Lipopolysaccharide Treatment Research Articles

Resistin alleviates lipopolysaccharide-induced inflammation in bovine alveolar macrophages by activating the AMPK/mTOR signaling pathway and autophagy

ObjectiveResistin (RETN) is an adipocyte-specific hormone that participates in metabolism and modulates cellular inflammation. Our study aimed to assess the effects of RETN treatment on autophagy and the underlying molecular and biological mechanisms in bovine alveolar macrophages (BAMs). MethodsThe optimal concentration of RETN + lipopolysaccharide (LPS) on macrophages was screened and then used to co-culture with alveolar macrophages. Autophagosomes in BAMs were examined using a transmission electron microscope (TEM). Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of microtubule-associated protein light chain 3 (LC3) and p62. Western blot (WB) was used to detect the protein expressions of LC3 and p62. The distribution of LC3 and p62 proteins in the cells was observed by immunofluorescence (IF). The concentrations of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The protein expression of adenosine-monophosphate-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), and p-mTOR was detected using WB. ResultsThe treatment of BAMs with RETN or LPS increased the number of autophagosomes and the ratio of LC3II/LC3I and decreased the expression level of p62 protein. RETN treatment significantly triggered autophagy compared to LPS treatment. Moreover, the ratios of p-AMPK/AMPK and p-mTOR/mTOR were upregulated and downregulated, respectively, after RETN treatment, suggesting that AMPK/mTOR signaling pathway activation is required for RETN-mediated autophagy in BAMs. Additionally, the ratio of LC3-II/LC3-I was lower, and the concentrations of IL-1β, IL-6, and TNF-α significantly decreased in the LPS and RETN co-treatment groups compared to the single LPS treatment group. However, both autophagy- and LPS-induced inflammation were partially alleviated by RETN treatment. ConclusionRETN can promote autophagy in BAMs by activating the AMPK/mTOR signaling pathway, it may help prevent LPS-induced inflammation.

Chicoric acid acts as an ALOX15 inhibitor to prevent ferroptosis in asthma

BackgroundChicoric acid (CA) is a crucial immunologically active compound found in chicory and echinacea, possessing a range of biological activities. Ferroptosis, a type of iron-dependent cell death induced by lipid peroxidation, plays a key role in the development and advancement of asthma. Targeting ferroptosis could be a potential therapeutic strategy for treating asthma. PurposeThe purpose of this study was to explore the screening of ALOX15, a pivotal target of ferroptosis in asthma, and potential therapeutic agents, as well as to investigate the promising potential of CA as an ALOX15 inhibitor for modulating ferroptosis in asthma. MethodsThrough high-throughput data processing of bronchial epithelial RNA from asthma patients using bioinformatics and machine learning, the key target of ferroptosis in asthma, ALOX15, was identified. An inhibitor of ALOX15 was then obtained through high-throughput molecular docking and molecular dynamics simulation tests. In vitro experiments were conducted using a 16HBE cell model induced by house dust mite (HDM) and lipopolysaccharide (LPS), which were treated with the ALOX15 inhibitor (PD146176), CA treatment, or ALOX15 knockdown. In vivo experiments were also carried out using a mouse model induced by HDM and LPS. ResultsThe composite model of ALOX15 and CA in molecular dynamics simulations shows good stability and flexibility. Network pharmacological analysis reveals that CA regulates ferroptosis through ALOX15 in treating asthma. In vitro studies show that ALOX15 is highly expressed in HDM and LPS treatments, while CA inhibits HDM and LPS-induced ferroptosis in 16HBE cells by reducing ALOX15 expression. Knockdown of ALOX15 has the opposite effect. Metabolomics analysis identifies key compounds associated with ferroptosis, including L-Targinine, eicosapentaenoic acid, 16-hydroxy hexadecanoic acid, and succinic acid. In vivo experiments demonstrate that CA suppresses ALOX15 expression, inhibits ferroptosis, and improves asthma symptoms in mice. ConclusionOur research initially identified CA as a promising asthma treatment that effectively blocks ferroptosis by specifically targeting ALOX15. This study not only highlights CA as a potential therapeutic agent for asthma but also introduces novel targets and treatment options for this condition, along with innovative approaches for utilizing natural compounds to target diseases associated with ferroptosis.

Dietary supplementation with N-acetylcysteine confers a protective effect on muscle and liver in lipopolysaccharide-challenged piglets.

