Comparative analysis of rabies pathogenic and vaccine strains detection by RIG-I-like receptors.

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.

BODIPY-Based Analogue of the TREM2-Binding Molecular Adjuvant Sulfavant A, a Chemical Tool for Imaging and Tracking Biological Systems.

Recently, we described synthetic sulfolipids named Sulfavants as a novel class of molecular adjuvants based on the sulfoquinovosyl-diacylglycerol skeleton. The members of this family, Sulfavant A (1), Sulfavant R (2), and Sulfavant S (3), showed important effects on triggering receptor expressed on myeloid cells 2 (TREM2)-induced differentiation and maturation of human dendritic cells (hDC), through a novel cell mechanism underlying the regulation of the immune response. As these molecules are involved in biological TREM2-mediated processes crucial for cell survival, here, we report the synthesis and application of a fluorescent analogue of Sulfavant A bearing the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene moiety (Me4-BODIPY). The fluorescent derivative, named PB-SULF A (4), preserving the biological activity of Sulfavants, opens the way to chemical biology and cell biology experiments to better understand the interactions with cellular and in vivo organ targets and to improve our comprehension of complex molecular mechanisms underlying the not fully understood ligand-induced TREM2 activity.

Functional diversification of a wild potato immune receptor at its center of origin.

Plant cell surface pattern recognition receptors (PRRs) and intracellular immune receptors cooperate to provide immunity to microbial infection. Both receptor families have coevolved at an accelerated rate, but the evolution and diversification of PRRs is poorly understood. We have isolated potato surface receptor Pep-13 receptor unit (PERU) that senses Pep-13, a conserved immunogenic peptide pattern from plant pathogenic Phytophthora species. PERU, a leucine-rich repeat receptor kinase, is a bona fide PRR that binds Pep-13 and enhances immunity to Phytophthora infestans infection. Diversification in ligand binding specificities of PERU can be traced to sympatric wild tuber-bearing Solanum populations in the Central Andes. Our study reveals the evolution of cell surface immune receptor alleles in wild potato populations that recognize ligand variants not recognized by others.

[Application of TIGIT combined with interleukin-6 detection in the evaluation of Crohn's disease status].

To explore the application of IL-6, PCT, T lymphocyte subsets and TIGIT expression on T lymphocytes in the evaluation of Crohn's disease status. Using a cross-sectional study, total of 119 confirmed patients with Crohn's disease who were treated in the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine from June 2020 to December 2022 were selected. The age range was 18-59 years old, and the median age (interquartile range) was 37 (29, 45) years old, including 57 cases in active disease group (30 males, 27 females), 62 cases in disease remission group (33 males, 29 females); 50 healthy control groups (27 males, 23 females), the age range was 19-60 years old, and the median age (interquartile range) was 38 (31, 46) years old. The level of IL-6 was detected by flow fluorescence microsphere method, the concentration of PCT was detected by immunochromatography, and the levels of T lymphocyte subsets and TIGIT were detected by flow cytometry. The differences and correlations between the detection indicators in each group were compared, logistic regression was used to analyze the factors influencing the progression of Crosne's disease and the clinical value of each detection indicator was analyzed by ROC curve. The results showed that there were no statistically significant differences in age and gender among the control group, the remission group, and the active group (H=1.422,χ2=0.020;P=0.491, P=0.990); in the active group, IL-6 was 17.55(9.67, 21.72)pg/ml, PCT was 0.38(0.14, 0.43)ng/ml, CD3+CD4+ was 35.47%±6.01%, CD3+CD8+ was 30.50%±5.20%, TIGIT was 25.08%±6.30%; in the remission group, IL-6 was 8.46(5.21, 10.04) pg/ml, PCT was 0.26(0.11, 0.35) ng/ml, CD3+CD4+ was 37.62%±4.86%, CD3+CD8+ was 28.30%±5.28%, TIGIT was 34.22%±5.45%; in the control group, IL-6 was 6.13(3.57, 8.12)pg/ml, PCT was 0.17(0.10, 0.21)ng/m, CD3+CD4+ was 39.74%±3.94%, CD3+CD8+ was 26.59%±4.50%, and TIGIT was 37.64%±6.22%.There were significant differences in IL-6, PCT, CD3+CD4+%, CD3+CD8+%, and TIGIT among the three groups(H=58.688, H=18.003, F=9.600, F=8.124, F=65.059;P<0.001, P<0.001, P<0.001, P<0.001, P<0.001), Among them, IL-6 and TIGIT in the active group were significantly different from those in the remission group (P<0.001, P<0.001), and only TIGIT was significantly different between the remission group and the control group (P=0.007);Spearman correlation analysis showed that the expression of TIGIT on T lymphocytes was negatively correlated with the levels of IL-6; the results of Logistic regression analysis showed that IL-6, PCT and TIGIT were independent factors affecting the progression of Crohn's disease;Comparing the ROC curves of the active group and the remission group, found that TIGIT was significantly different from PCT, CD3+CD4+, CD3+CD8+(Z=4.011, Z=4.091, Z=4.157; P<0.001, P<0.001, P<0.001), no statistical difference with IL-6 (Z=1.193, P=0.233). Selected the combined detection of IL-6 and TIGIT with the best AUC area and Youden index, which shows that the clinical value is improved, the AUC area of IL-6+TIGIT was significantly different from that of IL-6 (Z=2.674, P=0.008). In summary, TIGIT of T lymphocytes and IL-6 detection may be valuable in the diagnosis and treatment of Crohn's disease, and the combined detection of TIGIT and IL-6 may be meaningful for evaluating the status of Crohn's disease.

