Lipopolysaccharide Stimulation Research Articles

ADAR1 protects pulmonary macrophages from sepsis-induced pyroptosis and lung injury through miR-21/A20 signaling.

Acute lung injury is a serious complication of sepsis with high morbidity and mortality. Pyroptosis is a proinflammatory form of programmed cell death that leads to immune dysregulation and organ dysfunction during sepsis. We previously found that adenosine deaminase acting on double-stranded RNA 1 (ADAR1) plays regulatory roles in the pathology of sepsis, but the mechanism of ADAR1 in sepsis-induced pyroptosis and lung injury remains unclear. Here, we mainly investigated the regulatory effects and underlying mechanism of ADAR1 in sepsis-induced lung injury and pyroptosis of pulmonary macrophages through RNA sequencing of clinical samples, caecal ligation and puncture (CLP)-induced septic mouse models, and in vitro cellular experiments using RAW264.7 cells with lipopolysaccharide (LPS) stimulation. The results showed that pyroptosis was activated in peripheral blood mononuclear cells (PBMCs) from patients with sepsis. In the CLP-induced septic mouse model, pyroptosis was mainly activated in pulmonary macrophages. LPS-stimulated RAW264.7 cells showed significantly increased activation of the NLRP3 inflammasome. ADAR1 was downregulated in PMBCs of patients with sepsis, and overexpression of ADAR1 alleviated CLP-induced lung injury and NLRP3 inflammasome activation. Mechanistically, the regulatory effects of ADAR1 on macrophage pyroptosis were mediated by the miR-21/A20/NLRP3 signalling cascade. ADAR1 attenuated sepsis-induced lung injury and hindered the activation of pyroptosis in pulmonary macrophages in sepsis through the miR-21/A20/NLRP3 axis. Our study highlights the role of ADAR1 in protecting pulmonary macrophages against pyroptosis and suggests targeting ADAR1/miR-21 signalling as a therapeutic opportunity in sepsis-related lung injury.

Dishevelled Segment Polarity Protein 3 (DVL3) Induced by Bacterial LPS Promotes the Proliferation and Migration of Prostate Cancer Cells through the TLR4 Pathway.

Chronic inflammation is associated with various malignant tumors. Bacterial lipopolysaccharides (LPSs) play a significant part in the event and development of prostate cancer. Dishevelled segment polarity protein 3 (DVL3) is a shared component of the Wnt/β-catenin and Notch signaling pathways, which are involved in tumor progression, chemoresistance, and maintenance of stem cell-like properties. According to reports, prostatic cancer cell invasion and proliferation are mediated by toll-like receptor 4 (TLR4). However, the role and regulation of DVL3 in prostate cancer and its relationship with TLR4 remain unclear. Survival curves were plotted to evaluate the relationship between DVL3 expression and prognosis in patients with prostate cancer. DVL3 was silenced in PC3 and DU145 cells using small interfering RNAs (siRNAs). Subsequently, cell counting kit-8 (CCK-8) assay, colony formation assay, transwell migration assay, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed to investigate the role of DVL3 in cell proliferation and migration in vitro. The protein markers of potential pathways were analyzed via western blotting. DVL3 expression was linked to prognosis in patients with prostate cancer; In particular, patients with high DVL3 expression had a poor prognosis. LPS stimulation increased (p < 0.01) the expression of DVL3 in PC3 cells. DVL3 regulated tumor cell proliferation and migration by mediating the increase (p < 0.01) in TLR4 expression. Knockout of TLR4 validated that TLR4 played a crucial role in LPS-induced DVL3 expression. Silencing of DVL3 decreased (p < 0.01) the LPS-induced proliferation and migration of PC3 cells. Bacterial LPS-induced DVL3 promoted the multiplication and migration of prostate cancer cells through the TLR4 pathway. This study offers a valuable reference for the development and clinical application of targeted drugs for prostate cancer.