N-acetylcysteine (NAC) is a well-established antioxidant that offers exciting opportunities for intestinal health in weaned piglets, while the effects of NAC on muscle and liver has not been fully characterized. Therefore, the present study was performed to investigate the effects of dietary supplementation with NAC on muscle and liver in weaned piglets challenged with lipopolysaccharide (LPS). Twenty-four piglets (24-day-old) were randomly assigned to three treatment groups, the piglets in the control (CTR) and LPS- challenged (LPS) groups were fed the basal diet and those in the LPS+ NAC group was fed the basal diet supplemented with 500 mg/kg NAC. The animal trial lasted for 21 days. At the end of the trial, piglets in the LPS and LPS+ NAC groups were injected intraperitoneally with LPS (100 μg/kg body weight) and piglets in the CTR group were administrated with an equal volume of normal saline. 3 h later, the blood was collected and tissue samples were obtained after 6 h of LPS or normal saline treatment. The results showed that the level of IL-1β, and the mRNA levels of C-X-C motif chemokine receptor 3 (CXCR3) and interferon-γ (IFN-γ) in the liver were up-regulated, and the mRNA levels of insulin-like growth factor 1 (IGF-1), total glutathione (T-GSH), and the ratio of total protein to DNA in the liver were decreased under LPS challenge (P < 0.05). At the same time, LPS increased the level of H2O2 and decreased the content of T-GSH and DNA in the longissimus dorsi and gastrocnemius muscles (P < 0.05). In addition, the percentage of monocytes and the level of epidermal growth factor (EGF) were down-regulated in the LPS treatment (P < 0.05). Interestingly, dietary NAC supplementation reversed the above changes induced by LPS (P < 0.05). Furthermore, NAC might alleviate the muscle and liver injury in LPS-challenged piglets by regulating the expression of genes related to the type I interferon signaling pathway, as well as hypoxia inducible factor 1 (HIF1) and nuclear factor erythroid-2 related factor 2 (Nrf-2). Our findings suggested that dietary supplementation with NAC could benefit the health of muscle and liver in LPS-challenged weaned piglets.

Abstract B048: Lipopolysaccharide (LPS) mediated Toll like receptor-4 (TLR4) agonism immuno-modulates pancreatic cancer niche via CD4+ T cells-Myeloid cells axis

Abstract Introduction: Pancreatic cancer is a major cause of cancer-related mortality with a dismally low overall survival. Despite advancement in the cancer immunotherapy, pancreatic cancer remains unresponsive due to its immunosuppressives. We have established that lipopolysaccharide (LPS) reduces tumor burden in pancreatic cancer mouse-models by stimulating anti-tumor immunity and infiltrating cytotoxic CD4+ T-cells in the tumor-microenvironment. Here, we are investigating how LPS immuno-modulates pancreatic cancer niche and brings anti-cancerous effect. Methods: Pancreatic cancer-cells (KPCs) were implanted in C57BL/6, TRIF KO, MyD88 KO and TLR4 flox/flox LysmCre (TLR4f/f) mice subcutaneously and were treated with LPS (1mg/kg) weekly. The animals were sacrificed at endpoints and tumors were evaluated. Subcutaneous KPC-LPS model in BL/6 mice were also coupled with clodronate treatment to deplete cells of myeloid lineage. Tumor samples were analyzed using flow-cytometry and RT- PCR for immunocyte infiltration and expression of immune-associated genes. Dendritic cells (DCs) were isolated from BL/6 and TLR4f/f mice, pulsed with KPC cancer cell antigen, treated with LPS and taken to flow cytometry. Such DCs were also co-cultured with CD4+ T-cells, which were then subjected to cytotoxicity against cancer cells Results: LPS therapy in wild-type pancreatic cancer mouse-model significantly reduced the tumor burden by stimulating anti- tumor immunity in tumor-microenvironment and caused significant upregulation of activated DCs and macrophages. The tumor regressing effect of LPS disappeared in absence of TRIF as well as MyD88 receptors in TRIF KO and MyD88 KO mice respectively. Macrophage depletion by clodronate attenuated influence of LPS on tumor burden, which again proved futile in TLR4f/f model. On antigenic stimulation and LPS treatment, DCs from BL/6 mice expressed higher frequency of co-stimulatory molecules such as CD40, CD80 and CD86 whereas TLR4f/f mice derived DCs did not show such a trend. When such BL/6 DCs were co-cultured with CD4+ T- cells, they expressed higher cytotoxicity against cancer cells. Conclusions: LPS treatment reduces tumor burden in pancreatic cancer model by stimulating anti-tumor immunity in tumor- microenvironment wherein cells of the myeloid lineage are indispensable. This anti-cancer effect executed by CD4+ T-cells in an MHC II dependent manner, seems to be mediated through dendritic cells. Designer TLR4-agonists have the potential to emerges as a novel immunotherapeutic agent and could be combined with available immune-checkpoint blockade therapies. Citation Format: Utpreksha Vaish, Tejeshwar Jain, Dhanisha S Suresh, Maria F Salcedo-Noriega, Srikanth Iyer, Troy Randall, Vikas Dudeja. Lipopolysaccharide (LPS) mediated Toll like receptor-4 (TLR4) agonism immuno-modulates pancreatic cancer niche via CD4T cells-Myeloid cells axis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B048.

