NOD-like Receptor Research Articles

Effects of Clausena lansium leaves volatile oil emulsion against Staphylococcus aureus in mice via autophagy modulation

BackgroundVolatile oil from fresh Clausena lansium (Lour.) Skeels (Rutaceae) (common name Wampee) has been previously extracted by our group from fresh C. lansium leaf and its components were qualitative and quantitatively analyzed by GC-MS. It altered the cell membrane permeability of Staphylococcus aureus and reduced the levels of inflammation factors. However, previous in vivo reports on the anti-inflammatory and the antibacterial properties against S. aureus are scarce. Hypothesis/PurposeTo evaluate the protective in vivo effects of Wampee leaves volatile oil emulsion (WVOE) against S. aureus-induced pneumonia and elucidate the underlying mechanisms of action. MethodsWild-type and nucleotide oligomerization domain-like receptor protein 3 (NLRP3)-deficient mice were used. Mice were treated with WVOE for 7 days, and subjected to S. aureus infection by nasal administration on day 5 for 48 h. Lung and blood samples were collected for assessing lung damage and protein abundance. Lung bacterial load, wet/dry ratio, C-reactive protein (CRP) levels, inflammatory cytokines secretion, and lung histopathological injury were examined. ResultsWVOE effectively reduced lung bacterial load, wet/dry ratio, and CRP levels increased following S. aureus infection in mice. WVOE decreased the secretion of inflammatory cytokines (IL-6 and TNF-α) and lung histopathological injury, and suppressed the NF-κB pathway and NLRP3 inflammasome activation. NLRP3-/- mice exhibited lower bacterial load, inflammatory cytokines levels and lung histopathological injury compared with mice in the model group. Autophagy was enhanced in S. aureus-infected mice, with higher levels of p-mTOR, Beclin-1, Atg16L1, Atg7, p62, p-p62, and LC3II. WVOE administration restored the autophagy related protein levels. Autophagy was inhibited in NLRP3-/- mice of the control and model groups, and WVOE lost its ability to regulate the autophagy-related proteins enhanced upon S. aureus infection. WVOE enhanced autophagy to alleviate lung injury by inhibiting NLRP3-targeted P62. Furthermore, compared with the 3MA + model group, WVOE reduced the bacterial load and CRP levels, pulmonary septa narrowing, and congestion. NLRP3 protein expression increased due to autophagy inhibition. WVOE exerted a pharmacological effect through the PI3K/AKT/mTOR pathway. ConclusionWVOE regulated the PI3K/AKT/mTOR pathway and enhanced autophagy, with NLRP3 playing a crucial role. WVOE exhibited protective effects against S. aureus-induced pneumonia by inhibiting NLRP3 inflammasome activation and enhancing autophagy. These findings expand the understanding of antibacterial properties of WVOE, and provide novel insights into the therapeutic potential of WVOE in managing S. aureus infections.

4-Hydroxybenzoic acid restrains Nlrp3 inflammasome priming and activation via disrupting PU.1 DNA binding activity and direct antioxidation

Reactive oxygen species (ROS) production is considered central to triggering the nucleotide-binding domain-like receptor family pyrin domain containing 3 (Nlrp3) inflammasome activation and the subsequent inflammatory responses. Coenzyme Q10 (CoQ10) plays a critical role in maintaining intracellular ROS homeostasis and inhibiting excessive Nlrp3 inflammasome activation. However, direct supplementation of CoQ10 showed unsatisfactory clinical improvement due to its limited absorption and bioavailability. Therefore, stimulating endogenous CoQ10 biosynthesis by supplementing CoQ10 precursors may provide a more promising therapeutic approach. In this study, we described the role of 4-hydroxybenzoic acid (4-HBA), a precursor of CoQ10, in attenuating excessive inflammatory responses. We found that while supplementation of 4-HBA inhibited the priming and activation of Nlrp3 inflammasome, this effect was independent of its metabolic transformation into CoQ10. 4-HBA itself exhibits antioxidative activities. Furthermore, 4-HBA can disrupt the binding activity of PU.1 on the promoters of Tlr4 and Md2, thereby directly suppressing Nlrp3 inflammasome priming during LPS-induced inflammatory responses. Therefore, strategically utilizing 4-HBA or increasing 4-HBA intake may represent a potential strategy for reducing excessive inflammation.

