TLR4 Expression Research Articles

Chrysanthemum extract mitigates high-fat diet-induced inflammation, intestinal barrier damage and gut microbiota disorder.

An effective intervention for obesity without side effects is needed. Chrysanthemum may be the preferred choice due to its influence in the improvement of glycolipid metabolism. This study assessed the efficacy of chrysanthemum and its flavonoids in mitigating high-fat diet (HFD) induced obesity, focusing on the integrity of the intestinal barrier, inflammation, and gut microbiota. Fifty male C57BL/6J mice were divided into 5 groups randomly: normal control (NC), HFD, HFD with chrysanthemum aqueous extract (CM), HFD with a low-dose flavonoid extract of chrysanthemum (FLL), and HFD with a high-dose flavonoid extract of chrysanthemum (FLH). The results showed that after 9 weeks of intervention with CM, FLL and FLH, the body weight and blood lipid levels of mice were reduced. The chrysanthemum treatment regimens down-regulated the gene expression and protein levels of TLR4, MyD88, TRAF6 and NF-κB, upregulated the gene expression levels of ZO-1 and occludin, and decreased the levels of LPS and diamine oxidase (DAO) in the serum. With CM, FLL and FLH, the levels of the inflammatory factors IL-1β, TNF-α, and IL-6 were decreased, and the abundance of pernicious bacteria Lachnoclostridium, Streptococcus and Enterococcus was decreased. Notably, the purified chrysanthemum flavonoid extract showed greater effects as compared to the CM. The study demonstrated that chrysanthemum extracts could achieve anti-obesity effects by strengthening the intestinal barrier function, relieving inflammation and modulating the gut microbial composition.

CD20 and CD19 promote proliferation driven by the IgM-TLR9-L265P MyD88 complex.

The cancer driver mutation L265P MyD88 is found in approximately 30 % of cases in the activated B cell-like subgroup of diffuse large B cell-like lymphoma (ABC DLBCL). L265P MyD88 forms a complex with TLR9 and IgM, referred to as the My-T-BCR complex, to drive proliferation. We here show that the B cell surface molecules CD19 and CD20 enhance proliferation mediated by the My-T-BCR complex. Using the IL-3-dependent Ba/F3 line transduced to express the IgM complex (IgM, CD79a, and CD79b) and TLR9, we observed proliferation in the presence of anti-IgM antibody and the TLR9 ligand CpG-B. TLR9 was constitutively associated with IgM and L252P MyD88. CD19 promoted proliferation with anti-IgM and CpG-B specifically in L252P MyD88-expressing Ba/F3 cells, while CD20 enhanced the proliferation in both wild-type- and L252P MyD88-expressing Ba/F3 cells. Additionally, CD20 uniquely enabled IgM-mediated proliferation in L252P MyD88-expressing Ba/F3 cells. Although CpG-B was not required for this proliferation, TLR9 expression remained indispensable. In the ABC DLBCL line TMD8, anti-IgM Ab mediated growth was impaired by the lack of CD20 and CD19 or of TLR9. Mechanistically, CD19 promoted IgM-dependent AKT phosphorylation, whereas CD20 increased expression of cell surface IgM, thereby enhancing the formation of the IgM-TLR9 complex. These findings suggest that CD19 and CD20 differentially contribute to the proliferation driven by the My-T-BCR complex.

MiR-125b-5p ameliorates ox-LDL-induced vascular endothelial cell dysfunction by negatively regulating TNFSF4/TLR4/NF-κB signaling

BackgroundOxidized low-density lipoprotein (ox-LDL)-induced endothelial cell dysfunction plays a crucial role in the progression of atherosclerosis (AS). Although miR-125b-5p is known to be involved in cardiovascular and cerebrovascular disorders, its function in ox-LDL-induced endothelial injury is still not well understood.MethodsAn in vitro AS cell model was established by exposing human umbilical vein endothelial cells (HUVECs) to 100 µg/mL ox-LDL for 24 h. A series of functional assays, including CCK-8 assay, flow cytometry, MDA and SOD kits, capillary-like network formation assay and ELISA assay were performed in vitro. TNFSF4/TLR4/NF-κB pathway-related protein expressions were measured by Western blot. Molecular mechanisms were elucidated through quantitative real-time PCR, western blot analysis, and luciferase reporter assays.ResultsOur investigation revealed that exposure to ox-LDL led to a downregulation in miR-125b-5p, while upregulating the expression of tumor necrosis factor (ligand) superfamily, member 4 (TNFSF4), TLR4, p-p65 and p-IkBa in HUVECs in a dose-dependent manner. We confirmed TNFSF4 as a direct target of miR-125b-5p. Ox-LDL exposure led to decreased cell viability and angiogenic capacity, along with increased apoptosis, inflammation, and oxidative stress in HUVECs. These effects were reversed by overexpressing miR-125b-5p or knocking down TNFSF4. Overexpression of TNFSF4 significantly reversed the effects brought about by miR-125b-5p in HUVECs exposed to ox-LDL. Moreover, miR-125b-5p inactivated the TLR4/NF-κB signaling pathway by negatively regulating TNFSF4.ConclusionsIn summary, our findings demonstrate that miR-125b-5p possessed an anti-inflammatory and anti-apoptosis against ox-LDL-induced HUVEC injury by regulating the TNFSF4/TLR4/NF-κB signaling, indicating that miR-125b-5p may have an important therapeutic function for AS.