Huang-Lian-Jie-Du decoction alleviates depressive-like behaviors in dextran sulfate sodium-induced colitis mice via Trem2/Dap12 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du decoction (HLJD), a traditional Chinese medicine prescription, has been implicated as effective in treating colitis, depression and inflammation-related diseases. Whether HLJD decoction could ameliorate colitis-induced depression was still unknown and the underlying mechanism was needed to be clarified. AIM OF THE STUDY: Our study aimed to explore the effect and the underlying mechanism of HLJD treatment on colitis-induced depression and the involvement of the inflammatory factors and microglial-activated related genes. MATERIALS AND METHODS: The chronic colitis model was established by treating male mice with 1% dextran sulfate sodium (DSS) for 8 weeks. One week after DSS-treated, HLJD decoction was administered orally with 2 and 4 g/kg daily for 7 weeks. Behavior tests (Open field/Elevated plus maze/Novel object recognition) and TUNEL staining were then assessed. The expression of inflammatory-related genes and microglial dysregulation were measured by RT-PCR and the expression of Trem2, Danp12 and Iba1 were assessed by immunofluorescence methods. RESULTS: Depressive-like behaviors were observed in mice treated with DSS, which suffered colitis. Compared to normal control (NC-V) mice, the density of TUNEL + cells in the habenula (Hb), hippocampus (HIP), and cortex were significantly higher in colitis (DSS-V) mice, especially in Hb. Compared to NC-V and several brain regions, the expression levels of the Il-1ß, Il-10 and Dap12 mRNA were significantly increased in the lateral habenula (LHb) of colitis mice. Moreover, the expression of Trem2, Dap12 and Iba1 were increased in LHb of DSS-V mice. HLJD treatment could alleviate depressive-like behaviors, reduce the density of TUNEL + cells in Hb and the expression of Il-6, Il-10 and Dap12 mRNA in LHb of DSS-V mice. The overexpression of Trem2, Dap12 and Iba1 in LHb of DSS-V mice were reversed after HLJD treatment. CONCLUSION: These results reveal LHb is an important brain region during the process of colitis-induced depression. HLJD treatment could alleviates depressive-like behaviors in colitis mice via inhibiting the Trem2/Dap12 pathway in microglia of LHb, which would contribute to the precise treatment. It provides a potential mechanistic explanation for the effectiveness of HLJD treatment in colitis patients with depression.

Danggui-Shaoyao-San Promotes Amyloid-ß Clearance through Regulating Microglia Polarization via Trem2 in BV2 Cells.

