3 involving phosphory-
lases such as JUN N-terminal kinase 1 (JNK1), protein
kinase D (PKD), protein kinase A (PKA) and dephosphory-
lating enzymes such as protein tyrosine phosphatase non-
receptor type 22 (PTPN22), protein phosphatase 2A (PP2A)
which affect the activation of NLRP3 through phosphoryla-
tion [186]. 3. The sumolyzation of NLRP3 involving mito-
chondrial-anchored protein ligase (MAPL) and sentrin-
specific protease 6/7 (SENP6/7) which regulates the activity
of NLRP3 [187]. 4. Ubiquitination of ASC, spleen associated
tyrosine kinase (Syk), and linear ubiquitin chain assembly
complex (LUBAC) can regulate the ubiquitination of ASC to
affect the activation of NLRP3 [188]. Additionally, there are
other proteins, such as Bruton’s tyrosine kinase (BTK),
which interacts with NLRP3 and ASC to regulate NLRP3
activity [189,190]. Figure 2 depicts the effects of these
pathways and molecules on NLRP3 inflammasome.
For successful drug development, target validation and
evaluation of off-target effects are critical requirements. In
order to overcome the issues which could arise due to the
lack of specificity of inhibitors, some precautionary mea-
sures must be exercised while performing the inhibitor
screening process. For example, it should be ensured in
the first place that an inhibitor target only those compo-
nents which are unique to NLRP3 inflammasome to avoid
off-target binding. Targeting of upstream or downstream
effector molecules should be avoided because these effec-
tors can be involved in multiple pathways. The effects of
inhibitors must be evaluated on other inflammasomes as
well. Now that the structure of NLRP3 protein is available,
researchers should aim to design structure-based drugs
that will bind to NLRP3 protein exclusively to yield highly
specific inhibitors. Attaining specificity can be aided by
targeting differences in the dynamics of proteins.
Furthermore, if an inhibitor has the potential to bind to
multiple targets then its effect on NLRP3 inhibition must
be tested in an experimental setting where other targets
are absent so that it can be determined whether the
results are only due to NLRP3 inflammasome targeting.
Moreover, PTMs have emerged as an important factor
that significantly impacts the inflammasome assembly.
Future studies aimed at identifying the enzyme and their
co-factors which specifically carry out PTMs of NLRP3 may
provide novel specific druggable targets. Lastly, for most
NLRP3-associated diseases, multiple disease models are
available which all should be utilized for a given inhibitor
to check the specificity. Table 4 summarise models used to
test various inhibitors of NLRP3. The use of a combination
of animal models can effectively describe the potential
side effects as well.
5. Expert opinion
NLRP3 inflammasome has a vital role in the innate immune
system due to its immune sensing functions and its asso-
ciation with an overwhelming number of diseases which,
in turn, makes it an attractive target for therapeutic inter-
ventions through inhibition of its various components.
Inhibitors that target NLRP3 inflammasome are indispensa-
ble for the prevention and treatment of NLRP3 inflamma-
some-associated diseases. However, most of the reported
Table 4. A summary of models used to test various inhibitors of NLRP3.
Inhibitors Cell/animal model/patients Reference
Antroquinonol Glomerulonephritis mouse model [47]
MCC950 Autoimmune encephalomyelitis,
CAPS mouse model,
Muckle-Wells syndrome mouse model
Amyotrophic Lateral Sclerosis
[30,41]
MNS Bone marrow-derived macrophages [145]
CY-09 CAPS mouse model,
Type 2 diabetes mouse model,
Synovial fluid cells from gout patients
[40]
OLT1177 Human blood-derived macrophages,
Human blood neutrophils,
Monocytes isolated from patients with CAPS,
Spleen cells from mice
[43]
Glyburide Bone marrow-derived macrophages,
Familial cold-associated autoinflammatory
syndrome patients
[42]
16,673–34-0 Acute myocardial infarction mouse model [50]
JC124 Acute myocardial infarction mouse model,
Alzheimer’s disease mouse model
[121,147]
BHB Chronic mild stress mouse model for depression
Muckle-Wells syndrome mouse model
Familial cold autoinflammatory syndrome mouse model
Urate crystal-induced peritonitis model
[44]
Parthenolide Bone marrow-derived macrophages
Cystic fibrosis mouse model
[148,149]
Bay-117,082 Psoriasis-like dermatitis
Diabetic nephropathy
[150,151]
VX-740 and VX-765 Rheumatoid arthritis (RA) and osteoarthritis (OA) mouse model [67,68]
440 H. ZHANG ET AL.
inhibitors have poor specificity and have a risk of cross-
reaction by targeting other inflammasomes. There is a dire
need for the development of NLRP3 inhibitors which pos-
sess higher specificity and efficacy. Since NLRP3 inflamma-
some causes different diseases using different mechanisms,
and the signals that cause the activation of NLRP3 inflam-
masome are different under different circumstances, the
screening process of inhibitors requires various types of
cell and animal models. Appropriate models help to eval-
uate the specificity of the target and the therapeutic effect
and are conducive to the development and application of
NLRP3 inflammasome-targeted drugs. The principal objec-
tive of these models is to aid in the drug discovery process
to develop drugs that are safer and more effective.
Nonetheless, there are many concerns regarding the use-
fulness of these models. For example, the activation stimu-
lus used in some models may activate other
inflammasomes, which is not desired for determining the
efficacy of specific NLRP3 inflammasome inhibitors; there-
fore, these facts should be considered in advance when
selecting models for the experiments in order to have
accurate results. Moreover, many interventions that are
successful in these models are failed later on when tested
in clinical trials, therefore, the usefulness of these models
should be evaluated beforehand. Another pragmatic con-
strain in the development and validation of these models
is the required labor and time. If models depicting nearly
accurate disease conditions are not available, we will lack
the rapid and timely screening of a large number of inhi-
bitors and other drugs targeting NLRP3 inflammasome.
Furthermore, for many diseases appropriate models are
unavailable, therefore, the lack of drugs with proven effi-
cacy remains a challenge. Many of these models do not
replicate the exact nature of the disease and gender bias
in these models is another scrutiny. Most of the animal
models are limited to mice and due to the ethical issues
and financial constraints large animal models are rarely
used. In our opinion, for neurodegenerative diseases and
other diseases affecting the brain, it is important to eval-
uate the NLRP3 inhibitors and drugs in higher species
which have a closer match to humans. In order to achieve
a breakthrough in NLRP3 targeted drug discovery, current
research should focus on the following aspects: develop-
ment of highly specific inhibitors of NLRP3 inflammasome
since its structure is now available to prevent off-targets
effects; development of new efficient models; refinement
of preexisting models and optimization of dosing and tim-
ings of the drugs using these models.
Funding
T Jin is supported by the Strategic Priority Research Program of the
Chinese Academy of Sciences (XDB29030104), the National Natural
Science Fund (Grant No.: 31870731 and U1732109) as well as the
Fundamental Research Funds for the Central Universities. A Zahid is
supported by a CAS-TWAS President Fellowship. J Tao is supported by
the National Natural Science Foundation of China (81771774), the Natural
Science Foundation of Anhui Province (1708085MH191), and the Anhui
Key Research and Development Foundation (201904a07020103).
Declaration of interest
The authors have no other relevant affiliations or financial involvement
with any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the manuscript
apart from those disclosed.
Reviewer Disclosures
Peer reviewers on this manuscript have no relevant financial or other
relationships to disclose.
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EXPERT OPINION ON DRUG DISCOVERY 445
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446 H. ZHANG ET AL.