CD209d/e promotes inflammation and lung injury during influenza virus infection.

Influenza virus infects millions each year, contributing greatly to human morbidity and mortality. Upon viral infection, pathogen-associated molecular patterns activate pattern recognition receptors on host cells, triggering an immune response. The CD209 protein family, homologs of DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin), is thought to modulate immune responses to viruses. The effects of the mouse functional DC-SIGN homolog CD209d/e on the lung immune responses during influenza viral infection are not known. Therefore, we generated mice that lack both CD209d and e isoforms to determine the role in influenza viral infection. We infected wild-type and CD209d/e gene-deficient (CD209d/e-/-) mice with influenza virus and measured the cellular response in bronchoalveolar lavage, the expression of proinflammatory cytokines, antiviral genes, toll-like receptors (TLRs) in the lung, and lung pathology. We found CD209d/e-/- mice had decreased viral burden, TLR3 and TLR9 expression, interferon response, macrophages in bronchoalveolar lavage, and parenchymal lung inflammation compared with control mice. We also found less influenza viral uptake in alveolar macrophages and bone marrow-derived macrophages isolated from CD209d/e-/- mice when compared with control mice. We further investigated the role CD209d/e by treating bone marrow-derived macrophages from control and CD209d/e-/- mice with TLR agonists. We found that lacking CD209d/e decreased the expression of TLR3, TLR9, RIG1, STAT1, and STAT2 compared with controls. Collectively these results show that CD209d/e plays an important role in viral sensing/uptake and inflammatory immune responses during influenza viral infection.

P0180 Bifidobacterium longum alleviates dextran sulfate sodium-induced colitis through the differential regulation of Toll-like receptor 5 on enteric glial cells

Abstract Background Inflammatory bowel disease (IBD) is a chronic immune-associated disease arising from gastrointestinal dysbiosis and subsequent immunological dysfunction. Numerous studies have reported the beneficial effects of probiotics, such as Bifidobacterium longum (BL), on IBD, while the related mechanism behind these effects have not been fully elucidated. Enteric nervous system abnormalities have increasingly been tied to the pathogenesis of IBD, with a particular focus on the role of enteric glial cells (EGC) in this context. In this study, we aim to explore the mechanism of probiotics in the treatment of IBD from the perspective of EGC. Methods On base of C57BL/6 mouse, TLR5 of EGC knockdown mouse model (TLR5 KD mouse) was constructed by adeno-associated virus (serum type 6) (AAV6) which carried the interference sequence of TLR5 gene (AAV6-TLR5-RNAi). Experimental colitis was induced by dextran sulfate sodium (DSS). Mice were treated with phosphate buffer solution (PBS) or BL. Disease activity index (DAI) and histological score were applied to assess the severity of colitis. The level of GDNF、IL-1β of colonic tissue were measured by ELISA. The expression of glial fibrillary acidic protein (GFAP)、toll-like receptor 5 (TLR5)、cleaved caspase-3 of colonic tissue were tested by immunofluorescence staining and quantitative PCR (qPCR). EGC cell line, EGC CRL2690, was exposed to probiotic (Bifidobacterium longum; BL) bacteria and pathogenic (Enterohemorrhagic Escherichia coli; EHEC). Cell apoptosis rate was assessed by flow cytometry. TLRs expression in EGC CRL2690 were evaluated at both baseline and after exposure to bacteria by qPCR and western blot analysis. GDNF and IL-1β release from EGC CRL2690, following exposure to bacteria, were measured in the presence or absence of specific TLR and pathway inhibitors. Results The results showed that BL ameliorated DSS-induced colitis accompanied by increased expression of GDNF, while pathogenic EHEC aggravated DSS-induced colitis combined with decreased expression of GDNF. Mechanistically, BL could up-regulate the secretion of GDNF via TLR5/p38 MAPK pathway in EGC, which was different from EHEC, which could up-regulate the secretion of IL-1β through TLR2/NF-κB pathway. Conclusion Our results suggested a new possible mechanism whereby probiotics ameliorate DSS-induced colitis in mice by regulating EGC via TLR5, namely, BL promotes GDNF secretion in EGC through activation of p38 MAPK signaling pathway by TLR5. Meanwhile, our results suggested that probiotic, pathogenic bacteria appeared to influence the development of intestinal inflammation by differentially modulating the secretion function of EGC via different TLRs.