Effects of Omega-3 Polyunsaturated Fatty Acids on the Formation of Adipokines, Cytokines, and Oxylipins in Retroperitoneal Adi-Pose Tissue of Mice.

Oxylipins and specialized pro-resolving lipid mediators (SPMs) derived from polyunsaturated fatty acids (PUFAs) are mediators that coordinate an active process of inflammation resolution. While these mediators have potential as circulating biomarkers for several disease states with inflammatory components, the source of plasma oxylipins/SPMs remains a matter of debate but may involve white adipose tissue (WAT). Here, we aimed to investigate to what extent high or low omega (n)-3 PUFA enrichment affects the production of cytokines and adipokines (RT-PCR), as well as oxylipins/SPMs (liquid chromatography-tandem mass spectrometry) in the WAT of mice during lipopolysaccharide (LPS)-induced systemic inflammation (intraperitoneal injection, 2.5 mg/kg, 24 h). For this purpose, n-3 PUFA genetically enriched mice (FAT-1), which endogenously synthesize n-3 PUFAs, were compared to wild-type mice (WT) and combined with n-3 PUFA-sufficient or deficient diets. LPS-induced systemic inflammation resulted in the decreased expression of most adipokines and interleukin-6 in WAT, whereas the n-3-sufficient diet increased them compared to the deficient diet. The n-6 PUFA arachidonic acid was decreased in WAT of FAT-1 mice, while n-3 derived PUFAs (eicosapentaenoic acid, docosahexaenoic acid) and their metabolites (oxylipins/SPMs) were increased in WAT by genetic and nutritional n-3 enrichment. Several oxylipins/SPMs were increased by LPS treatment in WAT compared to PBS-treated controls in genetically n-3 enriched FAT-1 mice. Overall, we show that WAT may significantly contribute to circulating oxylipin production. Moreover, n-3-sufficient or n-3-deficient diets alter adipokine production. The precise interplay between cytokines, adipokines, and oxylipins remains to be further investigated.

UFMylation is involved in serum inflammatory cytokines generation and splenic T cell activation induced by lipopolysaccharide

UFMylation, a novel ubiquitin-like protein modification system, has been recently found to be activated in inflammation. However, the effects of UFMylation activation on inflammation in vivo remains unclear. In the present study, we generated a UFMylation activated mice using transgenic (TG) techniques. Lipopolysaccharide (LPS) was used to induce systemic inflammation in both TG and non-transgenic (NTG) mice. Serum cytokines were detected using a Mouse Cytokine Array, and the proportions of splenic NK, B and T cells were determined by using flow cytometry. We found that TG mice showed increased serum G-CSF, TNF RII and decreased serum TCA-3, CD30L, bFGF, IL-15 and MIG compared with NTG mice at baseline. Furthermore, serum cytokines in TG mice exhibited different responses to LPS compared to NTG mice. LPS up-regulated serum TNF RII, G-CSF, MCP-5, RANTES, KC, BLC, MIG and down-regulated IL-1b, IL-2, IL-3, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-15, IL-17, IFN-γ, TCA-3, Eotaxin-2, LIX, MCP-1, TNFα, GM-CSF in NTG mice, whereas LPS up-regulated G-CSF, MCP-5, RANTES, KC, BLC, MIG, ICAM-1, PF4, Eotaxin, CD30L, MIP-1a, TNFRI and down-regulated IL-1b, IL-3, LIX, MCP-1, TNFα, GM-CSF in TG mice. Data from flow cytometry indicated that LPS significantly reduced the percentages of NK and NKT cells in NTG mice, whereas UFMylation activation inhibited LPS-induced NKT cell decrease. The proportions of B cells, total CD4+ and total CD8+ T cells were comparable between TG and NTG mice in response to LPS treatment, whereas the percentages of CD4+CD69+ and CD8+CD69+T cells were lower in TG mice. These findings suggest that UFMylation may alter LPS-induced serum cytokine profile and participate in splenic T cell activation in vivo.

