Subarachnoid hemorrhage (SAH) is a devastating condition with high morbidity and mortality rates due to the lack of effective therapy. Nucleotide‐binding oligomerization domain‐like receptor family ...pyrin domain‐containing 3 (NLRP3) inflammasome activation associated with the upregulation of apoptotic signaling pathway has been implicated in various inflammatory diseases including hemorrhagic insults. Melatonin is reported to possess substantial anti‐inflammatory properties, which is beneficial for early brain injury (EBI) after SAH. However, the molecular mechanisms have not been clearly identified. This study was designed to investigate the protective effects of melatonin against EBI induced by SAH and to elucidate the potential mechanisms. The adult mice were subjected to SAH. Melatonin or vehicle was injected intraperitoneally 2 hr after SAH. Melatonin was neuroprotective, as shown by increased survival rate, as well as elevated neurological score, greater survival of neurons, preserved brain glutathione levels, and reduced brain edema, malondialdehyde concentrations, apoptotic ratio, and blood–brain barrier (BBB) disruption. Melatonin also attenuated the expressions of NLRP3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), cleaved caspase‐1, interleukin‐1β (IL‐1β), and interleukin‐6 (IL‐6); these changes were also associated with an increase in the anti‐apoptotic factor (Bcl2) and reduction in the pro‐apoptotic factor (Bim). In summary, our results demonstrate that melatonin treatment attenuates the EBI following SAH by inhibiting NLRP3 inflammasome‐associated apoptosis.
NOD1 {nucleotide-binding oligomerization domain 1; NLRC NOD-LRR (leucine-rich repeat) family with CARD (caspase recruitment domain) 1} and NOD2 (NLRC2) are among the most prominent members of the NLR ...(NOD-LRR) family -proteins that contain nucleotide-binding NACHT domains and receptor-like LRR domains. With over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules elaborated by tissue injury. NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG (peptidoglycan) constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB (nuclear factor-κB), stress kinases, IRFs (interferon regulatory factors) and autophagy. Hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been associated with an increasing number of chronic inflammatory diseases. In fact, potential roles for NOD1 and NOD2 in inflammatory disorders have been revealed by investigations using a series of animal models. In the present review, we describe recent experimental findings associating NOD1 and NOD2 with various autoimmune and chronic inflammatory disorders, and we discuss prospects for development of novel therapeutics targeting these NLR family proteins.
Neuroinflammation is closely linked to the pathogenesis of Alzheimer's disease (AD). Glucagon-like peptide-1 (GLP-1) analogs exhibit anti-inflammatory and neuroprotective effects; hence, we ...investigated whether they reduce cognitive impairment and protect astrocytes from oxidative stress. We found that 5 × FAD transgenic mice treated with the synthetic GLP-1 receptor agonist exenatide had improved cognitive function per the Morris water maze test. Immunohistochemistry, western blotting, and ELISAs used to detect inflammatory factors revealed reduced neuroinflammation in extracted piriform cortexes of exenatide-treated mice as well as lower amyloid β1-42-induced oxidative stress and inflammation in astrocytes treated with exendin-4 (the natural analog of exenatide). Adenovirus-mediated overexpression of nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 2 (NLRP2) revealed that exenatide/exendin-4 function may be attributed to NLRP2 inflammasome inhibition. Collectively, our results indicate that GLP-1 analogs improve cognitive dysfunction in vivo and protect astrocytes in vitro, potentially via the downregulation of the NLRP2 inflammasome.
•Exenatide reduces amyloid deposition and improves cognition in 5 × FAD mice.•Exenatide reduces astrocyte activation and NLRP2 levels in the piriform cortex.•Exenatide alleviates inflammatory responses in the piriform cortex of 5 × FAD mice.•Exendin-4 improves survival and reduces oxidative stress in astrocytes in vitro.•Exendin-4 mitigates the effects of Aβ- and NLRP2-overexpression in astrocytes.
Aim
To determine whether Fusobacterium nucleatum's ability to invade cells allows the bacteria to activate pro‐inflammatory response through cytosolic pattern recognition receptors, independent of ...surface Toll‐like receptors (TLRs).
Methodology
HEK293T cells, which lack endogenous TLRs, and overexpressing dominant negative myeloid differentiation primary response gene 88 (MyD88DN) protein, were infected with F. nucleatum and the production of interleukin‐8 (IL‐8) was determined. The necessity for intracellular invasion of the bacteria for cytokine production was also investigated by blocking bacterial invasion with cytochalasin D. The roles of NFĸB and p38 mitogen‐activated protein kinase (MAPK) and nucleotide‐binding oligomerization domain‐1 (NOD‐1) signalling pathways in F. nucleatum‐induced IL‐8 secretion were determined.
Results
Fusobacterium nucleatum‐infected HEK293T cells produced IL‐8 independent of the MYD88 signalling. This response was inhibited by preventing F. nucleatum invasion into HEK293T cells. p38 MAPK but not the NFĸB signalling pathway was required for F. nucleatum‐mediated IL‐8 production. HEK293T cells expressed NOD‐1 but not NOD‐2. Yet, inhibition of NOD‐1 signalling did not affect F. nucleatum‐induced IL‐8 secretion.