P0170 The presence of creeping fat in a TNBS-induced colitis model is associated with greater acute inflammation and bacterial translocation

Abstract Background The hyperplasia of the mesenteric adipose tissue in Crohn’s disease, known as creeping fat, is associated with transmural lesions and the translocation of microbial antigens. In a rat model of TNBS-induced colitis, 50% of the animals presented adipose hyperplasia. Macroscopic evaluations showed that these animals had a more advanced progression of the disease, and colonoscopy could predict the presence of fat two days before euthanasia (L Clua et al, ECCO, 2024). Methods Colitis was induced using TNBS (n=10; half female), with the animals being sacrificed after five days. Tissue from the lesion along with mesenteric adipose tissue was obtained for histopathological staining and evaluation (haematoxylin-eosin, Masson’s trichrome, and Gram). Samples of intestinal mucosa and mesenteric fat were preserved in RNAlater to assess the expression of inflammatory markers by qRT-PCR. Results were statistically compared between animals with and without creeping fat, including sex. Results In histopathological analysis, using a validated scoring system, animals with creeping fat showed significantly greater severity of acute inflammation at the transmural level, with an increase in cryptitis and a more pronounced and extensive fibrosis pattern. Additionally, colonies of Gram-positive cocci were detected in all animals with creeping fat. In contrast, animals without fat hyperplasia exhibited appreciable adipogenesis in only two individuals, with an average of 105 adipocytes per field, compared to 185 in animals with creeping fat. qRT-PCR revealed higher expression of Il1b, Il6, Il10, Nod2, and Tlr2 in the intestinal mucosa and hyperplastic mesenteric fat of animals with creeping fat. A significant increase in Tnfa was also observed in hyperplastic adipose tissue, while the expression of Tgfbr1 was higher in the intestinal mucosa of animals without creeping fat. No differences were found between sexes. Conclusion The results indicated a more intense acute inflammatory and fibrotic component in animals with creeping fat, consistent with the macroscopic observations. The hyperplasia and inflammation observed in the mesenteric adipose tissue may be provoked by bacterial translocation, whereas animals without hyperplasia present a component influenced by TGF-β1 activity that could help counteract the inflammation. References L Clua, R Suau, M Guasch, D Monfort-Ferré, I Vañó, C Serena, E Domènech, R Bartolí, J Manyé, P022 Development of a novel experimental model for studying creeping fat in Crohn’s Disease, Journal of Crohn’s and Colitis, Volume 18, Issue Supplement_1, January 2024, Pages i275–i276, https://doi.org/10.1093/ecco-jcc/jjad212.0152

Alleviation of Acute Heat Stress in Broiler Chickens by Dietary Supplementation of Polyphenols from Shredded, Steam-Exploded Pine Particles

Reducing the detrimental effects of heat stress (HS) in poultry is essential to minimize production losses. The present study evaluates the effects of dietary polyphenols prepared from underutilized wood byproducts on the growth, gut health, and cecal microbiota in broilers subjected to acute heat stress (AHS). One hundred eight one-day-old Indian River broilers were fed with 0%, 0.5%, or 1% polyphenols from shredded, steam-exploded pine particles (PSPP) in their diet. On the 37th day, forty birds were equally distributed among four groups containing either a control diet at thermoneutral temperatures (NT0%) or AHS temperatures with 0% (AHS0%), 0.5% (AHS0.5%), and 1% (AHS1%) PSPP-supplemented diets. The temperature in the NT room was maintained at 21.0 °C, while, in the AHS room, it was increased to 31 °C. AHS negatively influenced performance parameters and increased rectal temperature (RT) in broilers. The AHS0% group showed a higher expression of NOX4, HSP-70, and HSP-90 genes, while the expression was lower in PSPP-supplemented birds. In the jejunum, mRNA expression of SOD was increased in all the birds under AHS compared to NT. The expression of the CLDN1 and ZO2 genes was increased in AHS0%, while that of the ZO1 and MUC2 genes was increased in PSPP-supplemented birds. HS tends to increase TLR2 and TLR4 gene expression in chickens. The significantly modified genera were Bariatricus, Sporobacter, Sporanaerobacter, and Natranaerovirga. Concludingly, AHS negatively influences the performance parameters, RT, stress, gut-health-related genes, and pathogenic penetration, but PSPP supplementation reduces its bad impact by overcoming the stress and gut-health-related genes, increasing favorable bacterial abundance and reducing pathogenic penetration in chickens.