Enrichment of Cis-Acting Regulatory Elements in Differentially Methylated Regions Following Lipopolysaccharide Treatment of Bovine Endometrial Epithelial Cells.

Endometritis is an inflammatory disease that negatively influences fertility and is common in milk-producing cows. An in vitro model for bovine endometrial inflammation was used to identify enrichment of cis-acting regulatory elements in differentially methylated regions (DMRs) in the genome of in vitro-cultured primary bovine endometrial epithelial cells (bEECs) before and after treatment with lipopolysaccharide (LPS) from E. coli, a key player in the development of endometritis. The enriched regulatory elements contain binding sites for transcription factors with established roles in inflammation and hypoxia including NFKB and Hif-1α. We further showed co-localization of certain enriched cis-acting regulatory motifs including ARNT, Hif-1α, and NRF1. Our results show an intriguing interplay between increased mRNA levels in LPS-treated bEECs of the mRNAs encoding the key transcription factors such as AHR, EGR2, and STAT1, whose binding sites were enriched in the DMRs. Our results demonstrate an extraordinary cis-regulatory complexity in these DMRs having binding sites for both inflammatory and hypoxia-dependent transcription factors. Obtained data using this in vitro model for bacterial-induced endometrial inflammation have provided valuable information regarding key transcription factors relevant for clinical endometritis in both cattle and humans.

Effect of goose-derived adiponectin peptide gADP3 on LPS-induced inflammatory injury in goose liver

ABSTRACT 1. This study evaluated the effects and mechanisms of action of the peptide gADP3 on hepatic inflammatory injury induced by lipopolysaccharide (LPS). 2. Hepatic inflammatory injury was induced in geese by intraperitoneal injection of LPS and gADP3, and the adiponectin receptor agonist AdipoRon (positive control) was used for potential amelioration. Serum inflammatory factor levels, liver function-related biochemical indicators and oxidative stress-related biochemical parameters in the liver tissues were determined. The expression levels of adiponectin and its receptors, inflammation and oxidative stress-related genes and key signalling molecules involved in adiponectin, inflammation and oxidative stress signalling pathways in liver tissues were detected. 3. The peptide gADP3 alleviated inflammatory cell infiltration and hepatic inflammatory changes, reversed the decrease in serum albumin (ALB), total protein (TP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) content or activity induced by LPS and increased the activity of the antioxidant enzymes CAT (catalase), SOD (superoxide dismutase) and GSH-Px (glutathione peroxidase). 4. The peptide gADP3 upregulated the expression of antioxidant enzyme-related genes GCLC, HO-1 and NQO1 in liver tissues, decreased the levels of inflammatory factors like TNF-α, IL-1β, IL-6, IFN-γ and TGF-β and reduced mRNA expression levels of inflammatory-related genes TNF-α, IL-1β, iNOS and TGF-β. Additionally, it increased the mRNA and protein expression levels of adiponectin and its receptors, as well as key molecules in the adiponectin signalling pathway like AMPK and PPARα. In addition, gADP3 reversed the changes in mRNA or protein expression of inflammatory and oxidative stress signalling pathway-related genes P38MAPK, NF-κBP65, TLR4 and Nrf2 in liver tissues caused by LPS treatment. 5. In conclusion, goose-derived adiponectin peptide gADP3, similar to the adiponectin receptor agonist AdipoRon, attenuated LPS-induced hepatic inflammatory injury in geese.

Ameliorating effect of chotosan and its active component, Uncaria hook, on lipopolysaccharide-induced anxiety-like behavior in mice.

In this study, we aimed to examine the effects of chotosan, a traditional Japanese botanical drug, and its active component, Uncaria hook, on anxiety-like behaviors induced by systemic inflammation in mice. To induce systemic inflammation, the mice were treated with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS treatment, the mice were administered chotosan or Uncaria hook orally each day for 14days. Anxiety-like behavior of the mice was evaluated using the light-dark test 24h after LPS treatment. Repeated administration of chotosan prevented anxiety-like behavior in both normal and LPS-treated mice. Similarly, administration of Uncaria hook suppressed LPS-induced anxiety-like behavior in mice. Furthermore, treatment with tandospirone, a 5-HT1A receptor agonist, alleviated anxiety-like behavior in mice, whereas treatment with DOI, a 5-HT2A receptor agonist, enhanced anxiety-like behavior in mice. LPS treatment significantly increased serotonin (5-HT)2A receptor mRNA expression in the frontal cortex, whereas 5-HT1A receptor mRNA expression remained unchanged in the hippocampus. Notably, chotosan significantly suppressed the mRNA expression of 5-HT2A receptor. These findings indicate that chotosan exerts anxiolytic-like effects in the context of inflammation-induced anxiety, potentially mediated by the inhibition of 5-HT2A receptor hyperfunction in LPS-treated mice. Consequently, we postulate that chotosan may be effective in managing inflammation-induced anxiety-like behaviors.

