The soybean plasma membrane GmDR1 protein conferring broad-spectrum disease and pest resistance regulates several receptor kinases and NLR proteins.

Overexpression of Glycine max disease resistant 1 (GmDR1) exhibits broad-spectrum resistance against Fusarium virguliforme, Heterodera glycines (soybean cyst nematode), Tetranychus urticae (Koch) (spider mites), and Aphis glycines Matsumura (soybean aphids) in soybean. To understand the mechanisms of broad-spectrum immunity mediated by GmDR1, the transcriptomes of a strong and a weak GmDR1-overexpressor following treatment with chitin, a pathogen- and pest-associated molecular pattern (PAMP) common to these organisms, were investigated. The strong and weak GmDR1-overexpressors exhibited altered expression of 6098 and 992 genes, respectively, as compared to the nontransgenic control following chitin treatment. However, only 192 chitin- and 115 buffer-responsive genes exhibited over two-fold changes in expression levels in both strong and weak GmDR1-overexpressors as compared to the control. MapMan analysis of the 192 chitin-responsive genes revealed 64 biotic stress-related genes, of which 53 were induced and 11 repressed as compared to the control. The 53 chitin-induced genes include nine genes that encode receptor kinases, 13 encode nucleotide-binding leucine-rich repeat (NLR) receptor proteins, seven encode WRKY transcription factors, four ethylene response factors, and three MYB-like transcription factors. Investigation of a subset of these genes revealed three receptor protein kinases, seven NLR proteins, and one WRKY transcription factor genes that are induced following F. virguliforme and H. glycines infection. The integral plasma membrane GmDR1 protein most likely recognizes PAMPs including chitin and activates transcription of genes encoding receptor kinases, NLR proteins and defense-related genes. GmDR1 could be a pattern recognition receptor that regulates the expression of several NLRs for expression of PAMP-triggered immunity and/or priming the effector triggered immunity.

Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1ß-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1ß and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1ß, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1ß expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1ß (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1ß, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1ß, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1ß in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

An inulin-type fructan CP-A from Codonopsis pilosula attenuates experimental colitis in mice by promoting autophagy-mediated inactivation of NLRP3 inflammasome.

Inulin-type fructan CP-A, a predominant polysaccharide in Codonopsis pilosula, demonstrates regulatory effects on immune activity and anti-inflammation. The efficacy of CP-A in treating ulcerative colitis (UC) is, however, not well-established. This study employed an in vitro lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) and an in vivo dextran sulfate sodium (DSS)-induced colitis mouse model to explore CP-A's protective effects against experimental colitis and its underlying mechanisms. We monitored the clinical symptoms in mice using various parameters: body weight, disease activity index (DAI), colon length, spleen weight, and histopathological scores. Additionally, molecular markers were assessed through enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting assays. Results showed that CP-A significantly reduced reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and interleukins (IL-6, IL-1ß, IL-18) in LPS-induced cells while increasing IL-4 and IL-10 levels and enhancing the expression of Claudin-1, ZO-1, and occludin proteins in NCM460 cells. Correspondingly, in vivo findings revealed that CP-A administration markedly improved DAI, reduced colon shortening, and decreased the production of myeloperoxidase (MPO), malondialdehyde (MDA), ROS, IL-1ß, IL-18, and NOD-like receptor protein 3 (NLRP3) inflammasome-associated genes/proteins in UC mice. CP-A treatment also elevated glutathione (GSH) and superoxide dismutase (SOD) levels, stimulated autophagy (LC3B, P62, Beclin-1, and ATG5), and reinforced Claudin-1 and ZO-1 expression, thereby aiding in intestinal epithelial barrier repair in colitis mice. Notably, the inhibition of autophagy via chloroquine (CQ) diminished CP-A's protective impact against colitis in vivo. These findings elucidate that CP-A's therapeutic effect on experimental colitis possibly involves mitigating intestinal inflammation through autophagy-mediated NLRP3 inflammasome inactivation. Consequently, inulin-type fructan CP-A emerges as a promising drug candidate for UC treatment.

Conserved transcription factors NRZ1 and NRM1 regulate NLR receptor-mediated immunity.