PLGA co-loaded Salvia miltiorrhiza polysaccharide and Mn2+ as an adjuvant to induce potent immunity.

Developing a novel and potent adjuvant with excellent biocompatibility for immune response augmentation is crucial for enhancing vaccine efficacy. Here, we prepared a stable PLGA nanoparticle by encapsulating MnCl2/Salvia miltiorrhiza polysaccharide (MS-PLGA) and employed it as an adjuvant in the model antigen OVA (MS-PLGA-OVA) to elicit potent immunity. The biological experiments indicated that the MS-PLGA-OVA could effectively recruit APCs to the injection site and provoke long-term antibodies. Compared with the conventional Alum adjuvanted group, the MS-PLGA-OVA increased the IgG2a antibody titers and CD8+T cells maturation, triggering cytotoxic T lymphocyte response and inducing the activation of memory T cells. Importantly, the MS-PLGA could up-regulate the expression of TLRs and cGAS-STING pathway-related genes, thus increasing the DCs maturation, as well as the secretion of interleukin and IFN-β. Collectively, the MS-PLGA system may provide a novel and efficient adjuvant platform for various prophylactic vaccines and insights for the development of the next-generation nano adjuvant.

Afobazole alleviates streptozotocin-induced diabetic nephropathy in rats via hypoglycemic, antioxidant, anti-inflammatory, and anti-apoptotic properties: Role of the S1R/Nrf2 antioxidant axis.

Sigma-1 receptor (S1R) activation was recently identified as a promising target for preventing diabetic nephropathy (DN) by mitigating hypoxia, oxidative stress, and inflammation. This study aimed to investigate the potential reno-protective effect of the S1R agonist afobazole against streptozotocin (STZ)-induced DN in rats compared to metformin. Rats were split into six groups: the normal control group; the diabetic control group received STZ (55mg/kg i.p.); the other four groups received STZ and were treated with different doses of either afobazole (10, 15, and 20mg/kg) or metformin (200mg/kg). Metabolic parameters and renal function were assessed. Expression levels of oxidative stress markers and inflammatory cytokines were measured using ELISA, apoptosis-related proteins were evaluated using immunohistochemistry, and gene expression of S1R, Nrf2, NF-κB, and TLR-4 was determined. Histopathological analysis was performed on kidney tissues. Both afobazole and metformin exerted hypoglycemic effects, alleviating renal injury, reducing blood urea nitrogen (BUN) and serum creatinine, and restoring oxidant/antioxidant balance in diabetic rats. Both treatments boosted renal S1R and Nrf2 levels, suppressed inflammatory proteins and cytokines, and reduced apoptotic features. The study revealed that afobazole provided nephroprotection in STZ-induced DN through a hypoglycemic, antioxidant, anti-inflammatory, and anti-apoptotic potential mediated by activating the S1R/Nrf2 antioxidant axis. The 15mg/kg dose elicited the most pronounced nephroprotective effects, outperforming other treatment groups.