Suppressive Effects of β-Hydroxybutyrate Administration on Lipopolysaccharide-Induced Inflammation in Broiler Chickens.

This study aimed to evaluate the suppressive effects of β-hydroxybutyrate (BHB) administration on lipopolysaccharide (LPS)-induced inflammation in broiler chickens. Twenty-day-old male broiler chickens were randomly allocated to three groups, each of which was treated with saline (control), intraperitoneal administration of LPS [1.5 mg/kg body weight (BW), Escherichia coli O127:B8], or LPS plus BHB (3 mmol/kg BW). Plasma albumin and total protein concentration were significantly reduced by LPS administration, while BHB co-treatment partially attenuated the effects. The LPS treatment significantly induced plasma aspartate and alanine aminotransferase activities, and interleukin (IL)-6 concentration, with the increases suppressed by BHB co-treatment (p < 0.05). The LPS treatment significantly increased the gene expression levels of IL-1β, IL-6, and IL-18 in the spleen and peripheral blood monocytes (PBMC), while the increases were partially attenuated by BHB in the spleen. Relatively higher levels of BHB dehydrogenase 1 and succinyl-CoA:3-ketoacid CoA transferase were observed in the spleen and skeletal muscle, while these gene levels were lower in PBMC and the liver. The present results suggest that BHB can suppress LPS-induced inflammation, in which ketolytic enzyme expression levels may be involved in broiler chickens.

Bacterioruberin extract from Haloarchaea Haloferax marinum: Component identification, antioxidant activity and anti-atrophy effect in LPS-treated C2C12 myotubes.

Carotenoids are natural pigments utilized as colourants and antioxidants across food, pharmaceutical and cosmetic industries. They exist in carbon chain lengths of C30, C40, C45 and C50, with C40 variants being the most common. Bacterioruberin (BR) and its derivatives are part of the less common C50 carotenoid group, synthesized primarily by halophilic archaea. This study analysed the compositional characteristics of BR extract (BRE) isolated from 'Haloferax marinum' MBLA0078, a halophilic archaeon isolated from seawater near Yeoungheungdo Island in the Republic of Korea, and investigated its antioxidant activity and protective effect on lipopolysaccharide (LPS)-induced C2C12 myotube atrophy. The main components of BRE included all-trans-BR, monoanhydrobacterioruberin, 2-isopentenyl-3,4-dehydrorhodopin and all-trans-bisanhydrobacterioruberin. BRE exhibited higher antioxidant activity and DNA nicking protection activity than other well-known C40 carotenoids, such as β-carotene, lycopene and astaxanthin. In C2C12 myotubes, LPS treatment led to a reduction in myotube diameter and number, as well as the hypertranscription of the muscle-specific ubiquitin ligase MAFbx and MuRF1. BRE mitigated these changes by activating the Akt/mTOR pathway. Furthermore, BRE abolished the elevated cellular reactive oxygen species levels and the inflammation response induced by LPS. This study demonstrated that 'Hfx. marinum' is an excellent source of natural microbial C50 carotenoids with strong antioxidant capacity and may offer potential protective effects against muscle atrophy.

Co-exposure to different bacterial species’ lipopolysaccharides with the NASH diet exacerbates NASH and liver fibrosis progression in mice

Background and aimWith the obesity epidemic, nonalcoholic fatty liver disease (NAFLD) has become a public health concern, but its progression mechanism remains unclear. Experimental models mimicking human NAFLD/steatohepatitis (NASH) are crucial. This study simulates gut microbiota imbalance effects on NASH and liver fibrosis. MethodsWe used different bacterial sources of lipopolysaccharide (LPS), including Escherichia coli (GEC) and Salmonella abortus equi (GSE), combined with a Gubra Amylin NASH (GAN) diet to induce NASH and liver fibrosis. ResultsThe GSE group showed significantly higher serum alanine aminotransferase, hydroxyproline, CD68-positive cells, α-smooth muscle actin, glial fibrillary acidic protein, and TNF-α, COL1A1, TGF-β, and NLRP3 expressions compared to the the GAN group. The GSE group also had higher Erysipelotrichaceae, Akkermansiaceae, and Bacteroidaceae family numbers. ConclusionsThe GAN diet with LPS treatment successfully induced NASH and fibrosis making this model useful for preclinical NASH drug testing.