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative brain disorder currently without satisfactory therapeutic treatments. Triggering receptors expressed on a myeloid cells-2 (Trem2) gene mutation has been reported as a powerful AD risk factor that induces Trem2 gene deletion aggravated microglia disfunction and Amyloid-ß (Aß) aggregation in the brain. The traditional Chinese medicine (TCM) formula Danggui-Shaoyao-San (DSS) has shown therapeutic effect on alleviating the symptoms of AD. However, the neuroprotective effect and underlying mechanism of DSS against AD is still far from fully understood. METHODS: Double-label immunofluorescence and Western blotting were employed to evaluate the different polarization states of mouse BV2 microglial (BV2) cells after lipopolysaccharide (LPS) or interleukin (IL)-4 treatment. Trem2 over-expression lentiviral vector and Trem2 siRNA were used respectively to evaluate the effect of Trem2 on microglia polarization via detecting the proteins expression of iNOS and arginase1 (Arg1) by Western blotting while the Aß-scavenging capacity of BV2 cells was assessed by flow cytometry. Cell counting kit-8 (CCK8) assay was performed to assess the effect of DSS on the viability of BV2 cells. Flow cytometry was used to investigate the effect of DSS on the Aß-scavenging capacity of BV2 cells treated with corresponding concentration of DSS-containing serum. Protein of Trem2 and the gene expression of the M1 or M2 phenotype in BV2 cells treated with DSS after Trem2 over-expression or silence were detected by Western blot and RT-qPCR, respectively. RESULTS: In vitro experiments. DSS exhibited anti-inflammatory and neuroprotective functions. It was found that Trem2 had an effect on inducing a shift of M1 microglia towards the M2 phenotype and enhanced the Aß-scavenging capacity of BV2 cells, further that DSS administration relieved inflammation by engulfing Aß through the activities of Trem2. Importantly, DSS treatment effectively increased the Aß-scavenging capacity of BV2 cells through accelerating the shift of M1 microglia towards an M2 phenotype via increasing Trem2 expression. CONCLUSIONS: Results demonstrated that DSS promoted the clearance of Aß through the regulation of microglia polarization via increased expression of Trem2 in BV2 cells.

Regulation of TREM2 expression by transcription factor YY1 and its protective effect against Alzheimer's disease.

TREM2 encoding the transmembrane receptor protein TREM2 is a risk gene of Alzheimer's disease (AD), and the impairment of TREM2 functions in microglia due to mutations in TREM2 may significantly increase the risk of AD by promoting AD pathologies. However, how the expression of TREM2 is regulated and the transcription factors required for TREM2 expression are largely unknown. By luciferase assay, DNA pull-down, and in silico predictions, we identified Yin Yang 1(YY1) as a binding protein of the minimal promoter of the TREM2 gene, and the binding was further confirmed by EMSA and DNA pull-down assay. shRNA-mediated YY1 silencing significantly reduced the activity of the TREM2 minimal promoter and TREM2 protein levels in the microglial cell line BV2 and the neuroblastoma Neuro2A. Furthermore, we found that the levels of TREM2 and YY1 were both downregulated in lipopolysaccharide-treated BV2 cells and in the brain of AD model mice. These results demonstrated that YY1 plays a crucial role in the regulation of TREM2 expression. Our study suggests that microglial YY1 could be targeted to maintain TREM2 expression for AD prevention and therapy.

Electroacupuncture pretreatment induces ischemic tolerance by neuronal TREM2-mediated enhancement of autophagic flux.

The mechanism of electroacupuncture (EA) pretreatment-induced neuroprotection remains unclear. In this study, we found that neuronal Triggering receptor expressed on myeloid cells 2 (TREM2) expression was increased and peaked at 48 h and 72 h after ischemia/reperfusion. After specific knockdown of TREM2 in excitatory neurons, neurological function was damaged, and the infarct volume was enlarged. Furthermore, the expression of LC3II/LC3I and Beclin1 was decreased, while the expression of p62 was increased. EA pretreatment enhanced TREM2, LC3II/LC3I and Beclin1 expression while reducing p62 in the ischemic penumbra area. The EA-induced neuroprotective effects and improvements in autophagic flux were abolished by specific knockdown of TREM2 in excitatory neurons. Taken together, our findings provide novel mechanistic insight into EA-induced ischemic tolerance and suggest a promising therapeutic strategy of targeting neuronal TREM2 to treat brain ischemia.

