Nucleotide-binding Domain Leucine-rich Repeat Research Articles

Proteolysis in plant immunity

Abstract Compared with transcription and translation, protein degradation machineries can act faster and be targeted to different subcellular compartments, enabling immediate regulation of signaling events. It is therefore not surprising that proteolysis has been used extensively to control homeostasis of key regulators in different biological processes and pathways. Over the past decades, numerous studies have shown that proteolysis, where proteins are broken down to peptides or amino acids through ubiquitin-mediated degradation systems and proteases, is a key regulatory mechanism to control plant immunity output. Here, we briefly summarize the roles various proteases play during defense activation, focusing on recent findings. We also update the latest progress of ubiquitin-mediated degradation systems in modulating immunity by targeting plant membrane-localized pattern recognition receptors (PRRs), intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs), and downstream signaling components. Additionally, we highlight recent studies showcasing the importance of proteolysis in maintaining broad-spectrum resistance without obvious yield reduction, opening new directions for engineering elite crops that are resistant to a wide range of pathogens with high yield.

Influence of metallic particles and TNF on the transcriptional regulation of NLRP3 inflammasome-associated genes in human osteoblasts

IntroductionThe release of mature interleukin (IL-) 1β from osteoblasts in response to danger signals is tightly regulated by the nucleotide-binding oligomerization domain leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome. These danger signals include wear products resulting from aseptic loosening of joint arthroplasty. However, inflammasome activation requires two different signals: a nuclear factor-kappa B (NF-κB)-activating priming signal and an actual inflammasome-activating signal. Since human osteoblasts react to wear particles via Toll-like receptors (TLR), particles may represent an inflammasome activator that can induce both signals.MethodsTemporal gene expression profiles of TLRs and associated intracellular signaling pathways were determined to investigate the period when human osteoblasts take up metallic wear particles after initial contact and initiate a molecular response. For this purpose, human osteoblasts were treated with metallic particles derived from cobalt-chromium alloy (CoCr), lipopolysaccharides (LPS), and tumor necrosis factor-alpha (TNF) alone or in combination for incubation times ranging from one hour to three days. Shortly after adding the particles, their uptake was observed by the change in cell morphology and spectral data.ResultsExposure of osteoblasts to particles alone increased NLRP3 inflammasome-associated genes. The response was not significantly enhanced when cells were treated with CoCr + LPS or CoCr + TNF, whereas inflammation markers were induced. Despite an increase in genes related to the NLRP3 inflammasome, the release of IL-1β was unaffected after contact with CoCr particles.DiscussionAlthough CoCr particles affect the expression of NLRP3 inflammasome-associated genes, a single stimulus was not sufficient to prime and activate the inflammasome. TNF was able to prime the NLRP3 inflammasome of human osteoblasts.

Natural Compounds Oridonin and Shikonin Exhibit Potentially Beneficial Regulatory Effects on Select Functions of Microglia.

Accumulating evidence indicates that the adverse neuroimmune activation of microglia, brain immunocytes that support neurons, contributes to a range of neuroinflammatory disorders, including Alzheimer's disease. Correcting the abnormal functions of microglia is a potential therapeutic strategy for these diseases. Nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor (NLRP) 3 inflammasomes are implicated in adverse microglial activation and their inhibitors, such as the natural compounds oridonin and shikonin, reduce microglial immune responses. We hypothesized that some of the beneficial effects of oridonin and shikonin on microglia are independent of their suppression of NLRP3 inflammasomes. Murine and human microglia-like cells were stimulated with bacterial lipopolysaccharide (LPS) only, which did not induce NLRP3 inflammasome activation or the resulting secretion of interleukin (IL)-1β, allowing for the identification of other anti-inflammatory effects. Under these experimental conditions, both oridonin and shikonin reduced nitric oxide (NO) secretion and the cytotoxicity of BV-2 murine microglia towards HT-22 murine neuronal cells, but upregulated BV-2 cell phagocytic activity. Only oridonin inhibited the secretion of tumor necrosis factor (TNF) by stimulated BV-2 microglia, while only shikonin suppressed the respiratory burst response of human HL-60 microglia-like cells. This observed discrepancy indicates that these natural compounds may have different molecular targets in microglia. Overall, our results suggest that oridonin and shikonin should be further investigated as pharmacological agents capable of correcting dysfunctional microglia, supporting their potential use in neuroinflammatory disorders.

