Inflammasomes are newly recognized, vital players in innate immunity. The best characterized is the NLRP3 inflammasome, so-called because the NLRP3 protein in the complex belongs to the family of nucleotide-binding and oligomerization domain-like receptors (NLRs) and is also known as “pyrin domain-containing protein 3”. The NLRP3 inflammasome is associated with onset and progression of various diseases, including metabolic disorders, multiple sclerosis, inflammatory bowel disease, cryopyrin-associated periodic fever syndrome (CAPS), as well as other auto-immune and auto-inflammatory diseases. Several NLRP3 inflammasome inhibitors have been described, some of which show promise in the clinic. The present review will describe the structure and mechanisms of activation of the NLRP3 inflammasome, its association with various auto-immune and auto-inflammatory diseases, and the state of research into NLRP3 inflammasome inhibitors.

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NLRP3 inflammasome and its inhibitors: a review.

Inflammasomes play a crucial role in innate immunity by serving as signaling platforms which deal with a plethora of pathogenic products and cellular products associated with stress and damage. By far, the best studied and most characterized inflammasome is NLRP3 inflammasome, which consists of NLRP3 (nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3), ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and procaspase-1. Activation of NLRP3 inflammasome is mediated by highly diverse stimuli. Upon activation, NLRP3 protein recruits the adapter ASC protein, which recruits the procaspase-1 resulting in its cleavage and activation, inducing the maturation, and secretion of inflammatory cytokines and pyroptosis. However, aberrant activation of the NLRP3 inflammasome is implicated in various diseases including diabetes, atherosclerosis, metabolic syndrome, cardiovascular, and neurodegenerative diseases; raising a tremendous clinical interest in exploring the potential inhibitors of NLRP3 inflammasome. Recent investigations have disclosed various inhibitors of the NLRP3 inflammasome pathway which were validated through in vitro studies and in vivo experiments in animal models of NLRP3-associated disorders. Some of these inhibitors directly target the NLRP3 protein whereas some are aimed at other components and products of the inflammasome. Direct targeting of NLRP3 protein can be a better choice because it can prevent off target immunosuppressive effects, thus restrain tissue destruction. This paper will review the various pharmacological inhibitors of the NLRP3 inflammasome and will also discuss their mechanism of action.

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Pharmacological Inhibitors of the NLRP3 Inflammasome.

One of the most significant challenges of inflammatory bowel disease (IBD) research is to understand how alterations in the symbiotic relationship between the genetic composition of the host and the intestinal microbiota, under impact of specific environmental factors, lead to chronic intestinal inflammation. Genome-wide association studies, followed by functional studies, have identified a role for numerous autophagy genes in IBD, especially in Crohn disease. Studies using in vitro and in vivo models, in addition to human clinical studies have revealed that autophagy is pivotal for intestinal homeostasis maintenance, gut ecology regulation, appropriate intestinal immune responses and anti-microbial protection. This review describes the latest researches on the mechanisms by which dysfunctional autophagy leads to disrupted intestinal epithelial function, gut dysbiosis, defect in anti-microbial peptide secretion by Paneth cells, endoplasmic reticulum stress response and aberrant immune responses to pathogenic bacteria. A better understanding of the role of autophagy in IBD pathogenesis may provide better sub-classification of IBD phenotypes and novel approaches for disease management.Abbreviations: AIEC: adherent-invasive Escherichia coli; AMPK: AMP-activated protein kinase; ATF6: activating transcription factor 6; ATG: autophagy related; Atg16l1[ΔIEC] mice: mice with Atg16l1 depletion specifically in intestinal epithelial cells; Atg16l1[HM] mice: mice hypomorphic for Atg16l1 expression; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; CALCOCO2: calcium binding and coiled-coil domain 2; CASP: caspase; CD: Crohn disease; CGAS: cyclic GMP-AMP synthase; CHUK/IKKA: conserved helix-loop-helix ubiquitous kinase; CLDN2: claudin 2; DAPK1: death associated protein kinase 1; DCs: dendritic cells; DSS: dextran sulfate sodium; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK: eukaryotic translation initiation factor 2 alpha kinase; ER: endoplasmic reticulum; ERBIN: Erbb2 interacting protein; ERN1/IRE1A: ER to nucleus signaling 1; FNBP1L: formin binding protein 1-like; FOXP3: forkhead box P3; GPR65: G-protein coupled receptor 65; GSK3B: glycogen synthase kinase 3 beta; IBD: inflammatory bowel disease; IECs: intestinal epithelial cells; IFN: interferon; IL: interleukin; IL10R: interleukin 10 receptor; IRGM: immunity related GTPase M; ISC: intestinal stem cell; LAMP1: lysosomal-associated membrane protein 1; LAP: LC3-associated phagocytosis; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; LRRK2: leucine-rich repeat kinase 2; MAPK: mitogen-activated protein kinase; MHC: major histocompatibility complex; MIF: macrophage migration inhibitory factor; MIR/miRNA: microRNA; MTMR3: myotubularin related protein 3; MTOR: mechanistic target of rapamycin kinase; MYD88: myeloid differentiation primary response gene 88; NLRP3: NLR family, pyrin domain containing 3; NOD2: nucleotide-binding oligomerization domain containing 2; NPC: Niemann-Pick disease type C; NPC1: NPC intracellular cholesterol transporter 1; OMVs: outer membrane vesicles; OPTN: optineurin; PI3K: phosphoinositide 3-kinase; PRR: pattern-recognition receptor; PTPN2: protein tyrosine phosphatase, non-receptor type 2; PTPN22: protein tyrosine phosphatase, non-receptor type 22 (lymphoid); PYCARD/ASC: PYD and CARD domain containing; RAB2A: RAB2A, member RAS oncogene family; RELA: v-rel reticuloendotheliosis viral oncogene homolog A (avian); RIPK2: receptor (TNFRSF)-interacting serine-threonine kinase 2; ROS: reactive oxygen species; SNPs: single nucleotide polymorphisms; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; Th: T helper 1; TIRAP/TRIF: toll-interleukin 1 receptor (TIR) domain-containing adaptor protein; TLR: toll-like receptor; TMEM173/STING: transmembrane protein 173; TMEM59: transmembrane protein 59; TNF/TNFA: tumor necrosis factor; Treg: regulatory T; TREM1: triggering receptor expressed on myeloid cells 1; UC: ulcerative colitis; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type; XBP1: X-box binding protein 1; XIAP: X-linked inhibitor of apoptosis.