Mechanisms and network pharmacological analysis of Yangyin Fuzheng Jiedu prescription in the treatment of hepatocellular carcinoma.

OBJECTIVE: To identify the key drugs of Yangyin Fuzheng Jiedu prescription (YFJP) and investigate their therapeutic effects against hepatocellular carcinoma (HCC) and the potential mechanism using network pharmacology. METHODS: The H22 tumor-bearing mouse model was established. Thirty male BALB/c mice were divided randomly into five groups. The mice were orally treated with either disassembled prescriptions of YFJP or saline solution continuously for 14 days. The mice were weighed every 2 days during treatment and the appearance of tumors was observed by photographing. The tumor inhibition rate and the spleen and thymus indexes were calculated. Hematoxylin and eosin and immunohistochemical staining were performed to observe the histological changes and tumor-infiltrating lymphocytes. Cell apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. The proportion of CD8+ T cells and the expression of programmed cell death protein 1 (PD-1), T cell immunoglobulin domain and mucin domain-3 (Tim-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were analyzed using flow cytometry. The production of serum cytokines was detected using the Milliplex® MAP mouse high sensitivity T cell panel kit. The active components of the key drugs and HCC-related target proteins were obtained from the corresponding databases. The putative targets for HCC treatment were screened by target mapping, and potential active components were screened by constructing a component-target network. The interactive targets of putative targets were obtained from the STRING database to construct the protein-protein interaction network. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes pathway enrichment analyses were performed based on potential targets. The gene-gene inner and component-target-pathway networks were constructed and analyzed to screen the key targets. Western blotting was used to evaluate the protein expression of the key targets in the tumor-bearing mouse model. The binding activity of the key targets and compounds was verified by molecular docking. RESULTS: Among the three disassembled prescriptions of YFJP, the Fuzheng prescription (FZP) showed significant antitumor effects and inhibited weight loss during the treatment of H22 tumor-bearing mice. FZP increased the immune organ index and the levels of CD8+ and CD3+ T cells in the spleen and peripheral blood of H22 tumor-bearing mice. FZP also reduced the expression of PD-1, TIGIT, and TIM3 in CD8+ T cells and the production of IL-10, IL-4, IL-6, and IL-1ß. Network pharmacology and experimental validation showed that the key targets of FZP in the treatment of HCC were PIK3CA, TP53, MAPK1, MAPK3, and EGFR. The therapeutic effect on HCC was evaluated based on HCC-related signaling pathways, including the PIK3-Akt signaling pathway, PD-L1 expression, and PD-1 checkpoint pathway in cancer. GO enrichment analysis indicated that FZP positively regulated the molecular functions of transferases and kinases on the cell surface through membrane raft, membrane microarea, and other cell components to inhibit cell death and programmed cell death. CONCLUSION: FZP was found to be the key disassembled prescription of YFJP that exerted antitumor and immunoregulatory effects against HCC. FZP alleviated T cell exhaustion and improved the immunosuppressive microenvironment via HCC-related targets, pathways, and biological processes.