Cancer-derived exosomal Alu RNA promotes colorectal cancer progression

Inflammation plays a crucial role in cancer progression, but the relevance of the inflammasome remains unclear. Alu RNA was the first endogenous nucleic acid shown to activate the NLRP3 (nucleotide-binding domain leucine-rich repeat containing 3) inflammasome. Here, we showed that Alu RNA can induce epithelial-to-mesenchymal transition (EMT) through NLRP3 inflammasome activation and IL-1β release in colorectal cancer (CRC) cells. Alu RNA is stored, transported and transferred to CRC cells by exosomes. Exosomal Alu RNA promotes tumorigenesis by inducing invasion, metastasis and EMT via NLRP3 inflammasome activation. Consistent with these data, we found that significantly increased Alu RNA expression correlates with the induction of NLRP3 priming in human CRC patients. Furthermore, the level of Alu RNA in circulating exosomes correlates with CRC progression in a preclinical model. These findings reveal the direct involvement of Alu RNA in cancer pathogenesis, and its presence in CRC cell-derived exosomes could be used as a noninvasive diagnostic biomarker.

Isoliquiritigenin inhibits NLRP3 inflammasome activation with CAPS mutations by suppressing caspase-1 activation and mutated NLRP3 aggregation.

The nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome contributes to the development of inflammatory diseases. Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory disease caused by NLRP3 gene mutations that results in excessive IL-1β production. We previously identified isoliquiritigenin (ILG), a component of Glycyrrhiza uralensis extracts, as a potent inhibitor of the NLRP3 inflammasome. Here, we aimed to investigate whether ILG inhibits the activation of NLRP3 inflammasome caused by NLRP3 gene mutations. We demonstrated that ILG significantly inhibited NLRP3 inflammasome-mediated lactate dehydrogenase (LDH) release and IL-1β production in two CAPS model THP-1 cell lines, NLRP3-D303N and NLRP3-L353P, in a dose-dependent manner. Interestingly, the NLRP3 inhibitor MCC950 inhibited LDH release and IL-1β production in NLRP3-D303N cells, but not in NLRP3-L353P cells. Western blotting and caspase-1 activity assays showed that ILG, as well as caspase inhibitors, including Z-VAD and YVAD, suppressed caspase-1 activation. Notably, ILG prevented cryo-sensitive foci formation of NLRP3 without affecting the levels of intracellular Ca2+ . We concluded that ILG effectively prevents the constitutive activation of the inflammasome associated with NLRP3 gene mutations by inhibiting the aggregation of cryo-sensitive mutated NLRP3.

Chronic osteomyelitis risk is associated with NLRP3 gene rs10754558 polymorphism in a Chinese Han Population

BackgroundSingle nucleotide polymorphisms (SNPs) in the nucleotide-binding domain leucine-rich repeat protein-3 (NLRP3) gene are reported to be linked to many inflammatory disorders. However, uncertainty persists over the associations between these SNPs and susceptibilities to chronic osteomyelitis (COM). This study aimed to investigate potential relationships between NLRP3 gene SNPs and the risks of developing COM in a Chinese Han cohort.Methods The four tag SNPs of the NLRP3 gene were genotyped in a total of 428 COM patients and 368 healthy controlsusing the SNapShot technique. The genotype distribution, mutant allele frequency, and the four genetic models (dominant, recessive, homozygous, and heterozygous) of the four SNPs were compared between the two groups.ResultsA significant association was found between rs10754558 polymorphism and the probability of COM occurence by the heterozygous model (P = 0.037, odds ratio [OR] = 1.541, 95% confidence interval [CI] = 1.025–2.319), indicating that rs10754558 may be associated with a higher risk of developing COM.In addition, possible relationship was found between rs7525979 polymorphism and the risk of COM development by the outcomes of homozygous (P = 0.073, OR = 0.453, 95% CI = 0.187–1.097) and recessive (P = 0.093, OR = 0.478, 95% CI = 0.198–1.151) models, though no statistical differences were obtained.ConclusionsOutcomes of the present study showed, for the first time, that rs10754558 polymorphism of the NLRP3 gene may increase the risk of COM development in this Chinese Han population, with genotype CG as a risk factor. Nonetheless, this conclusion requires verification from further studies with a larger sample size.

