Abstract
COVID-19, the illness caused by SARS-CoV-2, has a wide-ranging clinical
spectrum that, in the worst-case scenario, involves a rapid progression
to severe acute respiratory syndrome and even death. Epidemiological
data show that “diabesity”, the association of obesity and diabetes,
is among the main risk factors associated with high morbidity and
mortality. The increased susceptibility to SARS-CoV-2 infection
documented in diabesity argues for initial defects in defense
mechanisms, most likely due to an elevated systemic metabolic
inflammation (“metaflammation”). The NLRP3 inflammasome is a master
regulator of metaflammation and has a pivotal role in the
pathophysiology of diabesity. Here we discuss the most recent findings
suggesting contribution of NLRP3 inflammasome to the increase in
complications in COVID-19 patients with diabesity. We also review
current pharmacological strategies for COVID-19, focusing on treatments
whose efficacy could be due, at least in part, to interference with the
activation of the NLRP3 inflammasome.
Metaflammation in “diabesity” and COVID-19
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a
novel member of human betacoronavirus (mutational ssRNA viruses) causing
the disease named COVID-19 (for coronavirus disease 2019), which is
recognized as a pandemic on March 11, 2020 by the World Health
Organization (WHO). COVID-19 is a syndrome with a wide clinical
spectrum: it can be asymptomatic or, in the majority of cases, cause
mild symptoms that are indistinguishable from other respiratory
infections, but it may also lead to a rapid progression to severe acute
respiratory syndrome (SARS), with respiratory failure and even death
(Zhou et al., 2020).
The activation of signalling receptors of the innate immune system is
the first step of the physiological response to virus infection.
However, this innate immune system activation, when excessive, may evoke
hyperinflammation and tissue damage in patients with severe COVID-19,
making it an important aspect of the pathophysiology of this syndrome
(Netea et al., 2020).
Older people are more likely to suffer from the most serious
complications of SARS-CoV-2 infection: this is likely due to the
physiological decrease in the efficacy of the immune system and the
simultaneous increase in age-related inflammatory response termed
‘inflammaging’ (Latz & Duewell, 2018). Moreover, aging and the modern
lifestyle typical of Western societies are both associated with an
increase in comorbidities, such as “diabesity”, the association of
obesity and diabetes, which defines a combination of primarily metabolic
disorders evoked by impairment of both lipids and sugars metabolisms
(Potenza, Nacci, De Salvia, Sgarra, Collino & Montagnani, 2017; Tschop
& DiMarchi, 2012). The close relationship existing between impaired
metabolism and immunity in diabesity is well documented, and the term
“metaflammation” has been coined to indicate this pathophysiologic
inflammatory response derived by metabolic alterations and dysfunctions
(Hotamisligil, 2017; Mastrocola, Aragno, Alloatti, Collino, Penna &
Pagliaro, 2018). An increasing number of studies report that patients
with severe obesity or type 2 diabetes mellitus (T2DM) exhibit an
increased circulating concentration of proinflammatory cytokines, such
as IL-1β, IL-6 and tumor necrosis factor (TNF)-α (Huang et al., 2020).
The metaflammation, induced in response to excess calories intake in the
adipose tissue, may subsequently involve other organs such as the
skeletal muscle, liver, heart and lung, leading to metabolic and
cardiovascular alterations.