Bioactive Constituents and the Molecular Mechanism of Melastoma dodecandrum Lour. in the Treatment of Inflammation Based on Network Pharmacology and Molecular Docking

Background: Melastoma dodecandrum Lour. (MD) is a component used in traditional Chinese medicine that is widely distributed in southern China. MD has long been used clinically to treat various diseases, such as inflammation. However, the potential anti-inflammatory mechanism of MD remains to be elucidated. Objective: In this study, network pharmacology and experimental validation have been used to explore the underlying mechanism of MD in inflammation. Methods: The chemical composition of MD was determined using ultra-high performance liquid chromatography-electrospray ionization-high resolution mass spectrometry (UHPLC-ESI-HRMS). The effects of MD on pro-inflammatory cytokines, such as NO, i-NOS, IL-1β, and TNF-α, in RAW264.7 cells stimulated by lipopolysaccharide (LPS) were determined by ELISA and QRT-PCR. Through the analysis of multiple databases, targets for the treatment of inflammation with MD were identified. Other extensive analyses included PPI, GO, and KEGG pathway enrichment, which were completed through the use of the STRING database, Cytoscape software, and the DAVID database. Key targets and key components have been selected for molecular docking. Results: A total of 33 active components were identified in MD, and 134 common targets were obtained and used to construct the networks. Of these, 10 core components and 10 core targets of MD in the treatment of inflammation were identified. The GO and KEGG enrichment analyses revealed that the common targets were involved in multiple signaling pathways, including the PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, chemokine signaling pathway, and IL-17 signaling pathway. The molecular docking methods confirmed the high affinity between bioactive molecules of MD and their targets in inflammation. Two core targets (PIK3CA and AKT) and three core components (asiatic acid, apigenin, and kaempferol) were found to be closely related to MD in the treatment of inflammation. In vitro, MD exerted a significant effect on LPS-stimulated NO, IL- 1β, and TNF-α secretion, and iNOS, IL-1β, and TNF-α expressions in macrophages. Conclusion: This study has demonstrated the bioactive constituents and mechanisms of MD in inhibiting the secretion of inflammatory factors and the multicomponent, multitarget, and multipathway treatment characteristics involved in inflammation, but this still needs further in vivo/in vitro experiments.

Golgi apparatus regulated pyroptosis through the miR-32-5p/Golga7/NLRP3 axis in chicken splenic lymphocytes exposure to ammonia

Ammonia, a common toxic gas, posed a hazard to both human and chickens. The Golgi apparatus, an essential organelle, helped maintain the internal environment of the organism and supported the protein foundation for the endoplasmic reticulum to be involved in pyroptosis. Thus, the Golgi apparatus has garnered significant attention. The purpose of our research was to explore the mechanisms of Golgin A7 (Golga7) involved in pyroptosis after chicken exposure to ammonia. To reach our goal, we first created an in vitro ammonia model to study the effect of ammonia on chicken splenic lymphocyte pyroptosis. Then, leveraging this model, we established Golga7 and miR-32-5p knockdown and overexpression models to investigate their roles in ammonia-induced pyroptosis. We found the ultrastructural changes in the nucleus, Golgi apparatus, and mitochondria of chicken splenic lymphocytes exposure to ammonia. The damage of mitochondria increased the level of Reactive Oxygen Species (ROS), which caused the down-regulation of miR-32-5p. The miR-32-5p inhibitor increased the expression of Golga7 and pyroptosis-related genes (NOD-like receptor protein 3 (NLRP3), Cysteine aspartase-1 (Caspase-1), Golgin A3 (Golga3), Nuclear Factor-kappa B (NF-κB), and Tumor Necrosis Factor-alpha (TNF-α)), which induced the pyroptosis, but when miR-32-5p mimic/si-Golga7 (Golga7 inhibitor) was utilized, these effects were reduced. Our research demonstrated that miR-32-5p/Golga7 regulated NLRP3 involving in the pyroptosis of chicken splenic cells exposed to ammonia. Our study provided a valuable foundation for the prevention and treatment chickens ammonia poisoning in the livestock production.

SARS-CoV-2 disrupts host gene networks: unveiling key hub genes as potential therapeutic targets for COVID-19 management

Although the end of COVID-19 as a public health emergency was declared on May 2023, still new cases of the infection are reported and the risk remains of new variants emerging that may cause new surges in cases and deaths. While clinical symptoms have been rapidly defined worldwide, the basic body responses and pathogenetic mechanisms acting in patients with SARS-CoV-2 infection over time until recovery or death require further investigation. The understanding of the molecular mechanisms underlying the development and course of the disease is essential in designing effective preventive and therapeutic approaches, and ultimately reducing mortality and disease spreading. The current investigation aimed to identify the key genes engaged in SARS-CoV-2 infection. To achieve this goal high-throughput RNA sequencing of peripheral blood samples collected from healthy donors and COVID-19 patients was performed. The resulting sequence data were processed using a wide range of bioinformatics tools to obtain detailed modifications within five transcriptomic phenomena: expression of genes and long non-coding RNAs, alternative splicing, allel-specific expression and circRNA production. The in silico procedure was completed with a functional analysis of the identified alterations. The transcriptomic analysis revealed that SARS-CoV-2 has a significant impact on multiple genes encoding ribosomal proteins (RPs). Results show that these genes differ not only in terms of expression but also manifest biases in alternative splicing and ASE ratios. The integrated functional analysis exposed that RPs mostly affected pathways and processes related to infection-COVID-19 and NOD-like receptor signaling pathway, SARS-CoV-2-host interactions and response to the virus. Furthermore, our results linked the multiple intronic ASE variants and exonic circular RNA differentiations with SARS-CoV-2 infection, suggesting that these molecular events play a crucial role in mRNA maturation and transcription during COVID-19 disease. By elucidating the genetic mechanisms induced by the virus, the current research provides significant information that can be employed to create new targeted therapeutic strategies for future research and treatment related to COVID-19. Moreover, the findings highlight potentially promising therapeutic biomarkers for early risk assessment of critically ill patients.