Nlrc3 signaling is indispensable for hematopoietic stem cell emergence via Notch signaling in vertebrates

Hematopoietic stem and progenitor cells generate all the lineages of blood cells throughout the lifespan of vertebrates. The emergence of hematopoietic stem and progenitor cells is finely tuned by a variety of signaling pathways. Previous studies have revealed the roles of pattern-recognition receptors such as Toll-like receptors and RIG-I-like receptors in hematopoiesis. In this study, we find that Nlrc3, a nucleotide-binding domain leucine-rich repeat containing family gene, is highly expressed in hematopoietic differentiation stages in vivo and vitro and is required in hematopoiesis in zebrafish. Mechanistically, nlrc3 activates the Notch pathway and the downstream gene of Notch hey1. Furthermore, NF-kB signaling acts upstream of nlrc3 to enhance its transcriptional activity. Finally, we find that Nlrc3 signaling is conserved in the regulation of murine embryonic hematopoiesis. Taken together, our findings uncover an indispensable role of Nlrc3 signaling in hematopoietic stem and progenitor cell emergence and provide insights into inflammation-related hematopoietic ontogeny and the in vitro expansion of hematopoietic stem and progenitor cells.

Role of NLRP3 Inflammasome in Stroke Pathobiology: Current Therapeutic Avenues and Future Perspective.

Neuroinflammation is a key pathophysiological feature of stroke-associated brain injury. A local innate immune response triggers neuroinflammation following a stroke via activating inflammasomes. The nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome has been heavily implicated in stroke pathobiology. Following a stroke, several stimuli have been suggested to trigger the assembly of the NLRP3 inflammasome. Recent studies have advanced the understanding and revealed several new players regulating NLRP3 inflammasome-mediated neuroinflammation. This article discussed recent advancements in NLRP3 assembly and highlighted stroke-induced mitochondrial dysfunction as a major checkpoint to regulating NLRP3 activation. The NLRP3 inflammasome activation leads to caspase-1-dependent maturation and release of IL-1β, IL-18, and gasdermin D. In addition, genetic or pharmacological inhibition of the NLRP3 inflammasome activation and downstream signaling has been shown to attenuate brain infarction and improve the neurological outcome in experimental models of stroke. Several drug-like small molecules targeting the NLRP3 inflammasome are in different phases of development as novel therapeutics for various inflammatory conditions, including stroke. Understanding how these molecules interfere with NLRP3 inflammasome assembly is paramount for their better optimization and/or development of newer NLRP3 inhibitors. In this review, we summarized the assembly of the NLRP3 inflammasome and discussed the recent advances in understanding the upstream regulators of NLRP3 inflammasome-mediated neuroinflammation following stroke. Additionally, we critically examined the role of the NLRP3 inflammasome-mediated signaling in stroke pathophysiology and the development of therapeutic modalities to target the NLRP3 inflammasome-related signaling for stroke treatment.

Novel Small-molecule Inhibitor of NLRP3 Inflammasome as Possible Treatment for Amyotrophic Lateral Sclerosis

Objectives: The nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor protein 3 (NLRP3) inflammasome is over activated in neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS), leading to chronic inflammation by activating several cytokines. A novel small-molecule inhibitor C77 was described to block NLRP3 inflammasome, therefore our study is focused in optimizing and understanding the capabilities of the small-molecule inhibitor for possible treatment of ALS. We hypothesize that C77 can cross the BBB and the muscle, therefore blocking NLRP3 inflammasome and slow disease progression and mortality.

