Exploring the Use of Medicinal Plants and Their Bioactive Derivatives as Alveolar NLRP3 Inflammasome Regulators during Mycobacterium tuberculosis Infection

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a successful intracellular pathogen that is responsible for the highest mortality rate among diseases caused by bacterial infections. During early interaction with the host innate cells, M. tuberculosis cell surface antigens interact with Toll like receptor 4 (TLR4) to activate the nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) canonical, and non-canonical inflammasome pathways. NLRP3 inflammasome activation in the alveoli has been reported to contribute to the early inflammatory response that is needed for an effective anti-TB response through production of pro-inflammatory cytokines, including those of the Interleukin 1 (IL1) family. However, overstimulation of the alveolar NLRP3 inflammasomes can induce excessive inflammation that is pathological to the host. Several studies have explored the use of medicinal plants and/or their active derivatives to inhibit excessive stimulation of the inflammasomes and its associated factors, thus reducing immunopathological response in the host. This review describes the molecular mechanism of the NLRP3 inflammasome activation in the alveoli during M. tuberculosis infection. Furthermore, the mechanisms of inflammasome inhibition using medicinal plant and their derivatives will also be explored, thus offering a novel perspective on the alternative control strategies of M. tuberculosis-induced immunopathology.     

Research Progress of Mitochondrial Mechanism in NLRP3 Inflammasome Activation and Exercise Regulation of NLRP3 Inflammasome

NLRP3 is an important pattern recognition receptor in the innate immune system, and its activation induces a large number of pro-inflammatory cytokines, IL-1β and IL-18 which are involved in the development of various diseases. In recent years, it has been suggested that mitochondria are the platform for NLRP3 inflammasome activation. Additionally, exercise is considered as an important intervention strategy to mediate the innate immune responses. Generally, chronic moderate-intensity endurance training, resistance training and high-intensity interval training inhibit NLRP3 inflammasome activation in response to various pathological factors. In contrast, acute exercise activates NLRP3 inflammasome. However, the mechanisms by which exercise regulates NLRP3 inflammasome activation are largely unclear. Therefore, the mechanism of NLRP3 inflammasome activation is discussed mainly from the perspective of mitochondria in this review. Moreover, the effect and potential mechanism of exercise on NLRP3 inflammasome are explored, hoping to provide new target for relevant research.     

NLRP3 Ubiquitination—A New Approach to Target NLRP3 Inflammasome Activation

In response to diverse pathogenic and danger signals, the cytosolic activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing (3)) inflammasome complex is a critical event in the maturation and release of some inflammatory cytokines in the state of an inflammatory response. After activation of the NLRP3 inflammasome, a series of cellular events occurs, including caspase 1-mediated proteolytic cleavage and maturation of the IL-1β and IL-18, followed by pyroptotic cell death. Therefore, the NLRP3 inflammasome has become a prime target for the resolution of many inflammatory disorders. Since NLRP3 inflammasome activation can be triggered by a wide range of stimuli and the activation process occurs in a complex, it is difficult to target the NLRP3 inflammasome. During the activation process, various post-translational modifications (PTM) of the NLRP3 protein are required to form a complex with other components. The regulation of ubiquitination and deubiquitination of NLRP3 has emerged as a potential therapeutic target for NLRP3 inflammasome-associated inflammatory disorders. In this review, we discuss the ubiquitination and deubiquitination system for NLRP3 inflammasome activation and the inhibitors that can be used as potential therapeutic agents to modulate the activation of the NLRP3 inflammasome.     

Anti-Inflammatory Effect ofEmodin via Attenuation of NLRP3 Inflammasome Activation

Emodin, an active constituent of oriental herbs, is widely used to treat allergy, inflammation, and other symptoms. This study provides the scientific basis for the anti-inflammasome effects of emodin on both in vitro and in vivo experimental models. Bone marrow-derived macrophages were used to study the effects of emodin on inflammasome activation by using inflammasome inducers such as ATP, nigericin, and silica crystals. The lipopolysaccharide (LPS)-induced endotoxin shock model was employed to study the effect of emodin on in vivo efficacy. Emodin treatment attenuated interleukin (IL)-1β secretion via the inhibition of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation induced by ATP, nigericin, and silica crystals. Further, emodin ameliorated the severity of NLRP3 inflammasome-mediated symptoms in LPS-induced endotoxin mouse models. This study is the first to reveal mechanism-based evidence, especially with respect to regulation of inflammasome activation, substantiating traditional claims of emodin in the treatment of inflammation-related disorders.     