The Effect of Lipopolysaccharide Challenge in RAW 264.7 Cells on Nitric Oxide Release and Cell Viability

Lipopolysaccharide (LPS) is a major component of a gram-negative bacterial wall that is widely used and well-established to induce inflammation in vitro. In addition, the in vitro model using RAW 264.7 cells is the most commonly applied in screening the anti-inflammatory and elucidating the pathophysiology of inflammation-based disease, as well. However, there is still limited data on the efficacy of different doses of LPS in inducing inflammation in RAW 264.7 cells. This study aimed to evaluate the effect and safety of LPS at various doses in RAW 264.7 cells. RAW 264.7 cells were exposed to LPS at different dose ranges (10ng/mL-10µg/mL) for 24 hours. The nitric oxide (NO) release as inflammatory responses and viability test were evaluated using Griess assay and CCK-8 assays, respectively. The result showed that NO production was increased at different doses of LPS compared to the control although not significant. Whereas, All LPS-treated RAW 264.7 cells tended to increase but not significantly compared to the control groups. This study showed that the LPS treatment effectively induced inflammation in RAW 264.7 cells as shown by NO production and was considerably safe as the viability was comparable between LPS and control group for RAW cells 264.7 at least up to 10µg/mL for 24 hours.

USP31 promotes the inflammatory response of human lung organoids to lipopolysaccharide treatment.

Acute lung injury (ALI) is a severe condition characterized by a high mortality rate, driven by an uncontrolled inflammatory response. Emerging evidence has underscored the crucial role of the ubiquitin system in ALI. However, due to their vast number, the specific functions of individual ubiquitination regulators remain unclear. In this study, we established human lung organoids (HLOs) derived from human embryonic stem cells and subjected them to lipopolysaccharide (LPS) treatment to induce an inflammatory response, mimicking ALI. Subsequently, we detected the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin 6, interleukin 18 (IL-18), and interleukin-1β (IL-1β), by qPCR experiments. We also detected changes in the mRNA expression of several USPs before and after HLOs treatment and thus screened for USPs that had significant changes in HLOs after LPS stimulation. After screening for USP, we silenced the USP in HLOs and then subjected them to LPS treatment, and TNF-α, IL-6, IL-18, and IL-1β expressions were detected using qPCR assays. Meanwhile, Western blot was used to detect changes in NOD-, LRR- and pyrin domain-containing 3 (NLRP3) and apoptosis-associated Speck-like protein containing a CARD (ASC) protein level in HLOs. Through screening the expression of 40 ubiquitin-specific proteases (USPs), which are responsible for removing ubiquitination, we identified several USPs that exhibited differential expression in LPS-treated HLOs compared to untreated HLOs. Notably, USP31 emerged as the most significantly upregulated USP, and the knockdown of USP31 markedly attenuated the inflammatory response of HLOs to LPS treatment. USP31 may play a facilitating role in the inflammatory response during ALI.

METTL14/IGF2BP2-mediated m6A modification of STEAP1 aggravates acute lung injury induced by sepsis.

Acute lung injury (ALI) is a severe complication of sepsis, characterized by inflammation, edema, and injury to alveolar cells, leading to high mortality rates. Septic ALI is a complex disease involving multiple factors and signaling pathways. STEAP family member 1 (STEAP1) has been reported to be upregulated in a sepsis-induced ALI model. However, the role of STEAP1 in the regulation of septic ALI is not yet fully understood. The study stimulated human pulmonary microvascular endothelial cells (HPMECs) using lipopolysaccharides (LPS) to establish an in vitro ALI model. The study used quantitative real-time polymerase chain reaction (qRT-PCR) to measure mRNA expression, and western blotting assay or immunohistochemistry (IHC) assay to analyze protein expression. Cell counting kit-8 (CCK-8) assay was performed to assess cell viability. Flow cytometry was conducted to analyze cell apoptosis. Tube formation assay was used to analyze the tube formation rate of human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). The levels of Fe2+ and reactive oxygen species (ROS) were determined using colorimetric and fluorometric assays, respectively. The glutathione (GSH) level was also determined using a colorimetric assay. m6A RNA immunoprecipitation assay, dual-luciferase reporter assay, and RNA immunoprecipitation assay were performed to identify the association of STEAP1 with methyltransferase 14, N6-adenosine-methyltransferase non-catalytic subunit (METTL14) and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2). The transcript half-life of STEAP1 was analyzed by actinomycin D assay. Finally, a rat model of polymicrobial sepsis was established to analyze the effects of STEAP1 knockdown on lung injury in vivo. We found that the mRNA expression levels of STEAP1 and METTL14 were upregulated in the blood of ALI patients induced by sepsis compared to healthy volunteers. LPS treatment increased the protein levels of STEAP1 and METTL14 in HPMECs. STEAP1 depletion attenuated LPS-induced promoting effects on HPMECs' apoptosis, inflammatory response, and ferroptosis, as well as LPS-induced inhibitory effect on tube formation. We also found that METTL14 and IGF2BP2 stabilized STEAP1 mRNA expression through the m6A methylation modification process. Moreover, METTL14 silencing attenuated LPS-induced effects by decreasing STEAP1 expression in HPMECs, and STEAP1 silencing ameliorated cecal ligation and puncture-induced lung injury of mice. METTL14/IGF2BP2-mediated m6A modification of STEAP1 aggravated ALI induced by sepsis. These findings suggest potential therapeutic targets for the treatment of this disease.