Nlrc3 Signaling Is Indispensable for Hematopoietic Stem Cell Emergence Via Notch Signaling in Vertebrates

Hematopoietic stem and progenitor cells (HSPCs) generate all the lineages of blood cells throughout the lifespan of vertebrates. The emergence of HSPCs occurs through the hemogenic endothelial (HE) to hematopoietic transition (EHT) process, which is finely tuned by a variety of signaling pathways. Previous studies have emerged the essential roles of pattern-recognition receptors such as Toll-like receptors and RIG-I-like receptors in EHT. However, whether the nucleotide-binding domain leucine-rich repeat (NLR) containing family members participate in vertebrate embryonic hematopoiesis remains unclear. To explore the potential pathways involved in hematopoietic ontogeny and differentiation in this stage, we screened the dynamic expression of NLR family genes and other genes related to inflammation in the published scRNA-seq data in vertebrates' embryos including zebrafish, mouse and human during HSPC development. And to verify the expression of NLR family genes in vitro by using HSC-like cells differentiation system of mouse and human embryonic stem cells (ESCs), these results suggest that NLRC3 was highly expressed during embryonic HSPC development in vivo and vitro and might play an important role in HSPC emergence in vertebrates. In order to validate this hypothesis, we synthesized probes from the full-length mRNA of nlrc3 and utilized them to observe the in-situ expression of nlrc3 in zebrafish at different developmental stages. The WISH experiment demonstrated that nlrc3 was expressed from the 1-cell stage and importantly, showed specific expression in the AGM region at the 24-28hpf and CHT region at 72hpf, which coincides with the onset and generation of HSPC generation. Then morpholino were utilized to knockdown nlrc3 in transgenic embryos Tg( runx1:EGFP/ kdrl:mCherry), Tg( cmyb:EGFP/ kdrl:mCherry) and Tg( CD41:GFP), and mutants generated by CRISPR-Cas9 were used to perform whole-mount in situ hybridization (WISH) and qPCR to detect specific markers such as runx1, cmyb(HSPCs), rag1(lymphocytes), gata1a(erythrocytes) and l-plastin(myelocytes), etc. The results showed that nlrc3 was required for HSPCs emergence, expansion and differentiation. In primitive hematopoietic wave, nlrc3 signaling may not impact primitive erythropoiesis but required for primitive neutrophils and macrophages. Mechanistically, we sorted EGFP + cells in Tg ( fli1a:EGFP) zebrafish embryos at 28 hpf to perform RNA-Seq, which was the stage where EHT and these EGFP + cells contained hemogenic endothelial cells. After bioinformatics analysis of sequencing data, we found that the effect of nlrc3 in zebrafish hematopoiesis is not based on affecting the development of arterial vessels or causing apoptosis. By using targeted inhibitor such as DAPT and JSH23, conditional rescue model like Tg ( hsp70:GAL4/ UAS:NICD), overexpression expression experiment in Tg ( tp1:EGFP/ kdrl:mCherry) and nlrc3 -/- mutants, etc, through Imaging systems and qPCR we found that NF-kB- nlrc3-Notch- hey1 axis triggers HSPCs emergence and differentiation in zebrafish. To further extend our findings in mammals, we utilized Nlrc3 knockout mice to explore whether Nlrc3 signaling was evolutionarily conserved in HSPCs emergence. Hematogenic endothelial cells (CD31 + CD41 - CD45 - TER119 -) from the embryonic AGM tissues at E10.5 were sorted and cultured in OP9 for 4 days and flow cytometry was performed to assess that c-Kit +CD45 + hematopoietic cells and CD31 +Sca-1 +CD201 + hematopoietic stem cells were notably decreased in these Nlrc3-/- embryos. Colony-forming unit-cell (CFU-C) assay and BM transplantation experiments were used to observe that fetal liver HSCs from E14.5 Nlrc3-/- embryos was decrease in the number and impaired in function in vitro and in vivo. Finally qPCR using mRNA from FL-derived LSK ( Lin-c-Kit+Sca-1+) cells further demonstrated that the NF-kB- Nlrc3-Notch- Hey1 axis orchestrated development of embryonic HSCs was highly conserved in vertebrates. Overall, by summarizing the results of the physiological and developmental roles in vivo and in vitro, we uncover an indispensable role of Nlrc3 signaling in vertebrates' HSPCs emergence, our findings provide new insights into inflammation-related hematopoietic ontogeny and Nlrc3 signal is of major importance since this investigation might be taken into consideration when recapitulation definitive hematopoiesis in vitro.