Plant innate immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors plays an important role in defense against various pathogens. Although key biochemical events involving NLR activation and signaling have been recently uncovered, we know very little about the transcriptional regulation of NLRs and their downstream signaling components. Here, we show that the Toll-Interleukin 1 receptor homology domain containing NLR (TNL) gene N (Necrosis), which confers resistance to Tobacco mosaic virus, is transcriptionally induced upon immune activation. We identified two conserved transcription factors, N required C3H zinc finger 1 (NRZ1) and N required MYB-like transcription factor 1 (NRM1), that activate N in an immune responsive manner. Genetic analyses indicated that NRZ1 and NRM1 positively regulate coiled-coil domain-containing NLR- and TNL-mediated immunity and function independently of the signaling component Enhanced Disease Susceptibility 1. Furthermore, NRZ1 functions upstream of NRM1 in cell death signaling, and their gene overexpression induces ectopic cell death and expression of NLR signaling components. Our findings uncovered a conserved transcriptional regulatory network that is central to NLR-mediated cell death and immune signaling in plants.

The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond.

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged AlphaFold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

Paclitaxel may inhibit migration and invasion of gastric cancer cells via nod-like receptor family pyrin domain-containing 3/caspase-1/Gasdermin E mediated pyroptosis pathway.

Gastric cancer (GC) is a gastric epithelium-derived malignancy insensitive to post-surgical radiotherapy. Paclitaxel, an anti-microtubule drug, has been proven to induce apoptosis of GC cells; however, its exact mechanism of action is unclear. Therefore, the molecular mechanism by which paclitaxel inhibits the proliferation, migration and invasion of GC cells was investigated in this study. First off, SNU-719 cells were co-cultured with paclitaxel and/or Caspase1 inhibitor VX765. Then the proliferation ability of the cells was detected by MTT after paclitaxel treatment (0, 10, 20, 40, and 80 nM), the migration ability by scratch assay, and the invasion ability by Transwell assay. Next, the levels of interleukin (IL)-1ß and IL-18 in cell culture supernatant were detected by the enzyme linked immunosorbent assay (ELISA). And the level of lactate dehydrogenase (LDH) in the supernatant was measured by a corresponding kit. Finally, western blot was performed to detect the concentrations of Gasdermin E (GSDME), GSDME-N, nod-like receptor family pyrin domain-containing 3 (NLRP3), caspase-1, cleaved caspase-1 protein in GC cells. As a result, paclitaxel inhibited the proliferation, migration, and invasion of SNU-719 cells in a concentration-dependent manner. Moreover, it induced the pyroptosis of SNU-719 cells. After cell co-culture with VX765 paclitaxel showed decreased inhibitory effect on the migration and invasion of SNU-719 cells. VX765, additionally, suppressed the NLRP3/caspase-1/GSDME mediated pyroptosis pathway activated by paclitaxel. In a nutshell, paclitaxel may inhibit the migration and invasion of GC cells SNU-719 through the NLRP3/caspase-1/GSDME mediated pyroptosis pathway.

Role of the NLRP1 inflammasome in skin cancer and inflammatory skin diseases.

Inflammasomes are cytoplasmic protein complexes that play a crucial role in protecting the host against pathogenic and sterile stressors by initiating inflammation. Upon activation, these complexes directly regulate the proteolytic processing and activation of proinflammatory cytokines interleukin (IL)-1ß and IL-18 to induce a potent inflammatory response, and induce a programmed form of cell death called pyroptosis to expose intracellular pathogens to the surveillance of the immune system, thus perpetuating inflammation. There are various types of inflammasome complexes, with the NLRP1 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-1) inflammasome being the first one identified and currently recognized as the predominant inflammasome sensor protein in human keratinocytes. Human NLRP1 exhibits a unique domain structure, containing both an N-terminal pyrin (PYD) domain and an effector C-terminal caspase recruitment domain (CARD). It can be activated by diverse stimuli, such as viruses, ultraviolet B radiation and ribotoxic stress responses. Specific mutations in NLRP1 or related genes have been associated with rare monogenic skin disorders, such as multiple self-healing palmoplantar carcinoma; familial keratosis lichenoides chronica; autoinflammation with arthritis and dyskeratosis; and dipeptidyl peptidase 9 deficiency. Recent research breakthroughs have also highlighted the involvement of dysfunctions in the NLRP1 pathway in a handful of seemingly unrelated dermatological conditions. These range from monogenic autoinflammatory diseases to polygenic autoimmune diseases such as vitiligo, psoriasis, atopic dermatitis and skin cancer, including squamous cell carcinoma, melanoma and Kaposi sarcoma. Additionally, emerging evidence implicates NLRP1 in systemic lupus erythematosus, pemphigus vulgaris, Addison disease, Papillon-Lefèvre syndrome and leprosy. The aim of this review is to shed light on the implications of pathological dysregulation of the NLRP1 inflammasome in skin diseases and investigate the potential rationale for targeting this pathway as a future therapeutic approach.