Compared with people of a normal weight, obese individuals have an
increased susceptibility to develop chronic diseases and infections
(Huttunen & Syrjanen, 2010; Milner & Beck, 2012; Wolowczuk et al.,
2008). Patients with T2DM developing COVID-19 are twice as likely to
require ventilation and intensive care unit (ICU) supports and their
mortality is 3-fold higher than in non-diabetic COVID-19 patients (Guan
et al., 2020; Yang et al., 2020). Obesity carries a 5-fold increase in
risk of developing severe pneumonia from COVID-19 (Cai et al., 2020) and
diabesity is a well-recognised risk factor for severe infections
(Almond, Edwards, Barclay & Johnston, 2013; Huttunen & Syrjanen,
2013), sleep apnoea (Dixon & Peters, 2018), poor immune response and
scant outcomes in patients with respiratory disease (Green & Beck,
2017). According to United States data, 37% of 3,615 hospitalized
COVID-19 cases were obese, and the obesity increased the odds of ICU
admission (Lighter et al., 2020). In a French study enrolling COVID-19
patients admitted to the ICU, the requirement for ventilation was
increased by 7-fold in patients with a body mass index (BMI)
> 35 kg/m2, and this was independent of
age and presence of diabetes or hypertension (Simonnet et al., 2020).
Adipocytes express angiotensin-converting enzyme-2 (ACE2), which is the
receptor for SARS-CoV-2 (Yan, Zhang, Li, Xia, Guo & Zhou, 2020) and,
like in human and simian immunodeficiency virus (HIV/SIV) infections,
adipose tissue may act as a reservoir for SARS-CoV-2 (Jia et al., 2020;
Wanjalla et al., 2019). As widely documented (Huttunen & Syrjanen,
2013; Singh, Gupta, Ghosh & Misra, 2020), obese people and individuals
with T2DM are more likely to develop a dysfunctional immune response
that provokes viral infection and makes failed the elimination of the
pathogen. Specific reasons for this are not still understood, even if
one reasonable explanation could be an excessive metaflammation, often
due to an over-activation of specific inflammatory pathways, including,
but not limited to, the NLRP3 inflammasome.
The NLRP3 inflammasome and its role in viral infection
NLRP3 inflammasome is a large, multimeric protein complex assembled
within the cell upon the recognition of unique molecular patterns by a
germline-encoded pattern recognition receptor (PRR) (Yang, Wang,
Kouadir, Song & Shi, 2019). Among the PRRs known to form inflammasomes,
NLRP3 belongs to the family containing both a nucleotide-binding
oligomerization domain (NOD) and leucine-rich repeat (LRR)-containing
proteins. NLRP3 is expressed in many cell types, as innate immunity,
endothelial, hematopoietic, lung epithelial, kidney and cardiac cells
(Ratajczak & Kucia, 2020). We and others have previously demonstrated
that NLRP3 inflammasome overactivation contributes to the pathogenesis
of cardio-metabolic disorders (Mastrocola et al., 2016; O’Riordan et
al., 2019; Wang et al., 2020; Zuurbier et al., 2019), whereas NLRP3
deficiency results in reduced systemic inflammation, along with
decreased immune cell activation and improved insulin resistance (Grant
& Dixit, 2013; Vandanmagsar et al., 2011). NLRP3 inflammasome is also a
crucial player in immune defence of the host against many pathogens,
including viruses (Xu et al., 2020; Zhao & Zhao, 2020). Once activated,
its amino-terminal pyrin domain (PYD) is able to recruit the downstream
adaptor protein apoptosis-associated speck-like protein containing a
caspase-recruitment domain (ASC) and to assemble the scaffold complex
important to recruit the inflammasome effector, pro-caspase-1, which
becomes activated. Active form of caspase-1 clivates both pro-IL-1β and
pro-IL-18 into their biologically active forms (Dinarello, 2009).