https://tandfonline.com/doi/pdf/10.1080/15548627.2019.1635384

New insights into the interplay between autophagy, gut microbiota and inflammatory responses in IBD.

Alzheimer’s disease (AD) is the most common cause of progressive dementia in the elderly. It is characterized by a progressive and irreversible loss of cognitive abilities and formation of senile plaques, composed mainly of amyloid β (Aβ), and neurofibrillary tangles (NFTs), composed of tau protein, in the hippocampus and cortex of afflicted humans. In brains of AD patients the metabolism of Aβ is dysregulated, which leads to the accumulation and aggregation of Aβ. Metabolism of Aβ and tau proteins is crucially influenced by autophagy. Autophagy is a lysosome-dependent, homeostatic process, in which organelles and proteins are degraded and recycled into energy. Thus, dysfunction of autophagy is suggested to lead to the accretion of noxious proteins in the AD brain. In the present review, we describe the process of autophagy and its importance in AD. Additionally, we discuss mechanisms and genes linking autophagy and AD, i.e., the mTOR pathway, neuroinflammation, endocannabinoid system, ATG7, BCL2, BECN1, CDK5, CLU, CTSD, FOXO1, GFAP, ITPR1, MAPT, PSEN1, SNCA, UBQLN1, and UCHL1. We also present pharmacological agents acting via modulation of autophagy that may show promise in AD therapy. This review updates our knowledge on autophagy mechanisms proposing novel therapeutic targets for the treatment of AD.

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Autophagy and Alzheimer's Disease: From Molecular Mechanisms to Therapeutic Implications.

Serotonin or 5-hydroxytryptamine (5-HT) is a neurotransmitter and hormone that contributes to the regulation of various physiological functions by its actions in the central nervous system (CNS) and in the respective organ systems. Peripheral 5-HT is predominantly produced by enterochromaffin (EC) cells of the gastrointestinal (GI) tract. These gut-resident cells produce much more 5-HT than all neuronal and other sources combined, establishing EC cells as the main source of this biogenic amine in the human body. Peripheral 5-HT is also a potent immune modulator and affects various immune cells through its receptors and via the recently identified process of serotonylation. Alterations in 5-HT signalling have been described in inflammatory conditions of the gut, such as inflammatory bowel disease. The association between 5-HT and inflammation, however, is not limited to the gut, as changes in 5-HT levels have also been reported in patients with allergic airway inflammation and rheumatoid arthritis. Based on searches for terms such as '5-HT', 'EC cell', 'immune cells' and 'inflammation' in pubmed.gov as well as by utilizing pertinent reviews, the current review aims to provide an update on the role of 5-HT in biological functions with a particular focus on immune activation and inflammation.