Analysis and identification of ferroptosis-related diagnostic markers in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune, inflammatory joint disease. There is growing evidence that ferroptosis is involved in the pathogenesis of RA. This study aimed to search for diagnostic markers of ferroptosis in RA and to analyse the potential mechanisms and clinical value. RA-associated datasets were used from the publicly available GEO database. Three methods of machine learning were applied to screen biomarkers. The diagnostic efficacy of the results was also verified by receiver operating characteristic (ROC) curve, external dataset, qRT-PCR and Western blot. Enrichment analysis was performed in this process, while protein-protein interaction (PPI) analysis and immune infiltration correlation analysis were performed using biomarkers, and competing endogenous RNA (ceRNA) networks were constructed to search for prospective therapeutic targets. MMP13 and GABARAPL1 can be used as ferroptosis diagnostic genes in RA. The ROC curve and PPI result demonstrated that MMP13 and GABARAPL1 had an excellent diagnostic value. The results of signature genes in the external dataset, qRT-PCR and Western blot further confirm our findings. The enrichment analysis showed that p53, MAPK and NOD-like receptor signalling pathways may be involved in the process of ferroptosis in RA. In addition, two ferroptosis diagnostic genes in RA participate in the occurrence of ferroptosis in RA via oxidative stress, metabolism and immune response. Immune infiltration analysis showed that RA extensively infiltrated B cells, T cells, macrophages and other immune cells. Persistent immune activation may be an essential reason for the progression of ferroptosis in RA. We also obtained five potential therapeutic agents for RA and some long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) regulating ferroptosis diagnostic genes. Our study suggests that MMP13 and GABARAPL1, which are closely linked with oxidative stress and immunological modulation, can be used as ferroptosis-related potential diagnostic markers in RA and provide new clues regarding the diagnostic and therapeutic targets of ferroptosis in RA.

Nuclear receptor subfamily 4 group a member 1 eases angiotensin II-arose oxidative stress in vascular smooth muscle cell by boosting nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 transcription

Objective: Hypertension significantly contributes to morbidity and mortality. Nuclear receptor subfamily 4 group a member 1 (Nur77) participates in regulating oxidative stress, but the mechanism in hypertension remains unclear. This study aimed to explore the function of Nur77 in oxidative stress induced by Angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) in hypertension. Material and Methods: First, models of VSMC with Nur77, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 3 (NLRC3) and tumor necrosis factor receptor-associated factor 6 (TRAF6) knockdown or overexpression were constructed using Short Hairpin RNA (Nur77) or pcDNA3.1 vector, respectively. Next, the putative-binding motifs between Nur77 and NLRC3 promoters were detected by dual luciferase assay. We conducted reverse transcription quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis to detect Nur77, NLRC3, and TRAF6 levels in VSMCs. Then, cell counting kit-8 assay, 5-ethynyl-2’-deoxyuridine assay, wound-healing assay, enzyme-linked immunosorbent assay, and 2’,7’-dichlorofluorescin diacetate were employed to examine the impact of the knockdown or overexpression of Nur77, NLRC3, and TRAF6 on VSMCs treated with Ang II. The assays measured cell viability and proliferation, cell migration, malondialdehyde levels, and reactive oxygen species levels. Results: The overexpression of Nur77 repressed cell growth (P < 0.001), migration (P < 0.01), and oxidative stress (P < 0.01) induced by Ang II in VSMCs. Nur77 transcriptionally promoted the expression of NLRC3 (P < 0.001), and the upregulation of NLRC3 suppressed cell proliferation (P < 0.05) and oxidative stress (P < 0.001) mediated by Ang II. Furthermore, NLRC3 negatively regulated the TRAF6/nuclear factor-kappa B (NF-κB) axis activated by Ang II, which resulted in the repression of hyperproliferation of VSMCs (P < 0.01) and oxidative stress (P < 0.001). Conclusion: Nur77 suppressed growth and oxidative stress induced by Ang II in VSMCs by promoting NLRC3 transcription, which, further, repressed the TRAF6/NF-κB axis. This understanding provides novel insights into the pathogenesis of hypertension.