Effects of Alpinae Oxyphyllae Fructus on microglial polarization in a LPS-induced BV2 cells model of neuroinflammation via TREM2.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of the important traditional Chinese medicines, Alpinia oxyphylla could warm and tonify the kidney and spleen. It has been used as anti-salivation, anti-diarrhea in various diseases. In recent years, many studies have reported the significant effect of Alpinia oxyphylla on improving cognitive ability, anti oxidative stress and protecting neurons. AIMS OF THE STUDY: In this paper, we studied whether AE and its main active components could improve M1 and M2 polarization, inhibit neuroinflammation through triggering receptor expressed on myeloid cells 2 (TREM2), and exert anti-inflammatory effects. MATERIALS AND METHODS: In this paper, the concentrations of inflammatory cytokines such as NO, TNF-α, IL-10 were assessed using detection kits respectively. Arg-1 and Iba-1, as polarized markers of M1 and M2, were detected by Immunofluorescence staining. CD86 and CD206 were tested by flow cytometry as surface markers of M1 and M2. Furthermore, RT-PCR was performed to determinate TNF-α, IL-10, Arg-1, and Iba-1. Western blot was used to test the activation of PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways. TREM2 siRNA treatment further verified the action target of Chrysin, the main active ingredient of Alpinia oxyphylla. Molecular docking study was performed to investigate the binding mode between Chrysin and the human TREM2. RESULTS: We found that AE could promote the phenotypic transformation of microglia from M1 to M2, and similar effects of Chrysin were observed. Furthermore, downregulation of TREM2 blocked the anti-neuroinflammation of Chrysin, and inhibited the shift of M1 phenotype to M2 phenotype. Additionally, TREM2-siRNA suppressed the effects of Chrysin on PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways. CONCLUSIONS: Our findings indicated that AE could improve the polarization response of microglia. TREM2 plays a vital role in the microglial repolarization effects of Chrysin through PI3K/AKT/GSK3ß and BDNF/TrkB/TLR4 signaling pathways regulated by neuroinflammation.

Vitamin D receptor and STAT6 interactome governs oesophageal epithelial barrier responses to IL-13 signalling.

OBJECTIVE: The contribution of vitamin D (VD) deficiency to the pathogenesis of allergic diseases remains elusive. We aimed to define the impact of VD on oesophageal allergic inflammation. DESIGN: We assessed the genomic distribution and function of VD receptor (VDR) and STAT6 using histology, molecular imaging, motif discovery and metagenomic analysis. We examined the role of VD supplementation in oesophageal epithelial cells, in a preclinical model of IL-13-induced oesophageal allergic inflammation and in human subjects with eosinophilic oesophagitis (EoE). RESULTS: VDR response elements were enriched in oesophageal epithelium, suggesting enhanced VDR binding to functional gene enhancer and promoter regions. Metagenomic analysis showed that VD supplementation reversed dysregulation of up to 70% of the transcriptome and epigenetic modifications (H3K27Ac) induced by IL-13 in VD-deficient cells, including genes encoding the transcription factors HIF1A and SMAD3, endopeptidases (SERPINB3) and epithelial-mesenchymal transition mediators (TGFBR1, TIAM1, SRC, ROBO1, CDH1). Molecular imaging and chromatin immunoprecipitation showed VDR and STAT6 colocalisation within the regulatory regions of the affected genes, suggesting that VDR and STAT6 interactome governs epithelial tissue responses to IL-13 signalling. Indeed, VD supplementation reversed IL-13-induced epithelial hyperproliferation, reduced dilated intercellular spaces and barrier permeability, and improved differentiation marker expression (filaggrin, involucrin). In a preclinical model of IL-13-mediated oesophageal allergic inflammation and in human EoE, VD levels inversely associated with severity of oesophageal eosinophilia and epithelial histopathology. CONCLUSIONS: Collectively, these findings identify VD as a natural IL-13 antagonist with capacity to regulate the oesophageal epithelial barrier functions, providing a novel therapeutic entry point for type 2 immunity-related diseases.

Bazi Bushen capsule attenuates cognitive deficits by inhibiting microglia activation and cellular senescence.