The role of serotonin and its receptors in activation of immune responses and inflammation.

SummaryAims
Activation of cannabinoid receptor 2 (CB2R) has been reported to ameliorate the pathogenesis of experimental autoimmune encephalomyelitis (EAE). In this study, we examined whether autophagy is involved in the beneficial effect of CB2R on EAE and explored the mechanism with a focus on inflammasome activation.

Methods
EAE severity was analyzed with clinical score and histological score stained by hematoxylin and eosin or luxol fast blue in spinal cord. Immunoblot analysis was conducted to detect proteins of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-related caspase-1 (Casp-1) and the maturation of interleukin (IL)-1β as well as autophagy-related light chain 3 (LC3), and Beciln 1 both in vivo and in vitro. Reverse transcription and real-time PCR were used to detect mRNA of NLRP3, IL-1β and Casp-1. Autophagy-related gene 5 (ATG5)-specific siRNA was transiently transfected in BV2 microglia, and immunofluorescence staining was carried out to detect the expression of NLRP3, caspase recruitment domain (ASC), and pro-caspase-1.

Results
The current data indicated that deleting CB2R decreased the expression of LC3-II/LC3-I ratio, Beclin 1 and increased caspase-1 activation and IL-1β production in the spinal cord of EAE mice, whereas activation of CB2R with a specific agonist HU-308 induced inverse effects. Further study indicated that HU-308 could promote autophagy and inhibit expression and activation of NLRP3 inflammasome in BV2 microglia. Blocking autophagy by ATG5-specific siRNA dismissed the effort of CB2R in mediating NLRP3 inflammasome in vitro.

Conclusion
Collectively, our results demonstrated for the first time that CB2R plays a protective role in EAE through promoting autophagy and inhibiting NLRP3 inflammasome activation.

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Activating cannabinoid receptor 2 alleviates pathogenesis of experimental autoimmune encephalomyelitis via activation of autophagy and inhibiting NLRP3 inflammasome.

Activation of cannabinoid receptor 2 (CB2R) ameliorates inflammation, but the underlying mechanism remains unclear. In the present study, we examined whether activation of CB2R could suppress the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome. In peritoneal macrophages isolated from C57BL/6 mice, LPS/DSS challenge for 24 h increased the expression of the components of NLRP3 inflammasome NLRP3, Casp-1 p20/Casp-1 p45 ratio, proIL-1β and IL-1β and also enhanced autophagy (LC3-II/LC3-I ratio, Beclin-1 and SQSTM1). Pretreatment of peritoneal macrophages with HU 308, a selective CB2R agonist, attenuated LPS/DSS-induced NLRP3 inflammasome activation, but further enhanced autophagy. In comparison with wild-type (WT) control, peritoneal macrophages from CB2R knockout (KO) mice had more robust NLRP3 inflammasome activation and attenuated autophagy upon LPS/DSS challenge. Knockdown autophagy-related gene 5 (Atg5) with a siRNA in peritoneal macrophages attenuated the inhibitory effects of HU 308 on LPS/DSS-induced NLRP3 inflammasome activation in vitro. In vivo, HU308 treatment attenuated DSS-induced colitis mice associated with reduced colon inflammation and inhibited NLRP3 inflammasome activation in wild-type mice. In CB2R KO mice, DSS-induced inflammation and NLRP3 inflammasome activation were more pronounced than those in WT control. Finally, we demonstrated that AMPK-mTOR-P70S6K signaling pathway was involved in this CB2R-mediated process. We conclude that activation of CB2R ameliorates DSS-induced colitis through enhancing autophagy that may inhibit NLRP3 inflammasome activation in macrophages.