Association and interaction analysis of NLRP3 gene polymorphisms with hypertension risk: a case-control study in China.

The NLRP3 inflammasome, a pivotal mechanism regulating inflammatory responses and featuring the pyrin domain containing 3 (NLRP3) within the NOD-like receptor family, is widely recognized as a central pathogenic factor in cardiovascular diseases. The present study endeavors to delve into the correlation and potential interplay between the rs10754558 polymorphism of NLRP3 and the predisposition to hypertension among the Chinese adult population. All the participants who came from a community in Bengbu, China were investigated by being interviewed with a questionnaire. Overall, 354 paired case-control participants were analyzed. Genomic DNA was extracted from 5ml venous blood using the Tiangen DNA extraction kit. The rs10754558 polymorphism of the NLRP3 gene was genotyped by TaqMan allelic discrimination real-time PCR.The association between the rs10754558 polymorphism and hypertension risk was investigated by a logistic regression analysis. Furthermore, an additive interaction analysis was conducted using related indicators, including the relative excess risk due to interaction (RERI), attributable proportion due to interaction (AP), and synergy index (SI). Participants carrying the GG genotype were more likely to develop hypertension than participants carrying the CC genotype (adjusted odds ratio [OR]: 2.16, 95% confidence interval [CI]: 1.33-3.52). A significant additive interaction between the NLRP3 polymorphism and obesity status concerning the risk of hypertension was observed, as estimated by all indicators: RERI (1.12, 95% CI: 0.70-1.5), AP (0.34, 95% CI: 0.14-0.53), and SI (1.92, 95% CI: 1.03-3.59). The values of RERI (1.74, 95% CI: 0.37-3.11), AP (0.46, 95% CI: 0.21-0.70), and SI (2.62, 95% CI: 1.18-5.83) showed that a significant interaction between the rs10754558 polymorphism and a family history of hypertension. Our findings indicate a significant association between the NLRP3 rs10754558 polymorphism and the risk of hypertension in Chinese adults. Moreover, a notable additive interaction emerges between NLRP3 polymorphisms and obesity status, further amplifying the risk of hypertension.

Pattern Recognition Receptors in Periodontal Disease: Molecular Mechanisms, Signaling Pathways, and Therapeutic Implications

Periodontal disease remains a significant global health concern, characterized by complex host–pathogen interactions leading to tissue destruction. This review explored the role of pattern recognition receptors (PRRs) in the pathogenesis of periodontal disease, synthesizing current knowledge on their molecular mechanisms and potential as therapeutic targets. We examined the diverse family of PRRs, focusing on toll-like receptors (TLRs) and NOD-like receptors (NLRs), elucidating their activation by periodontal pathogens and subsequent downstream signaling cascades. This review highlights the intricate interplay between PRR-mediated pathways, including NF-κB and MAPK signaling, and their impact on inflammatory responses and bone metabolism in periodontal tissues. We discussed the emerging concept of PRR crosstalk and its implications for periodontal homeostasis and disease progression. Furthermore, this review addressed the potential of PRR-targeted therapies, exploring both challenges and opportunities in translating molecular insights into clinical applications. By providing an overview of PRRs in periodontal health and disease, this review aims to stimulate future research directions and inform the development of novel diagnostic and therapeutic strategies in periodontology.

Thorase deficiency causes both Aβ accumulation and tau hyperphosphorylation in mouse brain.

The pathogenesis of two major pathogenic characters-amyloid beta (Aβ) accumulation and hyperphosphorylated tau protein-in the brains of patients with Alzheimer's disease (AD) remains unclear. Western blot and immunofluorescence staining were performed to detect the proteins in the brains of Thorase conditional knockout/transgenic mice and their littermates. A co-immunoprecipitation assay was applied to examine the Thorase-interacting proteins. Genetic deletion of Thorase resulted in tau hyperphosphorylation and promoted Aβ accumulation in the mouse brain. Conversely, Thorase overexpression alleviated the pathogenesis of AD. Thorase regulated the phosphorylation of tau by targeting specific kinases and theprotein phosphatase 2B (PP2B). Thorase deficiency also impaired microglial phagocytosis and induced neuroinflammation by the activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3)inflammasomes in microglia. Thorase may be a potential druggable target for developing therapeutic approaches to treat AD and other neurodegenerative diseases. Thorase deletion leads to elevated amyloid beta (Aβ) deposition and hyperphosphorylated tau accumulation in the brain. Thorase regulates the phosphorylation of tau protein via PP2B. Thorase deficiency impairs microglial phagocytosis and promotesNLRP3-mediated neuroinflammation. Overexpression of Thorase alleviates Aβ deposition and tau phosphorylation in the AD mouse model.