Platelet Inhibition Prevents NLRP3 Inflammasome Activation and Sepsis-Induced Kidney Injury

Platelets, cellular mediators of thrombosis, are activated during sepsis and are increasingly recognized as mediators of the immune response. Platelet activation is significantly increased in sepsis patients compared to ICU control patients. Despite this correlation, the role of activated platelets in contributing to sepsis pathophysiology remains unclear. We previously demonstrated NOD-like receptor protein 3 inflammasome (NLRP3) inflammasome activation in sepsis-induced platelets from cecal-ligation puncture (CLP) rats. Activated platelets were associated with increased pulmonary edema and glomerular injury in CLP vs. SHAM controls. In this study, we investigated whether inhibition of platelet activation would attenuate NLRP3 activation and renal and pulmonary injury in response to CLP. CLP was performed in male and female Sprague Dawley (SD) rats (n = 10/group) to induce abdominal sepsis and SHAM rats served as controls. A subset of CLP animals was treated with Clopidogrel (10 mg/kg/day, CLP + CLOP) to inhibit platelet activation. At 72 h post-CLP, platelet activation and NLRP3 inflammasome assembly were evaluated, IL-1β and IL-18 were measured in plasma, and tissues, renal and pulmonary pathology, and renal function were assessed. Activated platelets were 7.8 ± 3.6% in Sham, 22 ± 6% in CLP and significantly decreased to 14.5 ± 0.6% in CLP + CLOP (n = 8–10/group, p < 0.05). NLRP3 inflammasome assembly was inhibited in platelets of CLP + CLOP animals vs. CLP. Significant increases in plasma and kidney IL-1β and IL-18 in response to CLP were decreased with Clopidogrel treatment. Renal injury, but not lung histology or renal function was improved in CLP + CLOP vs. CLP. These data provide evidence that activated platelets may contribute to sepsis-induced renal injury, possibly via NLRP3 activation in platelets. Platelets may be a therapeutic target to decrease renal injury in septic patients.     

Exogenous Carbon Monoxide Decreases Sepsis-Induced Acute Kidney Injury and Inhibits NLRP3 Inflammasome Activation in Rats

Carbon monoxide (CO) has shown various physiological effects including anti-inflammatory activity in several diseases, whereas the therapeutic efficacy of CO on sepsis-induced acute kidney injury (AKI) has not been reported as of yet. The purpose of the present study was to explore the effects of exogenous CO on sepsis-induced AKI and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation in rats. Male rats were subjected to cecal ligation and puncture (CLP) to induce sepsis and AKI. Exogenous CO delivered from CO-releasing molecule 2 (CORM-2) was used intraperitoneally as intervention after CLP surgery. Therapeutic effects of CORM-2 on sepsis-induced AKI were assessed by measuring serum creatinine (Scr) and blood urea nitrogen (BUN), kidney histology scores, apoptotic cell scores, oxidative stress, levels of cytokines TNF-α and IL-1β, and NLRP3 inflammasome expression. CORM-2 treatment protected against the sepsis-induced AKI as evidenced by reducing serum Scr/BUN levels, apoptotic cells scores, increasing survival rates, and decreasing renal histology scores. Furthermore, treatment with CORM-2 significantly reduced TNF-α and IL-1β levels and oxidative stress. Moreover, CORM-2 treatment significantly decreased NLRP3 inflammasome protein expressions. Our study provided evidence that CORM-2 treatment protected against sepsis-induced AKI and inhibited NLRP3 inflammasome activation, and suggested that CORM-2 could be a potential therapeutic candidate for treating sepsis-induced AKI.     