Role for BLT1 in regulating inflammation within adipose tissue immune cells of aged mice

BackgroundAging is a complex biological process characterized by obesity and immunosenescence throughout the organism. Immunosenescence involves a decline in immune function and the increase in chronic-low grade inflammation, called inflammaging. Adipose tissue expansion, particularly that of visceral adipose tissue (VAT), is associated with an increase in pro-inflammatory macrophages that play an important role in modulating immune responses and producing inflammatory cytokines. The leukotriene B4 receptor 1 (BLT1) is a regulator of obesity-induced inflammation. Its ligand, LTB4, acts as a chemoattractant for immune cells and induces inflammation. Studies have shown that BLT1 is crucial for cytokine production during lipopolysaccharide (LPS) endotoxemia challenge in younger organisms. However, the expression patterns and function of BLT1 in older organisms remains unknown.ResultsIn this study, we investigated BLT1 expression in immune cell subsets within the VAT of aged male and female mice. Moreover, we examined how antagonizing BLT1 signaling could alter the inflammatory response to LPS in aged mice. Our results demonstrate that aged mice exhibit increased adiposity and inflammation, characterized by elevated frequencies of B and T cells, along with pro-inflammatory macrophages in VAT. BLT1 expression is the highest in VAT macrophages. LPS and LTB4 treatment result in increased BLT1 in young and aged bone marrow-derived macrophages (BMDMs). However, LTB4 treatment resulted in amplified Il6 from aged, but not young BMDMs. Treatment of aged mice with the BLT1 antagonist, U75302, followed by LPS-induced endotoxemia resulted in an increase in anti-inflammatory macrophages, reduced phosphorylated NFκB and reduced Il6.ConclusionsThis study provides valuable insights into the age- and sex- specific changes in BLT1 expression on immune cell subsets within VAT. This study offers support for the potential of BLT1 in modulating inflammation in aging.

Ribonuclease Inhibitor 1 (RNH1) Regulates Sperm tsRNA Generation for Paternal Inheritance through Interacting with Angiogenin in the Caput Epididymis.

Environmental stressors can induce paternal epigenetic modifications that are a key determinant of the intergenerational inheritance of acquired phenotypes in mammals. Some of them can affect phenotypic expression through inducing changes in tRNA-derived small RNAs (tsRNAs), which modify paternal epigenetic regulation in sperm. However, it is unclear how these stressors can affect changes in the expression levels of tsRNAs and their related endonucleases in the male reproductive organs. We found that Ribonuclease inhibitor 1 (RNH1), an oxidation responder, interacts with ANG to regulate sperm tsRNA generation in the mouse caput epididymis. On the other hand, inflammation and oxidative stress induced by either lipopolysaccharide (LPS) or palmitate (PA) treatments weakened the RNH1-ANG interaction in the epididymal epithelial cells (EEC). Accordingly, ANG translocation increased from the nucleus to the cytoplasm, which led to ANG upregulation and increases in cytoplasmic tsRNA expression levels. In conclusion, as an antioxidant, RNH1 regulates tsRNA generation through targeting ANG in the mouse caput epididymis. Moreover, the tsRNA is an epigenetic factor in sperm that modulates paternal inheritance in offspring via the fertilization process.