Treponema pallidum recombinant protein Tp47 activates NOD-like receptor family protein 3 inflammasomes in macrophages via glycolysis.

Glycolysis is a key pathway in cellular glucose metabolism for energy supply and regulates immune cell activation. Whether glycolysis is involved in the activation of NOD-like receptor family protein 3 (NLRP3) inflammasomes during Treponema pallidum (T. pallidum) infection is unclear. In this study, the effect of T. pallidum membrane protein Tp47 on NLRP3 inflammasome activation in rabbit peritoneal macrophages was analysed and the role of glycolysis in NLRP3 inflammasome activation was explored. The results showed that Tp47 promoted NLRP3, caspase-1, and IL-1ß mRNA expression in macrophages, enhanced glycolysis and glycolytic capacity of macrophage, and promoted the production of macrophage glycolytic metabolites citrate, phosphoenolpyruvate, and lactate. The M2 pyruvate kinase (PKM2) inhibitor shikonin down-regulated the Tp47-promoted NLRP3, caspase-1, and IL-1ß mRNA expression in macrophages, and suppressed the Tp47-enhanced glycolysis and glycolytic capacity. Similarly, si-PKM2 significantly inhibited Tp47-promoted NLRP3, caspase-1, and IL-1ß mRNA expression and the Tp47-enhanced glycolysis and glycolytic capacity in macrophages. In conclusion, Tp47 activated NLRP3 inflammasomes via PKM2-dependent glycolysis and provided a new perspective on the effect of T. pallidum infection on host macrophages, which would contribute to the understanding of the infection mechanism and host immune mechanism of T. pallidum.

Inhibition of circulating exosomes release with GW4869 mitigates severe acute pancreatitis-stimulated intestinal barrier damage through suppressing NLRP3 inflammasome-mediated pyroptosis.

Intestinal barrier dysfunction frequently occurs as a complication in cases of severe acute pancreatitis (SAP); however, no effective therapeutic methods are available because the precise mechanism remains obscure. Recent research has elucidated the role of circulating exosomes in the progression of SAP. Therefore, the present study explored whether inhibiting circulating exosomes release would improve intestinal barrier injury triggered via SAP and investigated the possible underlying mechanism. In vivo, we found that circulating exosomes release exhibited a considerable increase in SAP rats than in SO rats, and GW4869, a suppressor of exosomes release, significantly decreased exosomes release in SAP rats. We also observed that GW4869 suppressed NLRP3 inflammasome-mediated pyroptosis within the intestine and alleviated intestinal barrier injury within SAP. Moreover, the inflammatory response and remote organ (kidney and lung) injury associated with SAP improved after GW4869 treatment. In vitro, we confirmed that depletion of exosomes with GW4869 could partially abolish the destructive effects of SAP rat plasma on the viability and barrier function of IEC-6 cells. In summary, our findings show that the suppression of the release of circulating exosomes effectively inhibits the process of pyroptosis mediated by the NOD-like receptor protein 3 (NLRP3) inflammasome and, therefore, mitigates intestinal barrier dysfunction in SAP, suggesting that circulating exosomes may be a potential target for treating SAP.

RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation.

BACKGROUND AND AIMS: The pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. METHODS: For in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. RESULTS: PDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αß (PDGFRαß) rather than PDGFRαα or PDGFRßß in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. CONCLUSIONS: The results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.

Moxibustion intervention improves synovitis by down-regulating NLRP3/Caspase-1/IL-1ß signaling of synovial tissue in rats with adjuvant arthritis. / 基于NLRP3/Caspase-1/IL-1ßä¿¡å·é€šè·¯æŽ¢è®¨è‰¾ç¸æ”¹å–„ä½å‰‚æ€§å ³èŠ‚ç‚Žå¤§é¼ æ»‘è†œç‚Žçš„æœºåˆ¶.