Mitochondrial Modulations,Autophagy Pathways Shifts in Viral Infections: Consequences of COVID-19

Mitochondria are vital intracellular organelles that play an important role in regulating various intracellular events such as metabolism, bioenergetics, cell death (apoptosis), and innate immune signaling. Mitochondrial fission, fusion, and membrane potential play a central role in maintaining mitochondrial dynamics and the overall shape of mitochondria. Viruses change the dynamics of the mitochondria by altering the mitochondrial processes/functions, such as autophagy, mitophagy, and enzymes involved in metabolism. In addition, viruses decrease the supply of energy to the mitochondria in the form of ATP, causing viruses to create cellular stress by generating ROS in mitochondria to instigate viral proliferation, a process which causes both intra- and extra-mitochondrial damage. SARS-COV2 propagates through altering or changing various pathways, such as autophagy, UPR stress, MPTP and NLRP3 inflammasome. Thus, these pathways act as potential targets for viruses to facilitate their proliferation. Autophagy plays an essential role in SARS-COV2-mediated COVID-19 and modulates autophagy by using various drugs that act on potential targets of the virus to inhibit and treat viral infection. Modulated autophagy inhibits coronavirus replication; thus, it becomes a promising target for anti-coronaviral therapy. This review gives immense knowledge about the infections, mitochondrial modulations, and therapeutic targets of viruses.     

NLRP3 and Infections: β-Amyloid in Inflammasome beyond Neurodegeneration

Amyloid beta (Aβ)-induced abnormal neuroinflammation is recognized as a major pathological feature of Alzheimer’s disease (AD), which results in memory impairment. Research exploring low-grade systemic inflammation and its impact on the development and progression of neurodegenerative disease has increased. A particular research focus has been whether systemic inflammation arises only as a secondary effect of disease, or it is also a cause of pathology. The inflammasomes, and more specifically the NLRP3 inflammasome, are crucial components of the innate immune system and are usually activated in response to infection or tissue damage. Although inflammasome activation plays critical roles against various pathogens in host defense, overactivation of inflammasome contributes to the pathogenesis of inflammatory diseases, including acute central nervous system (CNS) injuries and chronic neurodegenerative diseases, such as AD. This review summarizes the current literature on the role of the NLRP3 inflammasome in the pathogenesis of AD, and its involvement in infections, particularly SARS-CoV-2. NLRP3 might represent the crossroad between the hypothesized neurodegeneration and the primary COVID-19 infection.     

Leptin Induces Apoptotic and Pyroptotic Cell Death via NLRP3 Inflammasome Activation in Rat Hepatocytes

Leptin, a hormone that is predominantly produced by adipose tissue, is closely associated with various liver diseases. However, there is a lack of understanding as to whether leptin directly induces cytotoxic effects in hepatocytes as well as the mechanisms that are involved. Inflammasomes, which are critical components in the innate immune system, have been recently shown to modulate cell death. In this study, we examined the effect of leptin on the viability of rat hepatocytes and the underlying mechanisms, with a particular focus on the role of inflammasomes activation. Leptin treatment induced cytotoxicity in rat hepatocytes, as determined by decreased cell viability, increased caspase-3 activity, and the enhanced release of lactate dehydrogenase. NLRP3 inflammasomes were activated by leptin both in vitro and in vivo, as determined by the maturation of interleukin-1β and caspase-1, and the increased expression of inflammasome components, including NLRP3 and ASC. Mechanistically, leptin-induced inflammasome activation is mediated via the axis of ROS production, ER stress, and autophagy. Notably, the inhibition of inflammasomes by treatment with the NLRP3 inhibitor or the IL-1 receptor antagonist protected the hepatocytes from leptin-induced cell death. Together, these results indicate that leptin exerts cytotoxic effects in hepatocytes, at least in part, via the activation of NLRP3 inflammasomes.     

Dieckol Attenuated Glucocorticoid-Induced Muscle Atrophy by Decreasing NLRP3 Inflammasome and Pyroptosis