Potential neuroprotective effects of anti‐cytokine antibodies against lipopolysaccharide‐induced microglial inflammation

Aims/Purpose: Microglia‐mediated neuroinflammation is involved in many neurodegenerative diseases, including glaucoma and diabetic retinopathy. This study examined the effects of anti‐cytokine antibodies on LPS‐induced inflammatory response of microglial cells, and explored the underlying molecular mechanisms by proteomic analysis.Methods: A lipopolysaccharide (LPS)‐induced neuroinflammation model was established in murine BV‐2 microglial cells in vitro. The expression of microglial cell markers (CD68 and CD206) were detected by immunocytochemistry. BV‐2 was stimulated with LPS (1 μg/mL) with or without anti‐tumour necrosis factor‐alpha (anti‐TNF‐α, 2 μg /ml) or anti‐interleukin‐1 beta (anti‐IL‐1β, 4 μg /ml) for 24 h. LPS‐induced inflammation level was analysed using the Griess assay to determine nitric oxide (NO) concentration. Proteome alterations were characterized employing the mass spectrometry‐based proteomics and bioinformatics analyses.Results: Immunocytochemistry confirmed the expression of microglial markers in BV‐2 cells. LPS stimulation induced excessive activation of BV‐2, with significant increase in NO generation (p &lt; 0.0001). Anti‐IL‐1β significantly attenuated 34% of the LPS‐induced NO generation (p &lt; 0.0001). Interestingly, anti‐TNF‐α showed a stronger anti‐inflammatory effect by reducing 57% of LPS‐induced NO generation (p &lt; 0.0001). Bioinformatics analyses revealed that differentially expressed proteins involved in inflammation (p = 3.8 × 10−5) and apoptotic (p = 3.2 × 10−11) signalling pathways were significantly activated by LPS. Particularly, LPS‐mediated upregulation of STAT1 (p = 8.6 × 10−6) was restored by anti‐IL‐1β and anti‐TNF‐α (p = 3.1 × 10−4 and p = 2.7 × 10−3, respectively). Anti‐IL‐1β induced proteome alterations involved in anti‐inflammatory signalling pathways and promote glial cell polarization towards neuroprotective M2 phenotype (p = 5.3 × 10−6), while anti‐TNF‐α treatment significantly regulated proteins involved in immune response of macrophages (p = 2.7 × 10−8).Conclusions: In summary, anti‐TNF‐α and anti‐IL‐1β antibodies alleviated LPS‐induced neuroinflammation and altered proteins involved in neuroprotective pathways. Hence, anti‐cytokine antibodies have therapeutic potential as neuroprotective agents.

Mitochondrial Genome Editing: Exploring the Possible Relationship of the Atherosclerosis-Associated Mutation m.15059G>A With Defective Mitophagy.

The aim of this study was to evaluate the effect of the m.15059G>A mitochondrial nonsense mutation on cellular functions related to atherosclerosis, such as lipidosis, pro-inflammatory response, and mitophagy. Heteroplasmic mutations have been proposed as a potential cause of mitochondrial dysfunction, potentially disrupting the innate immune response and contributing to the chronic inflammation associated with atherosclerosis. The human monocytic cell line THP-1 and cytoplasmic hybrid cell line TC-HSMAM1 were used. An original approach based on the CRISPR/Cas9 system was developed and used to eliminate mitochondrial DNA (mtDNA) copies carrying the m.15059G>A mutation in the MT-CYB gene. The expression levels of genes encoding enzymes related to cholesterol metabolism were analyzed using quantitative polymerase chain reaction. Pro-inflammatory cytokine secretion was assessed using enzyme-linked immunosorbent assays. Mitophagy in cells was detected using confocal microscopy. In contrast to intact TC-HSMAM1 cybrids, Cas9-TC-HSMAM1 cells exhibited a decrease in fatty acid synthase (FASN) gene expression following incubation with atherogenic low-density lipoprotein. TC-HSMAM1 cybrids were found to have defective mitophagy and an inability to downregulate the production of pro-inflammatory cytokines (to establish immune tolerance) upon repeated lipopolysaccharide stimulation. Removal of mtDNA harboring the m.15059G>A mutation resulted in the re-establishment of immune tolerance and the activation of mitophagy in the cells under investigation. The m.15059G>A mutation was found to be associated with defective mitophagy, immune tolerance, and impaired metabolism of intracellular lipids due to upregulation of FASN in monocytes and macrophages.