OBJECTIVES: To observe the effect of moxibustion on activities of NOD-like receptor family protein 3 (NLRP3)/cysteine aspartic acid specific protease-1 (Caspase-1)/interleukin-1ß (IL-1ß) signaling pathway in rats with adjuvant arthritis (AA), so as to explore its mechanisms underlying improvement of rheumatoid arthritis (RA). Me-thods Thirty male Wistar rats were randomly divided into normal control, AA model and moxibustion groups, with 10 rats in each group. The AA model was replicated by raising in wind, cold and damp environment combined with complete Freund's adjuvant injection. In the moxibustion group, moxibustion was applied to bilateral "Shenshu" (BL23) and "Zusanli"(ST36) for 20 min each time, once daily for 21 days. Changes of joint swelling degree (JSD) and arthritis index (AI) in each group were observed. The ultrastructural changes of synovial cells in each group were observed by transmission electron microscopy. The protein expression levels of NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, tumor necrosis factor-α (TNF-α) and IL-1ß in the synovial tissues of the knee joint were measured by Western blot. RESULTS: Compared with the normal control group, JSD, AI and the protein expressions of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß in the synovial tissues were significantly increased (P<0.01) in the model group. In comparison with the model group, JSD, AI and the protein expression levels of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß were significantly decreased (P<0.01) in the moxibustion group. Results of transmission electron microscope showed an irregular and vague nuclear membrane of synovial cells, and unclear mitochondrial membrane boundary with sparse, swelling crests in the model group, which was relatively milder in the damage degree in the moxibustion group. CONCLUSIONS: Moxibustion can relieve the inflammatory response in the synovial membrane of AA rats, which may be related to its function in down-regulating synovial NLRP3/Caspase-1/IL-1ß inflammatory signaling.

[Expressions of NLRP1 and NLRC4 inflammasomes in prostate cancer and their clinical value].

OBJECTIVE: To explore the clinical value of the inflammasomes NLRP1 and NLRC4 in the diagnosis and treatment of PCa. METHODS: Using immunohistochemical staining, we detected the expressions of the inflammasomes NLRP1 and NLRC4 and the inflammatory cytokines IL-18 and IL-1ß in 54 cases of BPH and 58 cases of PCa treated in Pinghu First People's Hospital from January 2022 to May 2022. We analyzed the characteristics of their expressions in the two groups of patients and the correlation of NLRP1 and NLRC4 expressions with tPSA, fPSA and Gleason scores in the PCa patients. Based on the Cancer Genome Atlas dataset, we compared the expressions of NLRP1 and NLRC4 in different stages of PCa. RESULTS: The NLRP1 and NLRC4 expressions were significantly increased in the PCa patients (P < 0.001). The expression of NLRP1 was linearly correlated with those of IL-1ß and IL-18 (P < 0.05), and so was the expression of NLRC4 with that of IL-18 (P < 0.05). The expressions of NLRP1 and NLRC4 were positively correlated with the Gleason scores of the PCa patients (P < 0.05), the former remarkably higher in T3 and T4 than in T1 (P > 0.05), and the latter markedly higher in T2, T3 and T4 than in T1 (P < 0.05). CONCLUSION: The inflammasomes NLRP1 and NLRC4 are highly expressed in PCa and facilitate tumorgenesis by promoting the maturation and release of the inflammatory cytokines IL-1ß and IL-18, which indicates their important role in the progression of tumors and clinical value in the risk assessment and prognosis of PCa.

NOD-like Receptor Signaling Pathway in Gastrointestinal Inflammatory Diseases and Cancers.

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are intracellular proteins with a central role in innate and adaptive immunity. As a member of pattern recognition receptors (PRRs), NLRs sense specific pathogen-associated molecular patterns, trigger numerous signaling pathways and lead to the secretion of various cytokines. In recent years, cumulative studies have revealed the significant impacts of NLRs in gastrointestinal (GI) inflammatory diseases and cancers. Deciphering the role and molecular mechanism of the NLR signaling pathways may provide new opportunities for the development of therapeutic strategies related to GI inflammatory diseases and GI cancers. This review presents the structures and signaling pathways of NLRs, summarizes the recent advances regarding NLR signaling in GI inflammatory diseases and GI cancers and describes comprehensive therapeutic strategies based on this signaling pathway.

Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

GSK-3α aggravates inflammation, metabolic derangement, and cardiac injury post-ischemia/reperfusion.

Reperfusion after acute myocardial infarction further exaggerates cardiac injury and adverse remodeling. Irrespective of cardiac cell types, loss of specifically the α isoform of the protein kinase GSK-3 is protective in chronic cardiac diseases. However, the role of GSK-3α in clinically relevant ischemia/reperfusion (I/R)-induced cardiac injury is unknown. Here, we challenged cardiomyocyte-specific conditional GSK-3α knockout (cKO) and littermate control mice with I/R injury and investigated the underlying molecular mechanism using an in vitro GSK-3α gain-of-function model in AC16 cardiomyocytes post-hypoxia/reoxygenation (H/R). Analysis revealed a significantly lower percentage of infarct area in the cKO vs. control hearts post-I/R. Consistent with in vivo findings, GSK-3α overexpression promoted AC16 cardiomyocyte death post-H/R which was accompanied by an induction of reactive oxygen species (ROS) generation. Consistently, GSK-3α gain-of-function caused mitochondrial dysfunction by significantly suppressing mitochondrial membrane potential. Transcriptomic analysis of GSK-3α overexpressing cardiomyocytes challenged with hypoxia or H/R revealed that NOD-like receptor (NLR), TNF, NF-κB, IL-17, and mitogen-activated protein kinase (MAPK) signaling pathways were among the most upregulated pathways. Glutathione and fatty acid metabolism were among the top downregulated pathways post-H/R. Together, these observations suggest that loss of cardiomyocyte-GSK-3α attenuates cardiac injury post-I/R potentially through limiting the myocardial inflammation, mitochondrial dysfunction, and metabolic derangement. Therefore, selective inhibition of GSK-3α may provide beneficial effects in I/R-induced cardiac injury and remodeling. KEY MESSAGES: GSK-3α promotes cardiac injury post-ischemia/reperfusion (I/R). GSK-3α regulates inflammatory and metabolic pathways post-hypoxia/reoxygenation (H/R). GSK-3α overexpression upregulates NOD-like receptor (NLR), TNF, NF-kB, IL-17, and MAPK signaling pathways in cardiomyocytes post-H/R. GSK-3α downregulates glutathione and fatty acid metabolic pathways in cardiomyocytes post-H/R.

Macrophage CAPN4 regulates CVB3-induced cardiac inflammation and injury by promoting NLRP3 inflammasome activation and phenotypic transformation to the inflammatory subtype.

Exploring the immune mechanism of coxsackievirus B3 (CVB3)-induced myocarditis may provide a promising therapeutic strategy. Here, we investigated the regulatory role of macrophage CAPN4 in the phenotypic transformation of macrophages and NOD-like receptor protein 3 (NLRP3) inflammasome activation. We found that CAPN4 was the most upregulated subtype of the calpain family in CVB3-infected bone marrow-derived macrophages (BMDMs) and Raw 264.7 cells after CVB3 infection and was upregulated in cardiac macrophages from CVB3-infected mice. Conditional knockout of CAPN4 (CAPN4flox/flox; LYZ2-Cre, CAPN4-cKO mice) ameliorated inflammation and myocardial injury and improved cardiac function and survival after CVB3 infection. Enrichment analysis revealed that macrophage differentiation and the interleukin signaling pathway were the most predominant biological processes in macrophages after CVB3 infection. We further found that CVB3 infection and the overexpression of CAPN4 promoted macrophage M1 polarization and NLRP3 inflammasome activation, while CAPN4 knockdown reversed these changes. Correspondingly, CAPN4-cKO alleviated CVB3-induced M1 macrophage transformation and NLRP3 expression and moderately increased M2 transformation in vivo. The culture supernatant of CAPN4-overexpressing or CVB3-infected macrophages impaired cardiac fibroblast function and viability. Moreover, macrophage CAPN4 could upregulate C/EBP-homologous protein (chop) expression, which increased proinflammatory cytokine release by activating the phosphorylation of transducer of activator of transcription 1 (STAT1) and 3 (STAT3). Overall, these results suggest that CAPN4 increases M1-type and inhibits M2-type macrophage polarization through the chop-STAT1/STAT3 signaling pathway to mediate CVB3-induced myocardial inflammation and injury. CAPN4 may be a novel target for viral myocarditis treatment.