Myoferlin alleviates pressure overload-induced cardiac hypertrophy and dysfunction by inhibiting NLRP3-mediated pyroptosis

Myoferlin (MYOF) is a muscle-derived secretory protein. Recent studies have found that MYOF protects against cell damage. However, the role of MYOF in cardiac hypertrophy remains unclear. Increasing evidence suggests that NLRP3 (NOD-like receptor protein 3) and the pyroptosis cascade play critical roles in the development of cardiac hypertrophy and inflammation. To investigate the role of MYOF in cardiac hypertrophy, we conducted a transverse aortic constriction (TAC) experiment in a mouse model. We found that MYOF can improve cardiac hypertrophy and cardiac function. Furthermore, our study confirmed a connection between cardiac hypertrophy and myocardial pyroptosis. Cardiac hypertrophy significantly increased the proportion of apoptotic cells and upregulated apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and gasdermin D (GSDMD). This suggests that pharmacological or genetic inhibition of NLRP3 can effectively reduce cardiac hypertrophy. An abnormal increase in NLRP3 can reverse the cardioprotective effects of MYOF. Our findings indicate that MYOF is a potential therapeutic agent for cardiac hypertrophy.

Mechanism of Resveratrol on LPS/ATP-induced pyroptosis and inflammatory response in HT29 cells

Pyroptosis plays an important role in maintenance of intestinal homeostasis, the abnormal activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome can promote the event and development of ulcerative colitis (UC). Its protective effects such as inhibiting pyroptosis in various inflammation-related diseases have been demonstrated, but whether resveratrol (RES) can also alleviate the progression of the disease by inhibiting pyroptosis in UC and the mechanism have rarely been studied. In this study, lipopolysaccharide (LPS) combined with adenosine triphosphate (ATP) was used to induce HT29 human colon cancer cells to construct an intestinal epithelial cell pyroptosis and inflammation model in vitro to investigate the anti-inflammatory effect of RES, reveal the regulatory mechanism of RES on pyroptosis, and provide a new theoretical basis for the treatment of UC. In vitro experiences, HT29 cells were dividing into control group, LPS/ATP group, RES low-dose group, RES high-dose group, NF-κB inhibitor pyrrolidine dithiocarbamate group (PDTC group), and LPS/ATP+PDTC group. The mRNA expressions of pyroptosis-related indicators such as NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-1(CASP1), IL-18, IL-1β, and inflammatory factors such as TNF-α and IL-6 were detected by qRT-PCR. The protein expressions of pyroptosis-related indicators NLRP3, ASC, CASP1, IL-18, IL-1β, NF-κB-p65 in the nucleus, and IκBα and p-IκBα in the cytoplasm were detected by Western blot. Immunofluorescence saw the distribution and expression of NLRP3, ASC and NF-κB-p65 protein in each group. The morphology and degree of pyroptosis in each group were observed by transmission electron microscope. The results showed that compared with the control group, the pyroptosis-related proteins including NLRP3, ASC, CASP1, IL-18, IL-1β, and inflammatory factors including TNF-α and IL-6 in the LPS/ATP group were increased, and LPS/ATP activated the activity of NF-κB signaling pathway. Compared with the LPS/ATP group, RES downregulated the expression of pyroptosis-related proteins and inflammatory factors in HT29 cells, and inhibited the activation of the NF-κB signaling pathway in HT29 cells pyroptosis. RES down-regulates the pyroptosis of HT29 cells induced by LPS/ATP and the expression of pyroptosis-related indicators NLRP3, ASC, CASP1, IL-18, IL-1β and inflammatory factors TNF-α and IL-6 in the inflammatory response and inhibits the occurrence of pyroptosis. The mechanism is related to the inhibition of NF-κB pathway activity.

Copper oxide nanoparticles exacerbate chronic obstructive pulmonary disease by activating the TXNIP-NLRP3 signaling pathway.