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Activation of Cannabinoid Receptor 2 Ameliorates DSS-Induced Colitis through Inhibiting NLRP3 Inflammasome in Macrophages

Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been reported to alleviate neuroinflammation Here we aimed to determine the role of autophagy in α7nAChR-mediated inhibition of neuroinflammation and its underlying mechanism Experimental autoimmune encephalomyelitis (EAE) mice and lipopolysaccharide (LPS)-stimulated BV2 microglia were used as in vivo and in vitro models of neuroinflammation, respectively The severity of EAE was evaluated with neurological scoring Autophagy-related proteins (Beclin 1, LC3-II/I, p62/SQSTM1) were detected by immunoblot Autophagosomes were observed using transmission electron microscopy and tandem ﬂuorescent mRFP-GFP-LC3 plasmid was applied to test autophagy flux The mRNA levels of interleukin-6 (IL-6), IL-1β, IL-18 and tumor necrosis factor-α (TNF-α) were detected by real-time PCR We used 3-methyladenine (3-MA) and autophagy-related gene 5 small interfering RNA (Atg5 siRNA) to block autophagy in vivo and in vitro, respectively Activating α7nAChR with PNU282987 ameliorates EAE severity and spinal inflammatory infiltration in EAE mice PNU282987 treatment also enhanced monocyte/microglia autophagy (Beclin 1, LC3-II/I ratio, p62/SQSTM1, colocalization of CD45 or CD68 positive cells with LC3) both in spinal cord and spleen from EAE mice The beneficial effects of PNU282987 on EAE mice were partly abolished by 3-MA, an autophagy inhibitor In vitro, PNU282987 treatment increased autophagy and promoted autophagy flux Blockade of autophagy by Atg5 siRNA or bafilomycin A1 attenuated the inhibitory effect of PNU282987 on IL-6, IL-1β, IL-18 and TNF-α mRNA Our results demonstrate for the first time that activating α7nAChR enhances monocyte/microglia autophagy, which suppresses neuroinflammation and thus plays an alleviative role in EAE

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Autophagy Plays an Important Role in Anti-inflammatory Mechanisms Stimulated by Alpha7 Nicotinic Acetylcholine Receptor.

Aims To evaluate whether activating α7 nicotinic acetylcholine receptor (α7nAChR) could inhibit the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome through regulation of β-arrestin-1 in monocyte/macrophage system, thus contributing to the control of neuroinflammation. Methods The protein levels of NLRP3, caspase-1 (Casp-1) p20 and proCasp-1, interleukin-1β (IL-1β) p17 and proIL-1β, IL-18 and proIL-18 were measured using Western blotting. The mRNA levels of Casp-1 and IL-1β were detected by real-time PCR (RT-PCR). The colocalization and interaction of NLRP3 protein and β-arrestin-1 were measured by immunofluorescence staining and immunoprecipitation. Results The expression of β-arrestin-1 was significantly increased and colocalized with CD45-positive cells in spinal cord of experimental auto-immune encephalomyelitis (EAE) mice when compared with the sham mice, which was attenuated by pretreatment with PNU282987, a specific α7nAChR agonist. PNU282987 also significantly inhibited the activation of NLRP3 inflammasome and thus decreased the production of IL-1β and IL-18 both in lipopolysaccharide (LPS)/ATP-stimulated BV2 microglia in vitro and spinal cord from EAE mice in vivo, while inverse effects were observed in α7nAChR knockout mice. Furthermore, overexpression of β-arrestin-1 attenuated the inhibitory effect of PNU282987 on NLRP3 inflammasome activation in LPS/ATP-stimulated BV2 microglia. PNU282987 inhibited the interaction between β-arrestin-1 and NLRP3 protein in vitro. Conclusions The present study demonstrates that activating α7nAChR can lead to NLRP3 inflammasome inhibition via regulation of β-arrestin-1 in monocyte/microglia system.

Activating α7 nicotinic acetylcholine receptor inhibits NLRP3 inflammasome through regulation of β-arrestin-1.

The protective action of ketanserin against lipopolysaccharide-induced shock in mice is mediated by inhibiting inducible NO synthase expression via the MEK/ERK pathway.

Wei Wei

Papers

Activating cannabinoid receptor 2 alleviates pathogenesis of experimental autoimmune encephalomyelitis via activation of autophagy and inhibiting NLRP3 inflammasome.

Activation of Cannabinoid Receptor 2 Ameliorates DSS-Induced Colitis through Inhibiting NLRP3 Inflammasome in Macrophages

Autophagy Plays an Important Role in Anti-inflammatory Mechanisms Stimulated by Alpha7 Nicotinic Acetylcholine Receptor.

Activating α7 nicotinic acetylcholine receptor inhibits NLRP3 inflammasome through regulation of β-arrestin-1.

The protective action of ketanserin against lipopolysaccharide-induced shock in mice is mediated by inhibiting inducible NO synthase expression via the MEK/ERK pathway.