The plant immune receptor SNC1 monitors helper NLRs targeted by a bacterial effector

Plants deploy intracellular receptors to counteract pathogen effectors that suppress cell-surface-receptor-mediated immunity. To what extent pathogens manipulate intracellular receptor-mediated immunity, and how plants tackle such manipulation, remains unknown. Arabidopsis thaliana encodes three similar ADR1 class helper nucleotide-binding domain leucine-rich repeat receptors (ADR1, ADR1-L1, and ADR1-L2), which are crucial in plant immunity initiated by intracellular receptors. Here, we report that Pseudomonas syringae effector AvrPtoB suppresses ADR1-L1- and ADR1-L2-mediated cell death. ADR1, however, evades such suppression by diversifying into two ubiquitination sites targeted by AvrPtoB. The intracellular sensor SNC1 interacts with and guards the CCR domains of ADR1-L1/L2. Removal of ADR1-L1/L2 or delivery of AvrPtoB activates SNC1, which then signals through ADR1 to trigger immunity. Our work elucidates the long-sought-after function of SNC1 in defense, and also how plants can use dual strategies, sequence diversification, and a multi-layered guard-guardee system, to counteract pathogen's attack on core immunity functions.

Genome-wide identification of nucleotide-binding domain leucine-rich repeat (NLR) genes and their association with green peach aphid (Myzus persicae) resistance in peach

Resistance genes (R genes) are a class of genes that are immune to a wide range of diseases and pests. In planta, NLR genes are essential components of the innate immune system. Currently, genes belonging to NLR family have been found in a number of plant species, but little is known in peach. Here, 286 NLR genes were identified on peach genome by using their homologous genes in Arabidopsis thaliana as queries. These 286 NLR genes contained at least one NBS domain and LRR domain. Phylogenetic and N-terminal domain analysis showed that these NLRs could be separated into four subfamilies (I-IV) and their promoters contained many cis-elements in response to defense and phytohormones. In addition, transcriptome analysis showed that 22 NLR genes were up-regulated after infected by Green Peach Aphid (GPA), and showed different expression patterns. This study clarified the NLR gene family and their potential functions in aphid resistance process. The candidate NLR genes might be useful in illustrating the mechanism of aphid resistance in peach.

Expression and clinical value of NLRP1 and NLRC4 inflammasomes in prostate cancer.

The present study explored the clinical value of the protein expression levels of nucleotide binding oligomerization-like receptor family pyrin domain containing 1 (NLRP1) and nucleotide-binding oligomerization domain leucine-rich repeat and caspase recruitment domain-containing 4 (NLRC4) inflammasomes in the diagnosis and treatment of prostate cancer. A total of 54 patients with prostatic hyperplasia and 58 patients with prostate cancer were recruited at The First People's Hospital of Pinghu between January and May 2022. Immunohistochemical staining was used to determine the protein expression levels of the NLRP1 and NLRC4 inflammasomes in addition to the proinflammatory cytokines IL-18 and IL-1β in the two groups of patients. The protein expression levels of NLRP1 and NLRC4 inflammasome were significantly increased in patients with prostate cancer compared with patients with prostate hyperplasia. The differences in expression of NLRP1 and NLRC4 inflammatory vesicles in prostate cancer of different stages were also compared based on data from The Cancer Genome Atlas. The protein expression level of NLRP1 demonstrated a significant positive correlation with IL-1β and IL-18 expression, and the protein expression level of the NLRC4 inflammasome was significantly positively correlated with IL-18 expression. The protein expression levels of both NLRP1 and NLRC4 demonstrated a significant positive correlation with the Gleason score of prostate cancer. The expression of NLRP1 in tumor (T)3/T4 was significantly higher compared with T1 and expression of the NLRC4 inflammasome in T2 and T3/T4 was significantly higher compared with T1. Expression of the NLRP1 and NLRC4 inflammasomes was significantly higher in patients with prostate cancer, compared with patients with prostatic hyperplasia. Therefore, expression of NLRP1 and NLRC4 may promote tumorigenesis by promoting the maturation and release of proinflammatory cytokines IL-1β and IL-18. Expression of the NLRP1 and NLRC4 inflammasomes demonstrated a significant positive correlation with the risk of prostate cancer. Expression of the NLRP1 and NLRC4 inflammasomes in middle- and advanced-stage tumors was higher compared with early-stage tumors. These results suggested that inflammasome expression may serve a significant role in the progression of tumors and could provide a fixed value for the risk assessment and prognosis prediction of prostate cancer.