Hypertonic saline induces inflammation in human macrophages through the NLRP1 inflammasome.

Nebulized hypertonic saline (3-7%) is commonly used to increase mucociliary clearance in patients with chronic airway disease and/or virus infections. However, altered salt concentrations may contribute to inflammatory responses. The aim of this study was to investigate whether 500 mM NaCl (3%) triggers inflammation in human macrophages and identify the molecular mechanisms involved. NaCl-induced pyroptosis, IL-1ß, IL-18 and ASC speck release were measured in primary human monocyte-derived macrophages. Treatment with the recombinant IL-1 receptor antagonist anakinra or the NLRP3 inhibitor MCC950 did not affect NaCl-mediated inflammasome assembly. Knock-down of NLRP1 expression, but not of NLRP3 and NLRC4, reduced NaCl-induced pyroptosis, pro-inflammatory cytokine and ASC speck release from human THP-1-derived macrophages. Data from this study suggest that 3% NaCl-induced inflammatory responses in human macrophages depend on NLRP1 and inflammasome assembly. Targeting inflammation in addition to inhalation with hypertonic saline may benefit patients with inflammatory airway disease.

The CXCL12-CXCR4-NLRP3 axis promotes Schwann cell pyroptosis and sciatic nerve demyelination in rats.

Studies have shown that the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome is detrimental to the functional recovery of the sciatic nerve, but the regulatory mechanisms of the NLRP3 inflammasome in peripheral nerves are unclear. C-X-C motif chemokine 12 (CXCL12) can bind to C-X-C chemokine receptor type 4 (CXCR4) and participate in a wide range of nerve inflammation by regulating the NLRP3 inflammasome. Based on these, we explore whether CXCL12-CXCR4 axis regulates the NLRP3 inflammasome in the peripheral nerve. We found that CXCR4/CXCL12, NLRP3 inflammasome-related components, pyroptosis-related proteins and inflammatory factors in the sciatic nerve injured rats were markedly increased compared with the sham-operated group. AMD3100, a CXCR4 antagonist, reverses the activation of NLRP3 inflammasome, Schwann cell pyroptosis and sciatic nerve demyelination. We further treated rat Schwann cells with LPS (lipopolysaccharide) and adenosine triphosphate (ATP) to mimic the cellular inflammation model of sciatic nerve injury, and the results were consistent with those in vivo. In addition, both in vivo and in vitro experiments demonstrated that AMD3100 treatment reduced the phosphorylation of nuclear factor κB (NF-κB) and the expression of thioredoxin interacting protein (TXNIP), which contributes to activating NLRP3 inflammasome. Therefore, our findings suggest that, after sciatic nerve injury, CXCL12-CXCR4 axis may promote Schwann cell pyroptosis and sciatic nerve demyelination through activating NLRP3 inflammasome and slow the recovery process of the sciatic nerve.

Transcriptome Revealed the Macrophages Inflammatory Response Mechanism and NOD-like Receptor Characterization in Siberian Sturgeon (Acipenser baerii).

Nucleotide-binding and oligomerization domain-like receptors (NOD-like receptors, NLRs) can regulate the inflammatory response to eliminate pathogens and maintain the host's homeostasis. In this study, the head kidney macrophages of Siberian sturgeon were treated with lipopolysaccharide (LPS) to induce inflammation by evaluating the expression of cytokines. The high-throughput sequencing for macrophages after 12 h treatment showed that 1224 differentially expressed genes (DEGs), including 779 upregulated and 445 downregulated, were identified. DEGs mainly focus on pattern recognition receptors (PRRs) and the adaptor proteins, cytokines, and cell adhesion molecules. In the NOD-like receptor signaling pathway, multiple NOD-like receptor family CARD domains containing 3-like (NLRC3-like) were significantly downregulated, and pro-inflammatory cytokines were upregulated. Based on the transcriptome database, 19 NLRs with NACHT structural domains were mined and named in Siberian sturgeon, including 5 NLR-A, 12 NLR-C, and 2 other NLRs. The NLR-C subfamily had the characteristics of expansion of the teleost NLRC3 family and lacked the B30.2 domain compared with other fish. This study revealed the inflammatory response mechanism and NLRs family characterization in Siberian sturgeon by transcriptome and provided basic data for further research on inflammation in teleost.