CONTEXT: Bazi Bushen capsule (BZBS) has anti-ageing properties and is effective in enhancing memory. OBJECTIVE: To find evidence supporting the mechanisms and biomarkers by which BZBS functions. MATERIALS AND METHODS: Male C57BL/6J mice were randomly divided into five groups: normal, ageing, ß-nicotinamide mononucleotide capsule (NMN), BZBS low-dose (LD-BZ) and BZBS high-dose (HD-BZ). The last four groups were subcutaneously injected with d-galactose (d-gal, 100 mg/kg/d) to induce the ageing process. At the same time, the LD-BZ, HD-BZ and NMN groups were intragastrically injected with BZBS (1 and 2 g/kg/d) and NMN (100 mg/kg/d) for treatment, respectively. After 60 days, the changes in overall ageing status, brain neuron morphology, expression of p16INK4a, proliferating cell nuclear antigen (PCNA), ionized calcium-binding adapter molecule 1 (Iba1), postsynaptic density protein 95 (PSD95), CD11b, Arg1, CD206, Trem2, Ym1 and Fizz1, and the senescence-associated secretory phenotype (SASP) factors were observed. RESULTS: Compared with the mice in the ageing group, the HD-BZ mice exhibited obvious improvements in strength, endurance, motor coordination, cognitive function and neuron injury. The results showed a decrease in p16INK4a, Iba1 and the upregulation of PCNA, PSD95 among brain proteins. The brain mRNA exhibited downregulation of Iba1 (p < 0.001), CD11b (p < 0.001), and upregulation of Arg1 (p < 0.01), CD206 (p < 0.05), Trem2 (p < 0.001), Ym1 (p < 0.01), Fizz1 (p < 0.05) and PSD95 (p < 0.01), as well as improvement of SASP factors. CONCLUSIONS: BZBS improves cognitive deficits via inhibition of cellular senescence and microglia activation. This study provides experimental evidence for the wide application of BZBS in clinical practice for cognitive deficits.

Exon 2-mediated deletion of Trem2 does not worsen metabolic function in diet-induced obese mice.

Triggering receptor expressed on myeloid cells 2 (Trem2) is highly expressed on myeloid cells and is involved in cellular lipid homeostasis and inflammatory processes. Trem2 deletion in mice (Trem2-/- ) evokes adipose tissue dysfunction, but its role in worsening obesity-induced metabolic dysfunction has not been resolved. Here we aimed to determine the causal role of Trem2 in regulating glucose homeostasis and insulin sensitivity in mice. Nine-week-old male and female littermate wild-type (WT) and Trem2-/- mice were fed a low- or high-fat diet for 18 weeks and phenotyped for metabolic function. Diet-induced weight gain was similar between genotypes, irrespective of sex. Consistent with previous reports, we find that loss of Trem2 causes massive adipocyte hypertrophy and an attenuation in the lipid-associated macrophage transcriptional response to obesity. In contrast to published data, we find that loss of Trem2 does not worsen metabolic function in obese mice. No differences in intraperitoneal glucose tolerance (ipGTT), oral GTT or mixed meal substrate control, including postprandial glucose, non-esterified fatty acids, insulin or triglycerides, were found between WT and Trem2-/- animals. Similarly, no phenotypic differences existed when animals were challenged with stressors on metabolic demand (i.e. acute exercise or environmental temperature modulation). Collectively, we report a disassociation between adipose tissue remodelling caused by loss of Trem2 and whole-body metabolic homeostasis in obese mice. The complementary nature of experiments conducted gives credence to the conclusion that loss of Trem2 is unlikely to worsen glucose homeostasis in mice.

Inhibitory Effects of Saururus chinensis Extract on Receptor for Advanced Glycation End-Products-Dependent Inflammation and Diabetes-Induced Dysregulation of Vasodilation.

Advanced glycation end-products (AGEs) and the receptor for AGEs (RAGE) are implicated in inflammatory reactions and vascular complications in diabetes. Signaling pathways downstream of RAGE are involved in NF-κB activation. In this study, we examined whether ethanol extracts of Saururus chinensis (Lour.) Baill. (SE) could affect RAGE signaling and vascular relaxation in streptozotocin (STZ)-induced diabetic rats. Treatment with SE inhibited AGEs-modified bovine serum albumin (AGEs-BSA)-elicited activation of NF-κB and could compete with AGEs-BSA binding to RAGE in a dose-dependent manner. Tumor necrosis factor-α (TNF-α) secretion induced by lipopolysaccharide (LPS)-a RAGE ligand-was also reduced by SE treatment in wild-type Ager+/+ mice as well as in cultured peritoneal macrophages from Ager+/+ mice but not in Ager-/- mice. SE administration significantly ameliorated diabetes-related dysregulation of acetylcholine-mediated vascular relaxation in STZ-induced diabetic rats. These results suggest that SE would inhibit RAGE signaling and would be useful for the improvement of vascular endothelial dysfunction in diabetes.

Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability remain. Molecular adjuvants targeting pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulates various PRRs, including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors. We hypothesized that targeting PRRs using molecular adjuvants on nanoparticles (NPs) along with a stabilized spike protein antigen could stimulate broad and efficient immune responses. Adjuvants targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer NPs were combined with the S1 subunit of spike protein and assessed in vitro with isogeneic mixed lymphocyte reactions (isoMLRs). For in vivo studies, the adjuvant-NPs were combined with stabilized spike protein or spike-conjugated NPs and assessed using a two-dose intranasal or intramuscular vaccination model in mice. Combination adjuvant-NPs simultaneously targeting TLR and RIG-I receptors (MPLA+PUUC, CpG+PUUC, and R848+PUUC) differentially induced T cell proliferation and increased proinflammatory cytokine secretion by APCs in vitro. When delivered intranasally, MPLA+PUUC NPs enhanced CD4+CD44+ activated memory T cell responses against spike protein in the lungs while MPLA NPs increased anti-spike IgA in the bronchoalveolar (BAL) fluid and IgG in the blood. Following intramuscular delivery, PUUC NPs induced strong humoral immune responses, characterized by increases in anti-spike IgG in the blood and germinal center B cell populations (GL7+ and BCL6+ B cells) in the draining lymph nodes (dLNs). MPLA+PUUC NPs further boosted spike protein-neutralizing antibody titers and T follicular helper cell populations in the dLNs. These results suggest that protein subunit vaccines with particle-delivered molecular adjuvants targeting TLR4 and RIG-I could lead to robust and unique route-specific adaptive immune responses against SARS-CoV-2.

TREM-2 mediates dendritic cell-induced NO to suppress Th17 activation and ameliorate chronic kidney diseases.

Chronic kidney disease (CKD) is a global public health issue. CKD is caused by the infiltration of various myeloid cell types into renal tissue, resulting in renal fibrosis and tubular atrophy. Unilateral ureteral obstruction (UUO) surgery in mice is a model of CKD and characterized by high expression of the anti-inflammatory receptor, Triggering receptor expressed on myeloid cells 2 (TREM-2), on myeloid cells in affected kidneys. Here, we show that iNOS expression and nitric oxide (NO) induction were decreased in Trem-2-/- bone marrow-derived DCs (BMDCs) and in Trem-2 knockdown DC2.4 cells stimulated in vitro with LPS. The nitration of RORγt was decreased in T cells co-cultured with LPS-stimulated Trem-2-/- BMDCs, enhancing IL-17 production. UUO-treated Trem-2-/- mice displayed aggravated renal pathogenesis accompanied by greater neutrophil infiltration and enhanced Th17 cells differentiation, phenotypes that could be rescued by the administration of L-arginine (a biological precursor of NO). Our data identify a key mechanism underlying TREM-2-mediated NO to modulate the cellular crosstalk between dendritic cells, Th17, and neutrophils. Furthermore, we also reveal TREM-2 as a potential novel target for the development of anti-inflammatory drugs in CKD treatment. KEY MESSAGES: The expression of TREM-2 is increased in nephritis TREM-2+ DCs maintain NO production to negatively regulate Th17 differentiation The severe pathologies of nephritis can be rescued by L-arginine supplementation.

Effects of electroacupuncture on DNA methylation of the TREM2 gene in senescence-accelerated mouse prone 8 mice.