Formononetin alleviates acute pancreatitis by reducing oxidative stress and modulating intestinal barrier

BackgroundAcute pancreatitis (AP) is a recurrent inflammatory disease. Studies have shown that intestinal homeostasis is essential for the treatment of AP. Formononetin is a plant-derived isoflavone with antioxidant properties that can effectively treat a variety of inflammatory diseases. This study aims to investigate the role of formononetin in protecting against AP and underlying mechanism.MethodsCaerulein was used to induce AP. The inflammatory cytokines were detected using Quantitative real-time PCR and commercial kits. Histological examination was applied with hematoxylin and eosin staining. Western blot was conducted to detect expression of intestinal barrier protein and signaling molecular. Molecular docking was performed to assess protein-ligand interaction.ResultsIn this study, we found formononetin administration significantly reduced pancreatic edema, the activities of serum amylase, lipase, myeloperoxidase, and serum endotoxin. The mRNA levels of inflammatory cytokines such as tumor necrosis factor α, monocyte chemoattractant protein-1, interleukin-6, and interleukin-1 beta (IL-1β) in pancreas were also significantly decreased by formononetin. The following data showed formononetin pretreatment up-regulated the expressions of tight junction proteins in the colon, and decreased Escherichia coli translocation in the pancreas. In addition, formononetin inhibited the activation of nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing 3 in pancreatic and colonic tissues of AP mice. Moreover, formononetin activated Kelch Like ECH Associated Protein 1 (Keap1) / Nuclear factor erythroid2-related factor 2 (Nrf2) signaling pathway to reduce reactive oxygen species (ROS) levels. Docking results showed that formononetin interact with Keap1 through hydrogen bond.ConclusionsThese findings demonstrate that formononetin administration significantly mitigate AP through reducing oxidative stress and restoring intestinal homeostasis, and provide insights into the new treatment for AP.

Translation Arrest: A Key Player in Plant Antiviral Response.

Plants evolved several mechanisms to protect themselves against viruses. Besides recessive resistance, where compatible host factors required for viral proliferation are absent or incompatible, there are (at least) two types of inducible antiviral immunity: RNA silencing (RNAi) and immune responses mounted upon activation of nucleotide-binding domain leucine-rich repeat (NLR) receptors. RNAi is associated with viral symptom recovery through translational repression and transcript degradation following recognition of viral double-stranded RNA produced during infection. NLR-mediated immunity is induced upon (in)direct recognition of a viral protein by an NLR receptor, triggering either a hypersensitive response (HR) or an extreme resistance response (ER). During ER, host cell death is not apparent, and it has been proposed that this resistance is mediated by a translational arrest (TA) of viral transcripts. Recent research indicates that translational repression plays a crucial role in plant antiviral resistance. This paper reviews current knowledge on viral translational repression during viral recovery and NLR-mediated immunity. Our findings are summarized in a model detailing the pathways and processes leading to translational arrest of plant viruses. This model can serve as a framework to formulate hypotheses on how TA halts viral replication, inspiring new leads for the development of antiviral resistance in crops.

Oral Hydroxychloroquine Mitigates Lipopolysaccharide-induced Lung Injury by Inhibiting Pyroptosis in Mice.