Caspase-1 can also activate another substrate, gasdermin D (GSDMD), that
is capable to mediate pyroptosis by creating pore channels in cell
membranes. Pyropoptosis is a lytic form of necrosis used by the innate
immune system to disrupt pathogen replication and intracellular
accumulation, thanks to the formation of pore-induced intracellular
traps (Jorgensen, Zhang, Krantz & Miao, 2016). Despite its role in
facilitating pathogen clearance, inflammasome activation can also be
detrimental to the host by enhancing viral dissemination (Lupfer, Malik
& Kanneganti, 2015). To date, it is still unclear whether SARS-CoV-2
activates the NLRP3 inflammasome (Yap, Moriyama & Iwasaki, 2020), but
this is likely, as SARS-CoV also activates the NLRP3 inflammasome by
modulating either ion channel activity or ASC ubiquitination. Moreover,
after binding to SARS-CoV-2, ACE2 is internalized, leading to high
cytosolic levels of angiotensin II, which is known to act as an
activator of the NLRP3 inflammasome in lung, kidney cells and
cardiomyocytes (Pinar, Scott, Huuskes, Tapia Caceres, Kemp-Harper &
Samuel, 2020; Sun et al., 2017; Zhao et al., 2018). At the same time,
given that caspase-1 affects expression of several innate immunity genes
and viral replication (Bauer et al., 2012), the NLRP3 inflammasome may
alter cellular physiology and homeostasis also modulating gene
expression, leading to altered innate antiviral defense pathways and
impacting therefore on host responsiveness to pathogens like SARS-CoV-2.
As diabesity is a high-risk factor for both influenza infections and
hospitalization for respiratory illness during seasonal influenza
(Karlsson, Sheridan & Beck, 2010; Kwong, Campitelli & Rosella, 2011),
we may speculate that a chronic over-activation of the NLRP3
inflammasome, due to metabolic impairments, may contribute to
alterations in the innate immune response to viral infection, making it
more permissive and severe (Figure 1).
The NLRP3 inflammasome and its involvement in the cytokine
storm
The activation of both innate and adaptive immunity after recognition of
viral antigens produces a large amount of pro-inflammatory cytokines and
chemokines even in moderate cases of COVID-19. In some patients this
activation is exacerbated, leading to a ‘cytokine storm’, which in turn
causes severe lung injury, multiple organ failure and death
(Sarzi-Puttini et al., 2020; Xu et al., 2020). The mechanisms by which
SARS-CoV-2 subverts the body’s innate antiviral cytokine responses are
yet to be elucidated. Nevertheless, most recent research on SARS-CoV
suggests that pyroptosis may have a pivotal role. Pyroptosis is a form
of programmed cell death with an high inflammatory scenario that is
commonly observed with cytopathic viruses (Fink & Cookson, 2005). As
mentioned before, NLRP3 inflammasome regulates pyroptosis by
GSDMD–mediated membrane rupture along with spontaneous release of
cytosolic contents into the extracellular spaces. The activation of
pyroptosis in alveolar macrophages and in recruited monocyte-derived
macrophages by SARS-CoV-2 aggravates pneumonia and ARDS. The release of
alarmins, including viral particles, ATP, reactive oxygen species (ROS)
as well as cytokines, chemokines and lactate dehydrogenase (LDH),
elicits an immediate reaction from surrounding immune cells, inducing a
pyroptotic chain reaction. Different studies have reported elevated
levels of LDH, a cytosolic enzyme that is measured for monitoring
pyroptosis (Rayamajhi, Zhang & Miao, 2013), in patients with the severe
form of the disease and worse outcomes (Lippi, Mattiuzzi, Bovo &
Plebani, 2020). In keeping with this and other observations, we can
suggest the NLRP3 inflammasome-mediated pyroptosis as one of the key
mechanisms involved inCOVID-19 (Figure 1). In accordance, a further
exacerbation of symptoms may derive from the pyroptosis induced release
of viral RNA and antigens that, by reaching the circulation, may
generate immune complex and depositions in other target organs,
initiating severe inflammatory cascades. Hence, in addition to local
damage, cytokine storm also has ripple effects across the body, evoking
organ damage and multi-organ failure (Ruan, Yang, Wang, Jiang & Song,
2020).