Dexamethasone (Dexa), frequently used as an anti-inflammatory agent, paradoxically leads to muscle inflammation and muscle atrophy. Receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) lead to nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome formation through nuclear factor-κB (NF-κB) upregulation. NLRP3 inflammasome results in pyroptosis and is associated with the Murf-1 and atrogin-1 upregulation involved in protein degradation and muscle atrophy. The effects of Ecklonia cava extract (ECE) and dieckol (DK) on attenuating Dexa-induced muscle atrophy were evaluated by decreasing NLRP3 inflammasome formation in the muscles of Dexa-treated animals. The binding of AGE or high mobility group protein 1 to RAGE or TLR4 was increased by Dexa but significantly decreased by ECE or DK. The downstream signaling pathways of RAGE (c-Jun N-terminal kinase or p38) were increased by Dexa but decreased by ECE or DK. NF-κB, downstream of RAGE or TLR4, was increased by Dexa but decreased by ECE or DK. The NLRP3 inflammasome component (NLRP3 and apoptosis-associated speck-like), cleaved caspase -1, and cleaved gasdermin D, markers of pyroptosis, were increased by Dexa but decreased by ECE and DK. Interleukin-1β/Murf-1/atrogin-1 expression was increased by Dexa but restored by ECE or DK. The mean muscle fiber cross-sectional area and grip strength were decreased by Dexa but restored by ECE or DK. In conclusion, ECE or DK attenuated Dexa-induced muscle atrophy by decreasing NLRP3 inflammasome formation and pyroptosis.     

Implication of the NLRP3 Inflammasome in Bovine Age-Related Sarcopenia

Sarcopenia is defined as the age-related loss of skeletal muscle mass, quality, and strength. The pathophysiological mechanisms underlying sarcopenia are still not completely understood. The aim of this work was to evaluate, for the first time, the expression of NLRP3 inflammasome in bovine skeletal muscle in order to investigate the hypothesis that inflammasome activation may trigger and sustain a pro-inflammatory environment leading to sarcopenia. Samples of skeletal muscle were collected from 60 cattle belonging to three age-based groups. Morphologic, immunohistochemical and molecular analysis were performed to assess the presence of age-related pathologic changes and chronic inflammation, the expression of NLRP3 inflammasome and to determine the levels of interleukin-1β, interleukin-18 and tumor necrosis factor alpha in muscle tissue. Our results revealed the presence of morphologic sarcopenia hallmark, chronic lymphocytic inflammation and a type II fibers-selective NLRP3 expression associated to a significant decreased number of immunolabeled-fibers in aged animals. Moreover, we found a statistically significant age-related increase of pro-inflammatory cytokines such as interleukin-1β and interleukin-18 suggesting the activation of NLRP3 inflammasome. Taken together, our data suggest that NLRP3 inflammasome components may be normally expressed in skeletal muscle, but its priming and activation during aging may contribute to enhance a pro-inflammatory environment altering normal muscular anabolism and metabolism.     

Toxoplasma gondii GRA9 Regulates the Activation of NLRP3 Inflammasome to Exert Anti-Septic Effects in Mice

Dense granule proteins (GRAs) are essential components in Toxoplasma gondii, which are suggested to be promising serodiagnostic markers in toxoplasmosis. In this study, we investigated the function of GRA9 in host response and the associated regulatory mechanism, which were unknown. We found that GRA9 interacts with NLR family pyrin domain containing 3 (NLRP3) involved in inflammation by forming the NLRP3 inflammasome. The C-terminal of GRA9 (GRA9C) is essential for GRA9–NLRP3 interaction by disrupting the NLRP3 inflammasome through blocking the binding of apoptotic speck-containing (ASC)-NLRP3. Notably, Q200 of GRA9C is essential for the interaction of NLRP3 and blocking the conjugation of ASC. Recombinant GRA9C (rGRA9C) showed an anti-inflammatory effect and the elimination of bacteria by converting M1 to M2 macrophages. In vivo, rGRA9C increased the anti-inflammatory and bactericidal effects and subsequent anti-septic activity in CLP- and E. coli- or P. aeruginosa-induced sepsis model mice by increasing M2 polarization. Taken together, our findings defined a role of T. gondii GRA9 associated with NLRP3 in host macrophages, suggesting its potential as a new candidate therapeutic agent for sepsis.     

Characterizing the NLRP3 Inflammasome in Mood Disorders: Overview,Technical Development,and Measures of Peripheral Activation in Adolescent Patients

The NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome is a node of intracellular stress pathways and a druggable target which integrates mitochondrial stress and inflammatory cascades. While a body of evidence suggests the involvement of the NLRP3 inflammasome in numerous diseases, a lack of reliable measurement techniques highlights the need for a robust assay using small quantities of biological samples. We present a literature overview on peripheral activation of the NLRP3 inflammasome in mood disorders, then outline a process to develop and validate a robust assay to measure baseline and activated intracellular levels of “apoptosis-associated speck-like protein containing a CARD” (ASC) as a key component of an inflammatory profile in peripheral blood mononuclear cells (PBMC). A consistent association between high NLRP3 mRNA levels and relevant cytokines was seen in the literature. Using our method to measure ASC, stimulation of PBMC with lipopolysaccharide and nigericin or adenosine triphosphate resulted in microscopic identification of intracellular ASC specks, as well as interleukin 1 (IL-1) beta and caspase-1 p10 in the periphery. This was abolished by dose-dependent pre-treatment with 100 nM MCC950. We also report the use of this technique in a small pilot sample from patients with bipolar disorder and depressive disorders. The results show that levels of intracellular ASC and IL-1 beta are sensitive to change upon activation and maintained over time, which may be used to improve the detection of NLRP3 activation and guide personalized therapeutic strategy in the treatment of patients.     

Activation of the NLRP3 Inflammasome Increases the IL-1β Level and Decreases GLUT4 Translocation in Skeletal Muscle during Insulin Resistance

Low-grade chronic inflammation plays a pivotal role in the pathogenesis of insulin resistance (IR), and skeletal muscle has a central role in this condition. NLRP3 inflammasome activation pathways promote low-grade chronic inflammation in several tissues. However, a direct link between IR and NLRP3 inflammasome activation in skeletal muscle has not been reported. Here, we evaluated the NLRP3 inflammasome components and their role in GLUT4 translocation impairment in skeletal muscle during IR. Male C57BL/6J mice were fed with a normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. The protein levels of NLRP3, ASC, caspase-1, gasdermin-D (GSDMD), and interleukin (IL)-1β were measured in both homogenized and isolated fibers from the flexor digitorum brevis (FDB) or soleus muscle. GLUT4 translocation was determined through GLUT4myc-eGFP electroporation of the FBD muscle. Our results, obtained using immunofluorescence, showed that adult skeletal muscle expresses the inflammasome components. In the FDB and soleus muscles, homogenates from HFD-fed mice, we found increased protein levels of NLRP3 and ASC, higher activation of caspase-1, and elevated IL-1β in its mature form, compared to NCD-fed mice. Moreover, GSDMD, a protein that mediates IL-1β secretion, was found to be increased in HFD-fed-mice muscles. Interestingly, MCC950, a specific pharmacological NLRP3 inflammasome inhibitor, promoted GLUT4 translocation in fibers isolated from the FDB muscle of NCD- and HFD-fed mice. In conclusion, we found increased NLRP3 inflammasome components in adult skeletal muscle of obese insulin-resistant animals, which might contribute to the low-grade chronic metabolic inflammation of skeletal muscle and IR development.     

NLRP3 Inflammasome Sequential Changes in Staphylococcus aureus-Induced Mouse Model of Acute Rhinosinusitis

The NLR pyrin domain containing 3 (NLRP3) inflammasome plays a crucial role in lung disease and may have a similar role in upper respiratory tract inflammation. We therefore constructed a C57BL/6 mouse model of acute rhinosinusitis induced by Staphylococcus aureus and investigated the role of the NLRP3 inflammasome in this model. Mice were classified as non-inoculated group (group A) and inoculated groups (groups B, C, D and E, sacrificed 1, 3, 7 and 14 days after inoculation, respectively). Hematoxylin-eosin staining showed that each group had inflammatory cell infiltration, except group A. The damage of the nasal mucosa was aggravated gradually over time. Western blot and immunofluorescence showed that the structural proteins of the NLRP3 inflammasome (NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), procaspase-1) in groups B, C, D and E were increased gradually. But they were reduced in group B compared with group A, except for NLRP3. Western blot showed that the cleavage fragment of procaspase-1, p20 in groups B, C, D and E was increased gradually. Real-time PCR showed that the corresponding mRNAs of the structural proteins were changed the same as their proteins. IL-1β mRNA and mature IL-1β protein were increased gradually in groups A, B, C, D and E. These results indicate that NLRP3 inflammasome activation was associated with the acute rhinosinusitis, and that there was a positive correlation between the expression level of the NLRP3 inflammasome and the severity of acute rhinosinusitis.     

Tonabersat Inhibits Connexin43 Hemichannel Opening and Inflammasome Activation in an In Vitro Retinal Epithelial Cell Model of Diabetic Retinopathy

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1β and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1β, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.