Although copper oxide nanoparticles (CuONPs) offer certain benefits to humans, they can be toxic to organs and exacerbate underlying diseases upon exposure. Chronic obstructive pulmonary disease (COPD), induced by smoking, can worsen with exposure to various harmful particles. However, the specific impact of CuONPs on COPD and the underlying mechanisms remain unknown. In this study, we investigated the toxic effects of CuONPs on the respiratory tract, the pathophysiology of CuONPs exposure-induced COPD, and the mechanism of CuONPs toxicity, focusing on thioredoxin-interacting protein (TXNIP) signaling using a cigarette smoke condensate (CSC)-induced COPD model. In the toxicity study, CuONPs exposure induced an inflammatory response in the respiratory tract, including inflammatory cell infiltration, cytokine production, and mucus secretion, which were accompanied by increased TXNIP, NOD-like receptor protein 3 (NLRP3), caspase-1, and interleukin (IL)-1β. In the COPD model, CuONPs exposure induced the elevation of various indexes related to COPD, as well as increased TXNIP expression. Additionally, TNXIP-knockout (KO) mice showed a significantly decreased expression of NLRP3, caspase-1, and IL-1β and inflammatory responses in CuONPs-exposed COPD mice. These results were consistent with the results of an in vitro experiment using H292 cells. By contrast, TNXIP-overexpressed mice had a markedly increased expression of NLRP3, caspase-1, and IL-1β and inflammatory responses in CuONPs-exposed COPD mice. We elucidated the exacerbating effect of CuONPs exposure on the respiratory tract with underlying COPD, as well as related signaling transduction via TXNIP regulation. CuONPs exposure significantly increased inflammatory responses in the respiratory tract, which was correlated with elevated TXNIP-NLRP3 signaling.

NLRP12/C1qA positive feedback in tumor-associated macrophages regulates immunosuppression through LILRB4/NF-κB pathway in lung adenocarcinoma.

The anti-tumor immune response is greatly hindered by the protumor polarization of tumor-associated macrophages (TAMs). Cancer-related inflammation plays a central role in TAMs protumor polarization. Our study explored the unique positive feedback loop between inflammasome and complement in TAMs. The present study identified NOD-like receptors family pyrin domain containing 12 (NLRP12) formed positive feedback with C1qA and drove TAMs protumor polarization via the LILRB4/NF-κB pathway. In addition, NLRP12 was predominantly expressed in TAMs and was associated with poorer prognosis in lung adenocarcinoma (LUAD) patients. Knocking down LILRB4 inhibited TAMs protumor polarization. NLRP12-overexpressing TAMs promoted tumor cells' malignant progression and inhibited T cells' proliferation and cytotoxic function. Lastly, NLRP12 knockout (NLRP12-/-) reversed macrophage polarization, enhanced T-cell anti-tumor immunity, and suppressed tumor growth. Our findings highlighted the essential role of NLRP12/C1qA positive feedback loop and the LILRB4/NF-κB pathway in promoting TAMs protumor polarization. Inhibition of NLRP12 suppressed tumor development and promoted immune response. NLRP12 may be a promising target for LUAD immunotherapy.

TMIC-70. CORRELATION OF LLT-1 AND NLRC4 INFLAMMASOME AND ITS EFFECT ON GLIOBLASTOMA PROGNOSIS

Abstract PURPOSE LLT-1 is a well-known ligand for the natural killer (NK) cell inhibitory receptor NKRP1A. Here, we examined NLRC4 inflammasome components and LLT-1 expression in glioblastoma (GBM) tissues to elucidate potential associations and interactions between these factors. METHODS Nine GBM tissues were collected for experiments and RNA sequencing, while fifty-two tumor core tissues were collected for RNA sequencing. Expression of LLT-1 and other proteins was assessed by immunofluorescence. Computational analyses utilized RNA-seq data from 296 and 52 patients from the Chinese Glioma Genome Atlas and CHA medical records, respectively, using survival, non-negative matrix factorization clustering, Gene Ontology enrichment, and protein-protein interaction analyses. Receptor-ligand interactions between tumor and immune cells were confirmed by single cell RNA sequencing analysis. RESULTS In GBM tissues, LLT-1 was predominantly colocalized with Glial fibrillary acidic protein (GFAP) -expressing astrocytes, but not with microglial markers like Iba-1. Additionally, LLT-1 and activated NLRC4 inflammasomes were co-expressed mainly in intratumoral astrocytes, suggesting an association between LLT-1, NLRC4, and glioma malignancy. High LLT-1 expression correlates with poor prognosis, particularly in the mesenchymal subtype, and is associated with TNF and NOD-like receptor signaling pathway enrichment, indicating a potential role in tumor inflammation and progression. At the single-cell level, mesenchymal-like malignant cells were highly enriched in the TNF, NLR, and IL-1 signaling pathways compared to other malignant cell types. CONCLUSION We revealed an association between NLRC4 inflammasome activity and LLT-1 expression, suggesting a novel regulatory pathway involving TNF, inflammasomes, and IL-1, potentially offering new anti-glioma approaches by enhancing NK cell-based treatments.