OBJECTIVE: To explore the mechanism by which electroacupuncture (EA) upregulates triggering receptor expressed on myeloid cells 2 (TREM2) protein in the hippocampus of Alzheimer's disease (AD) model animals from the perspective of TREM2 DNA methylation. METHODS: In total, 24 eight-month-old senescence-accelerated mouse prone 8 (SAMP8) mice were divided into an (untreated) AD group (n = 8), donepezil group (receiving donepezil treatment, n = 8) or EA group (receiving an EA intervention, n = 8). A healthy control group comprising 8-month-old senescence-accelerated mouse resistant 1 (SAMR1) mice (n = 8) was also included. Western blotting, bisulfite sequencing, and oxidative bisulfite sequencing were applied to test the relative expression of TREM2 protein and the methylation levels of the TREM2 gene. RESULTS: EA significantly upregulated the relative expression of TREM2 protein (p < 0.01), downregulated the 5-methylcytosine level (p < 0.01) and upregulated the 5-hydroxymethylcytosine level (p < 0.05) in the hippocampus. CONCLUSION: Downregulation of 5-methylcytosine levels and upregulation of 5-hydroxymethylcytosine levels in the TREM2 gene might be the mechanism by which EA promotes the expression of TREM2 protein.

Epimedii Folium and Curculiginis Rhizoma ameliorate lipopolysaccharides-induced cognitive impairment by regulating the TREM2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Neuroinflammation induced by microglia is closely related to a variety of neurodegenerative diseases including Alzheimer's disease (AD). Previous study has found that aqueous extract of Epimedii Folium and Curculiginis Rhizoma (EX) had anti-inflammatory effect on AD by activating the NLRP3 inflammasome and inhibiting NF-κB/MAPK pathway. However, whether the anti-neuroinflammatory effect of EX is related to microglia or not remains unclear. AIM OF THE STUDY: The present study aimed to investigate the protective effect of EX on cognitive impairment induced by LPS and explore the underlying mechanism of EX. MATERIALS AND METHODS: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was performed to qualify the major components of EX, EX in the serum and cerebrospinal fluid. To evaluate the anti-inflammatory effects of EX in vivo, the mice were orally administrated with EX (2.34, 4.68 g kg-1•d-1) for 28 days before cotreatment with LPS (1 mg kg-1•d-1, i.p.). The leaning and memory abilities of mice were examined by Morris water maze test. The expression of inflammatory related proteins and the activation of microglia were detected by ELISA, immunofluorescence, real-time PCR and Western blotting. RESULTS: HPLC-MS analysis confirmed and quantified 9 components in EX, 5 components in the serum and 4 components in the cerebrospinal fluid. In a LPS-induced neuroinflammatory mouse model, EX was found to exert anti-inflammatory activity by reducing the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), regulating the expression of different phenotypes of microglia, and increasing the expression of proteins related with TREM2 in the hippocampus tissue. Moreover, LPS-induced microglia activation was markedly attenuated in the hippocampus. CONCLUSIONS: These findings demonstrate that EX exerts anti-neuroinflammatory effects via reducing the production of inflammatory mediators, regulating the conversion of microglia and activating the proteins related with TREM2. EX might become a novel herb pairs to treat neuroinflammatory diseases.

1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis.

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)2D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)2D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1-/- deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

Phytochemicals in Cancer Immune Checkpoint Inhibitor Therapy.

The interaction of immune checkpoint molecules in the tumor microenvironment reduces the anti-tumor immune response by suppressing the recognition of T cells to tumor cells. Immune checkpoint inhibitor (ICI) therapy is emerging as a promising therapeutic option for cancer treatment. However, modulating the immune system with ICIs still faces obstacles with severe immunogenic side effects and a lack of response against many cancer types. Plant-derived natural compounds offer regulation on various signaling cascades and have been applied for the treatment of multiple diseases, including cancer. Accumulated evidence provides the possibility of efficacy of phytochemicals in combinational with other therapeutic agents of ICIs, effectively modulating immune checkpoint-related signaling molecules. Recently, several phytochemicals have been reported to show the modulatory effects of immune checkpoints in various cancers in in vivo or in vitro models. This review summarizes druggable immune checkpoints and their regulatory factors. In addition, phytochemicals that are capable of suppressing PD-1/PD-L1 binding, the best-studied target of ICI therapy, were comprehensively summarized and classified according to chemical structure subgroups. It may help extend further research on phytochemicals as candidates of combinational adjuvants. Future clinical trials may validate the synergetic effects of preclinically investigated phytochemicals with ICI therapy.