Sustained Release of HIF-2α Inhibitors Using Biodegradable Porous Silicon Carriers for Enhanced Immunogenic Cell Death of Malignant Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine skin cancer with limited treatment options, often associated with Merkel cell polyomavirus (MCPyV) and marked by hypoxic tumor microenvironments that promote resistance to therapies. Belzutifan, an FDA-approved hypoxia-inducible factor-2α (HIF-2α) inhibitor, has shown promise in inhibiting tumor growth; however, its clinical efficacy is hindered by its low solubility, rapid clearance, and limited bioavailability. In this study, we present a strategy using porous silicon (pSi) microparticles and nanoparticles as carriers for the sustained delivery of benzoate to MCC cells. The pSi carriers were engineered to securely encapsulate and gradually release belzutifan, overcoming the limitations of free drug administration. Microparticles provided sustained extracellular release, while nanoparticles enabled efficient intracellular delivery, enhancing HIF-2α inhibition. Moreover, the use of biodegradable silicon particles enables long-term consistent release of belzutifan over 10 days in vitro with a single dose administration in the tumor microenvironment, while free belzutifan is rapidly deactivated within 1 day postadministration. In vitro studies demonstrated significant immunogenic cell death (ICD) in MCC cells, marked by the cytosolic localization of HMGB1 and elevated expression of pro-inflammatory cytokines as well as strong upregulation of TLR9. Particularly, the increased TLR9 expression in both MCC cell lines with pSi carrier treatment reinforces immune activation through toll-like receptor signaling, enhancing both innate and adaptive immune responses within the tumor microenvironment. These findings indicate that pSi carriers not only enhance belzutifan's stability and release profile but also amplify antitumor immune responses within the tumor microenvironment. Our results suggest that belzutifan-loaded pSi carriers offer a potent and targeted therapeutic strategy for MCC, potentially addressing key challenges in cancer immunotherapy by combining HIF-2α inhibition with robust immune activation. This platform highlights the universal utility of pSi-based delivery systems to advance MCC treatment with implications for broader cancer therapies.

Methyl 3-Bromo-4,5-dihydroxybenzoate Attenuates Inflammatory Bowel Disease by Regulating TLR/NF-κB Pathways.

Inflammatory bowel disease (IBD) is characterized by uncontrolled, chronic relapsing inflammation in the gastrointestinal tract and has become a global healthcare problem. Here, we aimed to illustrate the anti-inflammatory activity and the underlying mechanism of methyl 3-bromo-4,5-dihydroxybenzoate (MBD), a compound derived from marine organisms, especially in IBD, using a zebrafish model. The results indicated that MBD could inhibit the inflammatory responses induced by CuSO4, tail amputation and LPS in zebrafish. Furthermore, MBD notably inhibited the intestinal migration of immune cells, enhanced the integrity of the gut mucosal barrier and improved intestinal peristalsis function in a zebrafish IBD model induced by trinitro-benzene-sulfonic acid (TNBS). In addition, MBD could inhibit ROS elevation induced by TNBS. Network pharmacology analysis, molecular docking, transcriptomics sequencing and RT-PCR were conducted to investigate the potential mechanism. The results showed that MBD could regulate the TLR/NF-κB pathways by inhibiting the mRNA expression of TNF-α, NF-κB, IL-1, IL-1β, IL6, AP1, IFNγ, IKKβ, MyD88, STAT3, TRAF1, TRAF6, NLRP3, NOD2, TLR3 and TLR4, and promoting the mRNA expression of IL4, IκBα and Bcl-2. In conclusion, these findings indicate that MBD could be a potential candidate for the treatment of IBD.

Pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

Hepatocellular carcinoma (HCC) is a global health concern, ranking as the fourth leading cause of cancer-related deaths worldwide. However, the role of piwi-interacting RNAs (piRNAs) in HCC processes has not been extensively explored. Through small RNA sequencing, our study identified a specific piRNA, pir-hsa-216911, which is highly expressed in HCC cells. This overexpression of pir-hsa-216911 promotes HCC cell invasion and inhibits cell death, particularly pyroptosis. Knocking out pir-hsa-216911 led to increased cell pyroptosis activity, resulting in the activation of caspase-1 and GSDMD. Further analysis revealed that pir-hsa-216911 targets and suppresses TLR4, a key gene associated with pyroptosis in HCC. In the Huh7 cell line, pir-hsa-216911 knockout confirmed its role in suppressing the TLR4/NFκB/NLRP3 pathway by silencing TLR4. Knocking out pir-hsa-216911 significantly inhibited the formation of Huh7 xenograft tumor. In HCC patients, pir-hsa-216911 was highly expressed in HCC tumor samples with steatosis, suppressing TLR4 expression and inhibiting GSDMD activation. This study introduces pir-hsa-216911 as a new high-expressing piRNA in HCC, which inhibits pyroptosis by silencing TLR4 to suppress GSDMD activation. These findings have significant implications for HCC molecular subtyping and as a potential target for cancer therapy.

Citronellal Alleviates Insulin Resistance in High-Fat Diet/Streptozocin Model: Role of Asprosin/Olfactory Receptor Axis.