The Knob Domain of the Fiber-1 Protein Affects the Replication of Fowl Adenovirus Serotype 4

Fowl adenovirus serotype 4 (FAdV-4) outbreaks have caused significant economic losses in the Chinese poultry industry since 2015. The relationships among viral structural proteins in infected hosts are relatively unknown. To explore the role of different parts of the fiber-1 protein in FAdV-4-infected hosts, we truncated fiber-1 into fiber-1-Δ1 (73–205 aa) and fiber-1-Δ2 (211–412 aa), constructed pEF1α-HA-fiber-1-Δ1 and pEF1α-HA-fiber-1-Δ2 and then transfected them into leghorn male hepatocyte (LMH) cells. After FAdV-4 infection, the roles of fiber-1-Δ1 and fiber-1-Δ2 in the replication of FAdV-4 were investigated, and transcriptome sequencing was performed. The results showed that the fiber-1-Δ1 and fiber-1-Δ2 proteins were the shaft and knob domains, respectively, of fiber-1, with molecular weights of 21.4 kDa and 29.6 kDa, respectively. The fiber-1-Δ1 and fiber-1-Δ2 proteins were mainly localized in the cytoplasm of LMH cells. Fiber-1-Δ2 has a greater ability to inhibit FAdV-4 replication than fiber-1-Δ1, and 933 differentially expressed genes (DEGs) were detected between the fiber-1-Δ1 and fiber-1-Δ2 groups. Functional analysis revealed these DEGs in a variety of biological functions and pathways, such as the phosphoinositide 3-kinase–protein kinase b (PI3K–Akt) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, cytokine–cytokine receptor interactions, Toll-like receptors (TLRs), the Janus tyrosine kinase–signal transducer and activator of transcription (Jak–STAT) signaling pathway, the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) signaling pathway, and other innate immune pathways. The mRNA expression levels of type I interferons (IFN-α and INF-β) and proinflammatory cytokines (IL-1β, IL-6 and IL-8) were significantly increased in cells overexpressing the fiber-1-Δ2 protein. These results demonstrate the role of the knob domain of the fiber-1 (fiber-1-Δ2) protein in FAdV-4 infection and provide a theoretical basis for analyzing the function of the fiber-1 protein of FAdV-4.

Klrb1 Loss Promotes Chronic Hepatic Inflammation and Metabolic Dysregulation

Background/Objectives: CD161, encoded by the KLRB1 gene, is an inhibitory receptor expresses on various immune cell and has gained attention in immune checkpoint research. In recent studies, KLRB1 has been found to be one of the potential markers of liver diseases such as cirrhosis. Therefore, it will be important to understand what process KLRB1 involved in the liver for the prevention of liver diseases. Methods: We compared KO mice with wild-type controls by routine blood analysis and RNA-seq, and additionally performed H&E staining and qPCR to validate the differentially expressed genes (DEGs). Results:KO mice had fewer lymphocytes compared to the wild-type mice. A transcriptomic analysis showed that Klrb1 loss causes the upregulation of immune-related genes and pathways like NOD-like receptor and p53 signaling, while causing the downregulation of lipid metabolism-related genes. A protein interaction analysis indicated a potential cancer risk under chronic inflammation. Histological examination with H&E staining reveals an inflammatory response around the central venous vessels in the liver tissue of the KO mice. Conclusions: We conclude that Klrb1 knockout disrupts the immune and metabolic functions in the liver, which may possibly lead to chronic inflammation and malignancy risks. These findings highlight the role of Klrb1 in hepatic health.

Evaluation of the Biological Effect of a Nicotinamide-Containing Broad-Spectrum Sunscreen on Photodamaged Skin.

UVA-UVB increases skin matrix metalloproteinases and breaks down extracellular proteins and fibrillar type1 collagen, leading to photodamage. Topical application of nicotinamide prevents UV-induced immunosuppression. Several studies have demonstrated the importance of protection against UV. This study aims to determine the biological effect of a high broad-spectrum UVB-UVA sunscreen containing nicotinamide and panthenol (SSNP) on photodamaged skin using linear confocal optical coherence tomography (LC-OCT), immunohistochemistry, and RNA profiling. Two areas of severely photodamaged forearm skin (L01 and L02) and one less sun-damaged (naturally protected) area on the inner part of the forearm (L03) were identified in 14 subjects. These areas were imaged using LC-OCT and L01 and L03 were biopsied at baseline. After 4weeks of treatment with SSNP, L02 was reimaged using LC-OCT, and biopsied. Histology, immunostaining with p21, p53, PCNA, and CPD, and RNA sequencing were performed in all samples. LC-OCT analysis showed that epidermis thickness and the number of keratinocytes is higher in the sun-exposed areas than in the non-exposed areas. Comparing before and after treatment, even though there is a trend towards normalization, the differences were not statistically significant. The expression of p21, PCNA, p53, and CPD increased in severely photodamaged skin compared to less-damaged skin. When comparing before and after treatment, only p21 showed a trend to decrease expression. RNA sequencing analysis identified 1552 significant genes correlating with the progression from non-visibly photodamaged skin to post-treatment and pre-treatment samples; in the analysis comparing pre- and post-treatment samples, 5429 genes were found to be significantly associated. A total of 1115 genes are common in these two analyses. Additionally, nine significant genes from the first analysis and eight from the second are related to collagen. Six of these collagen genes are common in the two analyses. MAPK and cGMP-PKG signalling pathways are upregulated in the progression to photodamage analysis. In the pre- and post-treatment analysis, 32 pathways are downregulated after treatment, the most statistically significant being the ErbB, Hippo, NOD-like receptor, TNF, and NF-kB signalling pathways. This study demonstrates the role of SSNP in collagen generation, highlights the relevance of the cGMP-PKG and MAPK signalling pathways in photodamage, and shows the ability of SSNP to downregulate pathways activated by UV exposure. Additionally, it deepens our understanding of the effect of SSNP on immune-related pathways.