Conclusions
Fusobacterium nucleatum invasion led to cytokine production, which is mediated by the p38 MAPK signalling but independent of TLRs, NOD‐1, NOD‐2 and NFĸB signalling.
Neutrophils make up an essential part of the innate immune system, and are involved both in the initial responses to pathogens, and in orchestrating later immune responses. Neutrophils recognize ...pathogens through pattern-recognition receptors (PRRs), which are activated by microbial motifs. The Nod-like receptors (nucleotide-binding domain leucine-rich repeat containing family; NLRs) constitute a recently discovered group of PRRs whose role in the neutrophil immune responses is not yet characterized. The present study aimed to investigate the expression and function of NLRs in neutrophils. Neutrophils were isolated from human peripheral blood, and the presence of nucleotide-binding oligomerization domain 1 (NOD1), NOD2 and NACHT-LRR-PYD-containing protein 3 (NLRP3) was evaluated with flow cytometry and immunohistochemistry. The expression of NOD1, NOD2 and NLRP3 messenger RNA was determined using real-time reverse transcription-polymerase chain reaction. Changes in neutrophil cytokine secretion, phenotype and migration following agonist-induced activation were studied using enzyme-linked immunosorbent assay, flow cytometry and a chemotaxis assay, respectively. No expression of NOD1 was found in isolated neutrophils and stimulation with the NOD1 ligand γ- d-glutamyl-meso-diaminopimelic acid induced no signs of activity. In contrast, a marked expression of NOD2 and NLRP3 was found. NOD2 activation with MurNAc- l-Ala- d-isoGln (MDP) resulted in interleukin-8 secretion, CD62 ligand down-regulation, CD11b up-regulation and increased migration towards an inflammatory stimulus. NLRP3 activation with alum caused interleukin-1β secretion and facilitated migration. Altogether, this suggests that NLRs may be a previously unknown pathway for neutrophil activation.
Background
Monosodium urate (MSU) crystals‐induced inflammation is a key initiator in gouty arthritis. Curcumin is an active ingredient possessing anti‐inflammatory efficacy. But the underlying ...mechanism is not fully understood and its effect on gouty arthritis remains elusive.
Methods
We evaluated the effects of curcumin on cell viability in primary rat abdominal macrophages with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT). Then supernatants of MSU crystals–stimulated cells were collected and subjected to enzyme‐linked immunosorbent assay for checking the modulation of curcumin on interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α. Meanwhile, cells were analyzed by using Western blot analysis and quantitative polymerase chain reaction (QPCR) to investigate the effects of curcumin on Nod‐like receptor 3 (NLRP3) inflammasome/nuclear factor‐kappa B (NF‐κB) signaling. We also investigated the in vivo efficacy of curcumin with MSU‐induced gouty arthritis rat models.
Results
Curcumin could reduce MSU crystals‐induced IL‐1β and TNF‐α in vitro. Western blot analysis and QPCR results revealed that curcumin regulated the production of these cytokines by suppressing the expression of inflammasome key components, including NLRP3, caspase‐1. Further studies showed that the suppressive efficacy of curcumin on inflammasome was mediated by inhibiting MSU‐induced NF‐κB signaling activation. Intraperitoneal administration of curcumin could ameliorate symptoms of MSU‐induced gouty arthritis, including the joint circumference, infiltration of neutrophils in knee joints, and production of IL‐1β, TNF‐α, and elastase. Western blot analysis revealed that the levels of NLRP3, procaspase‐1, caspase‐1, pro‐IL‐1β, and IL‐1β were downregulated by curcumin in vivo.
Conclusions
These results indicated that curcumin could effectively ameliorate MSU crystal‐induced gouty arthritis through NLRP3 inflammasome mediation via inhibiting NF‐κB signaling both in vitro and in vivo, suggesting a promising active ingredient for the prevention and treatment of gouty arthritis.
Curcumin inhibits Monosodium urate (MSU)‐induced inflammation in primary cultured macrophages. Curcumin ameliorates MSU‐induced gouty arthritis in Rattus norvegicus. Curcumin suppresses the activation of the NLRP3 inflammasome. Curcumin inhibits NLRP3 inflammasome via NF‐kB signaling.