NLRP3 inflammasome and resolution of inflammation
Resolution of inflammation is an active process triggered by the onset
of inflammation itself (Perretti & D’Acquisto, 2009). The main actors
of this process consist of specialized proresolving mediators (SPMs),
whose list is constantly updated, including annexin A1 (AnxA1),
formyl-peptide receptor 2 (FPR2) and lipoxin A4(LXA4) (Brancaleone et al., 2013; Dufton et al., 2010;
Norling, Dalli, Flower, Serhan & Perretti, 2012; Serhan, Chiang &
Dalli, 2015). Very recently, AnxA1 has been reported to be involved in
the regulation of NLRP3 inflammasome function (Galvao et al., 2020),
independently from the binding to its cognate receptor FPR2 (Galvao et
al., 2020). Indeed, lower levels of AnxA1 are associated to an increased
activation of NLRP3, thus raising IL-1β releasing (Kelley, Jeltema, Duan
& He, 2019). In addition, AnxA1 is known to trigger resolution pathway
by engaging FPR2 (Brancaleone, Dalli, Bena, Flower, Cirino & Perretti,
2011; Perretti & D’Acquisto, 2009) and (Machado et al., 2020)AnxA1/FPR2
signaling axis has been identified to control and limit viral infection
(Alessi, Cenac, Si-Tahar & Riteau, 2017; Lopategi et al., 2019).
Therefore, impairment of this axis could unbalance the immune response
to infections, thus generating an ineffective response which in turn
could lead to increased cytokine storm and exacerbation of COVID-19
symptoms.
Does NLRP3 inflammasome activation drive the exacerbation of
inflammation and disease pathology in obese/diabetic patients with
COVID-19?
The specific reasons for the higher susceptibility of obese and diabetic
patients to SARS-CoV2 are unclear, but chronic exposure to a low grade
NLRP3 inflammasome-dependent metaflammation may well be a key driver.
Diet-induced alterations in the gut microbiome and related increased gut
leakiness of bacterial wall lipopolysaccharides (endotoxins) are known
to promote organs and tissues NLRP3 inflammasome activation by Toll-like
receptor (TLR) pathways. This event is followed by the accumulation of
the cytokines of the IL-1 family, which are known to modulate the
insulin producing pancreatic 𝛽-cell function (Tack, Stienstra, Joosten
& Netea, 2012). We and others have demonstrated that NLRP3 inflammasome
overactivation is involved not only in the pathogenesis of diabesity,
but also in the exacerbation of related cardiovascular injuries,
including myocardial infarction, by increasing the local inflammatory
response and, at the same time, decreasing the efficiency of endogenous
protective responses (Mastrocola et al., 2016; Xu et al., 2013). NLRP3
inflammasome activation is involved in endothelial lysosome membrane
permeabilization, cathepsin B release, impaired glycocalyx thickness
(Ikonomidis et al., 2019), thus further contributing to the enhanced
susceptibility to cardiovascular injury. Similarly, the
diabesity-related basal activation of the NLRP3 inflammasome cascade,
leading to increase in either gastrointestinal and vascular
permeability, may contribute to exacerbate SARS-CoV-2 systemic diffusion
and enhance the intricate mechanisms of intracellular cross-talk
operational in the pathogenesis of COVID-19.
The NLRP3 inflammasome and pharmacologic treatments for
COVID-19
Till date, there are no specific medications available to treat
COVID-19. Clinical trials are in process on several drugs, mainly based
on the drug repurposing approach to redevelop a compound/drug for the
use in a different disease (COVID-19) other than that of its original
use. This review summarizes recent documentations on clinically approved
drugs, repurposed to counteract COVID-19 infection, whose potential
efficacy can be due, at least in part, to interference with the NLRP3
inflammasome cascade (Figure 2).
We systematically searched the PubMed and Google Scholar databases,
clinical trials.gov, chictr.org.cn/searchprojen.aspx and
https://www.clinicaltrialsregister.eu until June 25th, 2020 to prepare
this section of the narrative review on the role of NLRP3 inflammasome
in drugs repurposed in COVID-19. We also accessed the full text of the
relevant cross references from the search results.