Naringenin can Inhibit the Pyroptosis of Osteoblasts by Activating the Nrf2/HO-1 Signaling Pathway and Alleviate the Differentiation Disorder of Osteoblasts Caused by Microgravity.

Naringenin (4,5,7-trihydroxyflavone, NAR) is an effective active ingredient in Rhizoma Drynariae, which has many biological functions, encompassing anti-inflammatory and -oxidant functions. Prior research has shown that NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes possessed a significant contribution to osteoporosis. However, the NAR impact on bone loss caused by microgravity remains unclear. Classical microgravity simulation methods were used to induce simulated microgravity (SMG) in mice and cells. Microcomputed tomography, immunohistochemical examination, and hematoxylin and eosin staining were implemented to ascertain alterations in bone microstructure and morphology in mice subsequent to NAR gavage. Cellular investigations were implemented encompassing quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence labeling to investigate the molecular mechanism behind NAR resistance to microgravity-induced bone loss. Our research has shown that NAR can significantly enhance the SMG-stimulated alterations in bone microstructure and morphology in mice, mainly by increasing the trabecular thickness, bone volume fraction, and trabecular number while increasing the bone trabecula number. Cell experiments also showed that SMG caused the activation of inflammatory corpuscles of NLRP3 and induced pyroptosis simultaneously, which can be confirmed by the upregulation of protein and mRNA expression levels such as those of NLRP3, cleaved caspase-1, gasdermin D, and apoptosis-associated speck-like protein. The occurrence of pyroptosis further led to the disorder of osteogenic differentiation, which showed that the osteopontin, Runt-related transcription factor 2, bone morphogenetic protein 2, and alkaline phosphatase expression levels were decreased. The intervention of NAR can activate the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway, reverse this phenomenon via controlling the reactive oxygen species generation in cells and correcting mitochondrial malfunction, weaken the pyroptosis of osteoblasts (OBs), and promote osteogenic differentiation. In summary, NAR could hinder the pyroptosis of OBs caused by SMG and promote osteogenic differentiation via activating the Nrf2/HO-1 pathway. This provides a unique view for inhibiting bone loss under weightlessness and confirms the NAR capacity in treating microgravity-stimulated bone loss, giving new ideas and methods for future space medicine development.

Targeting GPR39 in structure-based drug discovery reduces Ang II-induced hypertension.

The endothelium-dependent vascular injury, a primary pathological feature of angiotensin II (Ang II)-induced hypertension. This study aimed to explore the role and underlying mechanisms of G protein-coupled receptor 39 (GPR39) in the pathogenesis of Ang II-induced hypertension. For in vivo studies, GPR39 knockout (KO) mice (C57BL/6 J, male) were generated and administered Ang II for 4 weeks. GPR39 expression was upregulated in the aorta of hypertensive patients and mice. The ablation of GPR39 mitigated vascular fibrosis, augmented endothelium-dependent vasodilation, and inhibited endothelial inflammation, oxidative stress, and apoptosis in mice. Additionally, GPR39 KO decreased NOD-like receptor protein 3 (Nlrp3) gene expression in Ang II-stimulated endothelial cells. Notably, Nlrp3 activation counteracted the therapeutic benefits of GPR39 KO. We identified the potential ligand of GPR39 using structure-based high throughput virtual screening (HTVS) and validated its antihypertensive function in vitro and in vivo. The small molecule ligand Z1780628919 of GPR39 can also reduce Ang II-induced hypertension and improve vascular function. GPR39 KO and the small molecule ligand Z1780628919 potentially downregulates Nlrp3, thereby mitigating vascular fibrosis, endothelial inflammation, oxidative stress, and apoptosis. This effect contributes to the alleviation of Ang II-induced hypertension and the rectification of vascular dysfunctions. These findings suggest new avenues for therapeutic intervention.