Ectopic olfactory receptors are expressed in nonolfactory tissues and perform diverse roles including regulation of glucose homeostasis. We explored the effect of citronellal treatment on olfactory receptor 4M1 subtype (OR4M1) signaling in insulin resistance and Type II diabetes in rats. We aimed to validate the anti-diabetic effect of citronellal through Asprosin/OR4M1 modulation. Exploring new antidiabetics and pharmacological targets is important to improve quality of life and limit complications. The model was established in Sprague-Dawley rats by a high-fat diet for 4weeks followed by a single low-dose streptozotocin (STZ) (35 mg/kg/ip). One week after STZ injection, oral citronellal (100mg/kg) was administered for 4weeks. Citronellal lowered serum glucose and triglycerides and ameliorated OGTT and HOMA-IR results. Docking results revealed that citronellal blocked the Asprosin binding site at OR4M1. The hepatic expression of OR4M1 and Asprosin was reduced. Citronellal lowered cAMP levels causing attenuated levels of protein kinase A and downstream gluconeogenic enzymes: glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Citronellal also inhibited the expression of hepatic TLR-4 and inhibited JNK phosphorylation. Citronellal attenuated hepatic levels of NF-κB, p-NF-κB, and downstream proteins MCP-1 and TNF-α. These results suggest that citronellal alleviates insulin resistance by mitigating Asprosin/OR4M1 and Asprosin/TLR4/JNK signaling.

Bacillus subtilis MZ-01 alleviates diarrhea caused by ETEC K88 by reducing inflammation and promoting intestinal health.

The purpose of this study was to investigate the effects of Bacillus subtilis supplementation on the health of weaned piglets and whether Bacillus subtilis supplementation can reduce the damage of piglets induced by ETEC K88. The experiment was designed with a 2×2 factorial arrangement, comprising the control (CON) group, Bacillus subtilis (PRO) group, Escherichia coli K88 (ETEC) group, and Bacillus subtilis+ETEC (PRO+ETEC) group. Regardless of the presence of ETEC, the addition of PRO increased the piglets' final body weight (FW), average daily gain (ADG), and daily feed intake (ADFI). Additionally, PRO primarily achieves a reduction in heat-stable enterotoxin (ST) levels, suppresses the expression of NF-κB, TLR4, and MyD88 mRNA in the jejunum and ileum, lowers pro-inflammatory factors in the blood and small intestine, enhances the expression of tight junction proteins in the small intestine, improves the composition of the colonic microbiota, increases colonic short-chain fatty acids (SCFAs) contents, thereby alleviates diarrhea and mitigates bodily damage caused by ETEC K88 infection. The addition of Bacillus subtilis MZ-01 alleviated ETEC K88-induced piglet diarrhoea by reducing heat-stable enterotoxin levels, decreasing pro-inflammatory factors in the blood and intestine, and enhancing the intestinal barrier and tight junction proteins.

TRAIL agonists rescue mice from radiation-induced lung, skin or esophageal injury.

Radiotherapy can be limited by pneumonitis which is impacted by innate immunity, including pathways regulated by TRAIL death receptor DR5. We investigated whether DR5 agonists could rescue mice from toxic effects of radiation and found two different agonists, parenteral PEGylated trimeric-TRAIL (TLY012) and oral TRAIL-Inducing Compound (TIC10/ONC201) could reduce pneumonitis, alveolar-wall thickness, and oxygen desaturation. Lung protection extended to late effects of radiation including less fibrosis at 22-weeks in TLY012-rescued survivors versus un-rescued surviving irradiated-mice. Wild-type orthotopic breast tumor-bearing mice receiving 20-Gy thoracic radiation were protected from pneumonitis with disappearance of tumors. At the molecular level, radioprotection appeared due to inhibition of CCL22, a macrophage-derived chemokine previously associated with radiation pneumonitis and pulmonary fibrosis. Treatment with anti-CCL22 reduced lung injury in vivo but less so than TLY012. Pneumonitis severity was worse in female versus male mice, and this was associated with increased expression of X-linked TLR7. Irradiated mice had reduced esophagitis characterized by reduced epithelial disruption and muscularis externa thickness following treatment with ONC201 analogue ONC212. The discovery that short-term treatment with TRAIL pathway agonists effectively rescues animals from pneumonitis, dermatitis and esophagitis following high doses of thoracic radiation exposure has important translational implications.

Inhibition of RIPK1-driven necroptosis ameliorates inflammatory hyperalgesia caused by lipopolysaccharide: involvement of TLR-, NLRP3-, and caspase-11-mediated signaling pathways.