Sex-dependent effects of antimicrobials and lipopolysaccharide on blood-brain-barrier permeability in pubertal male and female CD1 mice

Exposure to stressors during puberty can disrupt normal development and possibly increase susceptibility to neurodegenerative disorders later in life. However, the mechanisms underlying the relationship between pubertal stress exposure and neurodegeneration remain unclear. As such, the current study was designed to examine the effects of pubertal antimicrobial (AMNS) and lipopolysaccharide (LPS) treatments on intestinal and blood-brain-barrier (BBB) permeability in male and female mice. Moreover, we also examined the sex-specific effects of pubertal AMNS and LPS treatments on gross motor activity, heart rate, and core body temperature. At four weeks of age, male and female CD1 mice were implanted with the G2 HR E-Mitter telemetry system. At five weeks of age, mice received 200 μL of broad-spectrum antimicrobial or water, through oral gavage, twice daily for seven days. Mice received an intraperitoneal injection of either saline or LPS at six weeks of age. BBB and intestinal permeability were examined 24 h, 72 h, and one week post-LPS/saline treatment. Telemetric data was collected for 48 h post-LPS/saline treatment. The results showed that pubertal AMNS and LPS treatments increased sickness behaviours and decreased body temperature and heart rate, in a sex-dependent manner. Furthermore, pubertal AMNS and LPS treatments resulted in sex-dependent regional increases in BBB permeability 24 h and 72 h post-LPS/saline treatment, while global increases in BBB permeability were only observed one week post-LPS/saline treatment. These results further our understanding of the combined effects of AMNS and LPS treatments on physiology and on the enduring negative changes observed following pubertal exposure to stressors.

Flaxseed Oil Attenuates Intestinal Damage by Regulating Ferroptosis Signaling Pathway Following LPS Challenge in Piglets.

Ferroptosis has been demonstrated to play an important role in various tissue injuries and diseases. Flaxseed oil (FO) has been proven to have benefits for intestinal health. This study aims to explore whether FO relieved lipopolysaccharide (LPS)-induced intestinal injury through modulating ferroptosis signaling pathway. A total of 120 weaned piglets are fed diets with 3% soybean oil (SO) or 3% FO for 4 weeks. At the end of the trial, 24 piglets selected from two dietary treatment groups are used in a 2 × 2 factorial design with oil treatment (3% SO versus 3% FO) and LPS challenge (saline versus LPS). At 4h postinjection with LPS, 24 piglets are slaughtered and intestinal samples are collected. FO improves growth performance of pigs. After LPS treatment, FO mitigates intestinal morphological damage and functional damage. Notably, FO reverses the typical ultra-morphology and biochemical indexes of ferroptosis involving glutathione, malondialdehyde, and 4-hydroxynonenal contents. Mechanistically, FO ameliorates the changes on mRNA or protein abundance of key ferroptosis signals including transferrin receptor protein 1 (TFR1), recombinant iron responsive element binding protein 2 (IREB2), FTL, HSPB1, ferritin heavy chain 1 (FTH1), ferroportin 1 (FPN1), SLC7A11, solute carrier family 3 member 2 (SLC3A2), glutathione peroxidase 4 (GPX4), and arachidonate-15-lipoxygenase (ALOX15). FO improves growth performance and mitigates intestinal structural and functional damage, which is involved in regulating ferroptosis signaling pathway.

Suppressed testicular macrophage M1 polarization by HDAC5 enforces insensitivity to LPS-elicited blood-testis barrier damage

Infertility caused by lipopolysaccharide (LPS) exposure due to infection is endangering male fertility worldwide, but the mechanism remains unclear. The blood-testis barrier (BTB) is essential for maintaining spermatogenesis and male fertility. In the present study, we showed that LPS (5.0 mg/kg) treatment markedly down-regulated the expression of BTB-related proteins, expanded the biotin penetration distance and caused histopathological injury in seminiferous tubules in mouse testes. Notably, testicular macrophage M1 polarization induced by LPS seems to be related to BTB damage, which was well confirmed by co-culture of RAW264.7 and TM4 cells in vitro. Interestingly, a low-dose LPS (0.1 mg/kg) pretreatment attenuated down-regulation of BTB-related proteins expression and histopathological injury and shorten biotin penetration distance in seminiferous tubules caused by LPS. Correspondingly, a low-dose LPS pretreatment suppresses testicular macrophage M1 polarization induced by LPS in mouse testes. Further experiments revealed that histone deacetylase 5 (HDAC5) was markedly down-regulated at 2 h and slightly down-regulated at 8 h, but up-regulated at 24 h in mouse testes after LPS treatment. Additionally, low-dose LPS pretreatment against the down-regulation of HDAC5 protein caused by LPS treatment. Notably, the suppressed testicular macrophage M1 polarization by low-dose LPS pretreatment was broken by BRD4354, a specific inhibitor of HDAC5 in vitro. These results suggest suppressed testicular macrophage M1 polarization by HDAC5 enforces insensitivity to LPS-elicited BTB damage.