Hydroxychloroquine (HCQ) is a molecule derived from quinacrine; it displays a wide range of pharmacological properties, including anti-inflammatory, immunomodulatory, and antineoplastic. However, little is known about this molecule's role in lung injury. This study aimed to identify HCQ's regulatory role of HCQ in sepsis-induced lung injury and its molecular mechanism. To test the protective properties of HCQ, we established an in vivo model of lipopolysaccharide (LPS)-induced lung injury in mice. The extent of the injury was determined by evaluating histopathology, inflammatory response, oxidative stress, and apoptosis. Mechanistically, conventional nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing 3 (NLRP3) knockout mice were employed to investigate whether HCQ exerted pulmonary protection by inhibiting NLRP3-mediated pyroptosis. Our findings revealed that HCQ pretreatment significantly mitigated LPS-induced lung injury in mice in terms of histopathology, inflammatory response, oxidative stress, and apoptosis, while inhibiting LPS-induced NLRP3 inflammasome activation and pyroptosis. Additionally, the indicators of lung injury, including histopathology, inflammatory response, oxidative stress, and apoptosis, were still reduced drastically in LPS-treated NLRP3 (-/-) mice after HCQ pretreatment. Notably, HCQ pretreatment further decreased the levels of pyroptosis indicators, including IL-1β, IL-18 and Cle-GSDMD, in LPS-treated NLRP3 (-/-) mice. Taken together, HCQ protects against lung injury by inhibiting pyroptosis, maybe not only through the NLRP3 pathway but also through non-NLRP3 pathway; therefore, it may be a new therapeutic strategy in the treatment of lung injury.

Immune receptor mimicking hormone receptors: a new guarding strategy

Plant intracellular nucleotide-binding domain leucine-rich repeat (NLR) receptors play crucial roles in immune responses against pathogens. How diverse NLRs recognize different pathogen effectors remains a significant question. A recent study published in Nature uncovered how pepper NLR Tsw detects phytohormone receptors’ interference caused by tomato spotted wilt virus (TSWV) effector, triggering a robust immune response, showcasing a new manner of NLR guarding.

Homocysteine-induced Barrier Dysfunction and Hyperpermeability in the Blood-Brain and Blood-Retinal Barrier: Role of NLRP3 Inflammasome Pathway

Prolonged exposure to microgravity during long- and short-duration spaceflight has been found to produce many pathophysiological changes. Of these, a set constellation of changes called “Spaceflight Associated Neuro-ocular Syndrome” (SANS) induces neuro-ophthalmic changes. Recent evidence suggests astronauts who experienced SANS generally had higher concentrations of one-carbon pathway intermediate homocysteine (Hcy) prior to spaceflight. Hcy is a known inducer of inflammatory mediators and vascular endothelial dysfunction, leading to blood-brain barrier (BBB) breakdown. Mechanisms behind this dysfunction/hyperpermeability are not clearly known, but recent studies show evidence for loss of BBB integrity following microgravity/simulated microgravity. Recent studies from our lab suggest that activation of the nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome plays a role in mediating BBB dysfunction and related microvascular hyperpermeability. Our main objective was to understand the mechanism(s) by which Hcy could induce BBB dysfunction/hyperpermeability, and potential association with SANS. We hypothesized that Hcy-mediated dysfunction/BBB hyperpermeability occurs via NLRP3 inflammasome activation and subsequent proteolytic breakdown of tight junction proteins. Understanding this pathway is critical to devise protection against SANS at a microvascular level. To study this relationship, human brain microvascular endothelial cells (HBMECs) were treated with various concentrations of Hcy. The monolayer permeability was evaluated using Transwell permeability assays with fluorescence tracer FITC-dextran (10-kDa). Matrixmetalloproteinase (MMP-9) and calpain activities were measured fluorometrically. Hcy induced endothelial monolayer hyperpermeability dose-dependently (p<0.05). NLRP3 inhibitor MCC950 decreased Hcy-induced hyperpermeability (p<0.05). Hcy treatment showed no significant effect on cell viability at lower concentration but decreased cell viability at higher concentrations. MCC950 inhibited Hcy-induced MMP-9 and calpain 1 activity. These results suggest a possible mechanism of BBB dysfunction/hyperpermeability by Hcy via activation of the NLRP3 inflammasome pathway. The proteolytic enzymes MMP-9 and calpain 1 are potential downstream mediators of Hcy-induced BBB dysfunction/hyperpermeability. Seeing that the blood-retinal barrier (BRB) shares structural-functional similarity with the BBB, human retinal microvascular endothelial cells (HRMEC) were examined for comparisons of barrier permeability. The results suggest Hcy-induced hyperpermeability in HRMEC monolayers and decreased expression of tight junction protein zonula occludens-1. Expression and effects were attenuated by NLRP3 inhibition. Our results suggest that NLRP3 inflammasome signaling may play an important role in promoting Hcy-induced barrier dysfunction and hyperpermeability in the blood-brain and blood-retinal barriers. Morehouse School of Medicine, Grady Hospital Surgery Dept, NASA This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Puncta-localized TRAF domain protein TC1b contributes to the autoimmunity of snc1.