Increasing evidence suggests that inhibition of receptor-interacting serine/threonine-protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain-like pseudokinase (MLKL) necrosome has protective effects in vivo models of painful conditions seen in humans associated with inflammation and demyelination in the central nervous system. However, the contribution of RIPK1-driven necroptosis to inflammatory pain remains unknown. Therefore, this study aims to determine the effect of necrostatin (Nec) -1s, a selective RIPK1 inhibitor, on lipopolysaccharide (LPS)-induced inflammatory pain and related underlying mechanisms. In the saline-, LPS-, and/or Nec-1s-injected male mice, thermal hyperalgesia was evaluated by hot plate test. Alterations in the expression of proteins involved in the RIPK1, toll-like receptor (TLR) 4, myeloid differentiation factor (MyD) 88/toll-interleukin (IL)-1 receptor domain-containing adapter-inducing interferon-β (TRIF)/nuclear factor (NF)-kB, nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing (NLRP) 3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1, and caspase-11/gasdermin D (GSDMD) signaling pathways, as well as proteins related to demyelination and remyelination in the brain and spinal cord were determined by the immunoblotting method. The LPS-induced alleviation of thermal hyperalgesia was prevented by necrostatin-1s. Necrostatin-1s reversed (1) increased activity of RIPK1, RIPK3, MLKL, and NF-kB p65, (2) enhanced expression of TLR4, MyD88, TRIF, NF-kB p65, HMGB1, NLRP3, ASC, caspase-1 p20, IL-1β, caspase-11 p20, p30-GSDMD, and semaphorin 3A, and (3) diminished myelin PLP expression induced by LPS. These findings suggest that the use of RIPK1 inhibitors could be a therapeutic approach in the management of inflammatory pain associated with necroptosis, pyroptosis, and demyelination.

Effect of rifampicin on Toll-like receptor system gene expression in brain structures of rats with prenatal alcohol exposure

Alcohol intake during pregnancy may affect the normal course of fetal development, causing the symptoms of fetal alcohol spectrum disorders (FASD). There is evidence that a number of prenatal pathologic conditions exhibit altered expression of several pro- and anti-inflammatory cytokines. In our study, we focused on studying expression of numerous pro- and anti-inflammatory cytokine genes in the forebrain and temporal regions of rat brain during the postnatal development, thus modeling prenatal alcohol exposure effects (PAE). We also evaluated expression of genes associated with regulation of genes controlling expression of pro- and anti-inflammatory cytokines. Moreover, the objectives of our study included pharmacological correction of the observed changes by rifampicin (Rif), a potential pharmacological agent which had a neuroprotective effect shown by other studies,. The experimental model of PAE was produced by oral intake of 15% ethanol solution to pregnant female rats throughout pregnancy. The drug injections were performed to the pups from the 1 to the 7 postnatal days. Brain structures were sampled for gene expression analysis on the 8 postnatal day. The results of the study showed distinct changes in Tlr3 and Tlr4 gene expression in anterior and temporal lobes of brain on the 8 day of postnatal development. Expression of Myd88 and Ticam genes showed multidirectional changes among the studied brain structures of PAE rats. The increased mRNA level of proinflammatory genes was noted. Usage of Rif in experiments showed the ability of Rif (50 mg/kg) to correct the observed long-term pathological changes in the expression of the genes under study. It is of interest to study the dose-dependent effect in the future, as well as to investigate the revealed changes at the level of protein analysis. In future studies, it seems important to evaluate TLR signaling system in other brain structures with PAE, as well as at different terms of postnatal development in ontogenesis.

Necrotizing enterocolitis causes increased ileal goblet cell loss in Wnt2b KO mice.

WNT2B is Wnt ligand which is able to support intestinal stem cells (ISC) in culture and support the intestinal epithelium in vivo. We have previously shown that WNT2B is critical for resistance to colitis, but not small intestinal injury, in the adult mouse. WNT2B is thought to coordinate with WNT3 in supporting ISC, and we have also shown that WNT3 expression is low in the early postnatal ileum in mice. Here, we hypothesized that WNT2B may be more critical in the small intestine during early development, and we challenged Wnt2b KO mice and controls with experimental necrotizing enterocolitis (NEC) on postnatal days 5-8. Wnt2b KO mice had similar ileum histology and injury scores to control mice. Molecular analyses showed that Wnt2b KO mice have differences in Lgr5 and Tlr4 expression compared to wild type controls in untreated conditions, but under experimental NEC expression of epithelial markers and inflammatory genes associated with NEC were similar to wild type. Periodic acid Schiff positive cells were lower in the villi of Wnt2b KO mice during NEC, however expression of goblet cell markers was not different compared to wild type mice. We also used an organoid-based NEC model to highlight the epithelium in isolation and also found no impact of WNT2B KO in the setting of NEC. These data further affirm that WNT2B is critical for inflammation responses in the mouse colon, but does not appear to play a major role in the small intestine, no matter the developmental period.

