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.