Immune receptors play important roles in the perception of pathogens and initiation of immune responses in both plants and animals. Intracellular nucleotide-binding domain leucine-rich repeat (NLR)-type receptors constitute a major class of receptors in vascular plants. In the Arabidopsis thaliana mutant suppressor of npr1-1, constitutive 1 (snc1), a gain-of-function mutation in the NLR gene SNC1 leads to SNC1 overaccumulation and constitutive activation of defense responses. From a CRISPR/Cas9-based reverse genetics screen in the snc1 autoimmune background, we identified that mutations in TRAF CANDIDATE 1b (TC1b), a gene encoding a protein with four tumor necrosis factor receptor-associated factor (TRAF) domains, can suppress snc1 phenotypes. TC1b does not appear to be a general immune regulator as it is not required for defense mediated by other tested immune receptors. TC1b also does not physically associate with SNC1, affect SNC1 accumulation, or affect signaling of the downstream helper NLRs represented byACTIVATED DISEASE RESISTANCE PROTEIN 1-L2 (ADR1-L2), suggesting that TC1b impacts snc1 autoimmunity in a unique way. TC1b can form oligomers and localizes to punctate structures of unknown function. The puncta localization of TC1b strictly requires its coiled-coil (CC) domain, whereas the functionality of TC1b requires the four TRAF domains in addition to the CC. Overall, we uncovered the TRAF domain protein TC1b as a novel positive contributor to plant immunity.

Eugenol alleviates neuronal damage via inhibiting inflammatory process against pilocarpine-induced status epilepticus.

Neuroinflammation is one of the most common pathological outcomes in various neurological diseases. A growing body of evidence suggests that neuroinflammation plays a pivotal role in the pathogenesis of epileptic seizures. Eugenol is the major phytoconstituent of essential oils extracted from several plants and possesses protective and anticonvulsant properties. However, it remains unclear whether eugenol exerts an anti-inflammatory effect to protect against severe neuronal damage induced by epileptic seizures. In this study, we investigated the anti-inflammatory action of eugenol in an experimental epilepsy model of pilocarpine-induced status epilepticus (SE). To examine the protective effect of eugenol via anti-inflammatory mechanisms, eugenol (200 mg/kg) was administrated daily for three days after pilocarpine-induced SE onset. The anti-inflammatory action of eugenol was evaluated by examining the expression of reactive gliosis, pro-inflammatory cytokines, nuclear factor-κB (NF-κB), and the nucleotide-binding domain leucine-rich repeat with a pyrin-domain containing 3 (NLRP3) inflammasome. Our results showed that eugenol reduced SE-induced apoptotic neuronal cell death, mitigated the activation of astrocytes and microglia, and attenuated the expression of interleukin-1β and tumor necrosis factor α in the hippocampus after SE onset. Furthermore, eugenol inhibited NF-κB activation and the formation of the NLRP3 inflammasome in the hippocampus after SE. These results suggest that eugenol is a potential phytoconstituent that suppresses the neuroinflammatory processes induced by epileptic seizures. Therefore, these findings provide evidence that eugenol has therapeutic potential for epileptic seizures.