Xixin Decoction's novel mechanism for alleviating Alzheimer's disease cognitive dysfunction by modulating amyloid-β transport across the blood-brain barrier to reduce neuroinflammation.

Xixin Decoction (XXD) is a classical formula that has been used to effectively treat dementia for over 300 years. Modern clinical studies have demonstrated its significant therapeutic effects in treating Alzheimer's disease (AD) without notable adverse reactions. Nevertheless, the specific mechanisms underlying its efficacy remain to be elucidated. This investigation sought to elucidate XXD's impact on various aspects of AD pathology, including blood-brain barrier (BBB) impairment, neuroinflammatory processes, and amyloid-β (Aβ) deposition, as well as the molecular pathways involved in these effects. In vitro experiments were conducted using hCMEC/D3 and HBVP cell coculture to establish an in vitro blood-brain barrier (BBB) model. BBB damage was induced in this model by 24-h exposure to 1μg/mL lipopolysaccharide (LPS). After 24, 48, and 72h of treatment with 10% XXD-medicated serum, the effects of XXD were assessed through Western blotting, RT-PCR, and immunofluorescence techniques. In vivo, SAMP8 mice were administered various doses of XXD via gavage for 8weeks, including high-dose XXD group (H-XXD) at 5.07gkg-1·d-1, medium-dose XXD group (M-XXD) at 2.535gkg-1·d-1, and low-dose XXD group (L-XXD) at 1.2675gkg-1·d-1. Cognitive function was subsequently evaluated using the Morris water maze test. BBB integrity was evaluated using Evans blue staining, and protein expression levels were analyzed via ELISA, Western blotting, and immunofluorescence. In vitro experiments revealed that XXD-containing serum, when cultured for 24, 48, and 72h, could upregulate the expression of P-gp mRNA and protein, downregulate CB1 protein expression, and upregulate CB2 and Mfsd2a protein expression. In vivo studies demonstrated that XXD improved spatial learning and memory abilities in SAMP8 mice, reduced the amount of Evans blue extravasation in brain tissues, modulated the BBB-associated P-gp/ECS axis, RAGE/LRP1 receptor system, as well as MRP2 and Mfsd2a proteins, and decreased the accumulation of Aβ in the brains of SAMP8 mice. Additionally, XXD upregulated the expression of TREM2, downregulated IBA1, TLR1, TLR2, and CMPK2 expression, and reduced the levels of pro-inflammatory factors NLRP3, NF-κB p65, COX-2, TNF-α, and IL-1β in the hippocampal tissues. XXD may exert its effects by regulating the P-gp/ECS axis, the RAGE/LRP1 receptor system, and the expression of MRP2 and Mfsd2a proteins, thereby modulating the transport function of the BBB to expedite the clearance of Aβ, reduce cerebral Aβ accumulation, and consequently inhibit the activation of microglia induced by Aβ aggregation. This process may suppress the activation of the CMPK2/NLRP3 and TLRs/NF-κB pathways, diminish the production of inflammatory cytokines and chemokines, alleviate neuroinflammation associated with microglia in the brain of AD, and ultimately improve AD pathology.

Ferulic acid mediates microbial fermentation of arabinoxylan to enhance host immunity by suppressing TLR4/NF-κB signaling.

The study was conducted to explore the relationship between arabinoxylan (AX) structure and microbial fermentation characteristics, and reveal molecular mechanism of AX on regulating immune function of the host. Results indicated that the group of wheat bran AX showed greater activity of feruloyl esterase, production of short chain fatty acids and ferulic acid compared with the blank group (P < 0.05). The AX increased sIgA concentration and protein expression of protein expression of TLR4 and NF-κB (p65), but decreased mRNA expression of pro-inflammatory cytokines in the ileum of weaned pig model, leading to the reduced diarrhea (P < 0.05). The AX increased an abundance of Bifidobacterium pseudocatenulatum, production of butyric acid and ferulic acid in the ileal digesta of pigs (P < 0.05). In a LPS-treated mouse model, butyric acid and ferulic acid combination increased IL-10 concentration and abundance of Bifidobacterium pseudocatenulatum, but reduced mRNA expression of IL-6 and gene expression of TLR4 and NF-κB (p65) in the jejunum. In summary, AX is fermented by gut microbiota to produce ferulic acid, as well as butyric acid, which improved host immunity by promoting relative abundance of Bifidobacterium pseudocatenulatum and suppressing activation of TLR4/NF-κB signaling.