Impaired mitochondrial autophagy (mitophagy) and NLRP3 inflammasome activation have been incriminated in the pathogenesis of T2DM. Metformin besides being an insulin sensitizer also induces ...autophagy; however, its effect on mitophagy and NLRP3 activation in patients with T2DM still remains elusive. Forty‐five drug‐naïve T2DM patients with HbA1C 7%‐9% (53‐75 mmol/mol) were randomly assigned to receive either metformin, voglibose, or placebo for 3 months, and were also recommended for lifestyle intervention programme (n = 15 each). Mitochondrial oxidative stress (MOS) parameters, qPCR and immunoblotting of mitophagy‐related markers (PINK1, PARKIN, MFN2, NIX, LC3‐II, LAMP2), p‐AMPKα (T172), and NLRP3 proteins, as well as transmission electron microscopy (TEM) for assessing mitochondrial morphology were performed in the mononuclear cells of study patients. Both metformin and voglibose showed a similar efficacy towards the reduction in HbA1c and MOS indices. However, multivariate ANCOVA divulged that mRNA and protein expression of mitophagy markers, NLRP3 and p‐AMPKα (T172), were significantly increased only with metformin therapy. Moreover, PINK1 expression displayed a significant positive association with HOMA‐β indices, and TEM studies further confirmed reduced distortions in mitochondrial morphology in the metformin group only. Our observations underscore that metformin upregulates mitophagy and subsequently ameliorates the altered mitochondrial morphology and function, independent of its glucose‐lowering effect. Further, restoration of normal mitochondrial phenotype may improve cellular function, including β‐cells, which may prevent further worsening of hyperglycaemia in patients with T2DM.
Severe, steroid-resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. ...Excessive nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and concomitant IL-1β responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid-resistant asthma.
To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL-1β in severe, steroid-resistant asthma.
We developed mouse models of Chlamydia and Haemophilus respiratory infection-mediated, ovalbumin-induced severe, steroid-resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL-1β responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1β responses in experimental severe, steroid-resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase-1 inhibitor Ac-YVAD-cho; and neutralizing anti-IL-1β antibody. Roles for IL-1β-induced neutrophilic inflammation were examined using IL-1β and anti-Ly6G.
Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1β responses that drive steroid-resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL-1β expression. Treatment with anti-IL-1β, Ac-YVAD-cho, and MCC950 suppressed IL-1β responses and the important steroid-resistant features of disease in mice, whereas IL-1β administration recapitulated these features. Neutrophil depletion suppressed IL-1β-induced steroid-resistant airway hyperresponsiveness.
NLRP3 inflammasome responses drive experimental severe, steroid-resistant asthma and are potential therapeutic targets in this disease.
Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide‐binding oligomerization domain‐containing protein (NOD)‐like receptor pathways. However, ...the function of most innate immune receptors in atherosclerosis remains unclear. Here, we show that NOD2 is a crucial innate immune receptor influencing vascular inflammation and atherosclerosis severity. 10‐week stimulation with muramyl dipeptide (MDP), the NOD2 cognate ligand, aggravated atherosclerosis, as indicated by the augmented lesion burden, increased vascular inflammation and enlarged lipid‐rich necrotic cores in Ldlr−/− mice. Myeloid‐specific ablation of NOD2, but not its downstream kinase, receptor‐interacting serine/threonine‐protein kinase 2, restrained the expansion of the lipid‐rich necrotic core in Ldlr−/− chimeric mice. In vitro stimulation of macrophages with MDP enhanced the uptake of oxidized low‐density lipoprotein and impaired cholesterol efflux in concordance with upregulation of scavenger receptor A1/2 and downregulation of ATP‐binding cassette transporter A1. Ex vivo stimulation of human carotid plaques with MDP led to increased activation of inflammatory signaling pathways p38 MAPK and NF‐κB‐mediated release of proinflammatory cytokines. Altogether, this study suggests that NOD2 contributes to the expansion of the lipid‐rich necrotic core and promotes vascular inflammation in atherosclerosis.
Summary
The pattern‐recognition receptor (PRR) family includes Toll‐like receptors (TLRs), nucleotide‐binding oligomerization domain (NOD) ‐like receptors (NLRs), RIG‐I‐like receptors (RLRs), C‐type ...lectin receptors (CLRs) and the receptor for advanced glycation end products (RAGE). They recognize various microbial signatures or host‐derived danger signals and trigger an immune response. Eosinophils are multifunctional leucocytes involved in the pathogenesis of several inflammatory processes, including parasitic helminth infection, allergic diseases, tissue injury and tumour immunity. Human eosinophils express several PRRs, including TLR1–5, TLR7, TLR9, NOD1, NOD2, Dectin‐1 and RAGE. Receptor stimulation induces survival, oxidative burst, activation of the adhesion system and release of cytokines (interleukin‐1β, interleukin‐6, tumour necrosis factor‐α and granulocyte–macrophage colony‐stimulating factor), chemokines (interleukin‐8 and growth‐related oncogene‐α) and cytotoxic granule proteins (eosinophil cationic protein, eosinophil‐derived neurotoxin, eosinophil peroxidase and major basic protein). It is also evident that eosinophils play an immunomodulatory role by interacting with surrounding cells. The presence of a broad range of PRRs in eosinophils indicates that they are not only involved in defence against parasitic helminths, but also against bacteria, viruses and fungi. From a clinical perspective, eosinophilic PRRs seem to be involved in both allergic and malignant diseases by causing exacerbations and affecting tumour growth, respectively.
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