JunD functions as a transcription factor of IL-10 to regulate bacterial infectious inflammation in grass carp (Ctenopharyngodon idella)

IL-10 is a key anti-inflammatory mediator ensuring the protection of a host from excessive inflammation in response to pathogen infections, whose transcription or expression levels are tightly linked to the onset and progression of infectious diseases. An AP-1 family member called CiJunD was shown to be a transcription factor of IL-10 in grass carp (Ctenopharyngodon idella) in the current study. CiJunD protein harbored the conserved Jun and bZIP domains. Mutant experiments demonstrated that CiJunD bound to three specific sites on IL-10 promoter, i.e., 5′-ATTATTCATA-3′, 5′-AGATGAGACATCT-3′, and 5′-ATTATTCATC-3′, mainly relying on the bZIP domain, and initiated IL-10 transcription. Expression data from the grass carp spleen infected by Aeromonas hydrophila and lipopolysaccharide (LPS) challenged spleen leukocytes indicated that the expressions of CiJunD and IL-10 were positively correlated, while the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-8, IFN-γ, and TNF-α, showed an overall downward trend when CiJunD and IL-10 peaked. The ability of CiJunD to down-regulate the production of pro-inflammatory cytokines and up-regulate the expression of IL-10, both with and without LPS stimulation, was confirmed by overexpression experiments. Meanwhile, the subcellular fractionation assay revealed that the nuclear translocation of CiJunD was significantly enhanced after the LPS challenge. Moreover, in vivo administration of grass carp with Oxamflatin, a potent agonist of JunD activity, could promote IL-10 but suppress the expression of pro-inflammatory cytokines. Intriguingly, tissue inflammation lesions and the survival rates of grass carp infected with A. hydrophila were also significantly improved by Oxamflatin administration. This work sheds light on the regulation mechanism by JunD of IL-10 expression and bacterial infectious inflammation for the first time, and it may present a viable method for preventing infectious diseases in fish by regulating IL-10 expression and inflammatory response.

Early growth response 1/Krüppel-like factor 5 pathway inhibitor alleviates lipopolysaccharide-induced lung injury by promoting autophagy

Early transcription factors play critical roles in the development of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Early growth response 1 (EGR1) is a transcription factor essential for various biological processes, including regulation of metabolism, differentiation, and inflammation. However, its role in ALI has been poorly reported. In this study, we aimed to determine the effect of EGR1 on ALI to gain insights into the theoretical basis for further treatment of ALI. By employing concerted molecular biology techniques, we showed that EGR1 protein was upregulated in mice. EGR1 protein was upregulated in mice and human lung epithelial cells in response to lipopolysaccharide (LPS) stimulation. EGR1 knockdown promoted autophagy and reduced LPS-induced pro-inflammatory mediator production. EGR1 was preferentially bound to the GCGTGGGCG motif region and EGR1-binding peak-related genes were mainly enriched in autophagy and injury stress-related pathways. Additionally, EGR1 promoted Krüppel-like factor 5 (KLF5) transcription by binding to the KLF5 promoter region, and KLF5 knockdown significantly decreased inflammatory damage, suggesting that EGR1 promotes ALI progression by regulating KLF5 expression. Furthermore, ML264, an inhibitor of the EGR1/KLF5 pathway axis, displayed a protective role in ALI to reduce inflammation. In conclusion, our findings demonstrate the potential of EGR1 knockdown to inhibit KLF5 and promote autophagy, further reducing the inflammatory response to mitigate ALI/ARDS. The EGR1/KLF5 pathway axis may be a valuable therapeutic target for the treatment of ALI/ARDS.

The potential use of ascorbic acid to recover the cellular senescence of lipopolysaccharide-induced human apical papilla cells: an in vitro study.

To examine the effect of lipopolysaccharide (LPS) on cellular senescence induction of human apical papilla cells (hAPCs) and evaluate the potential use of 50 μg/ml ascorbic acid to recover cellular senescence and regenerative functions. hAPCs were treated with LPS at 1 and 10 μg/ml either with or without 50 μg/ml ascorbic acid for 48 h. The cellular senescence biomarkers were analyzed by senescence-associated β-galactosidase (SA-β-gal) staining and senescence-related gene expression, p16 and p21. Cell migration, at 12 h and 24 h, was evaluated using a scratch wound assay. Mineralization potential was assessed at 21 days using Alizarin red S staining and dentine sialophosphoprotein (DSPP) and bone sialoprotein (BSP) gene expression. 1 μg/ml and 10 μg/ml LPS stimulation for 48 h induced cellular senescence, as shown by remarkable SA-β-gal staining and p16 and p21 gene expression. The percentage of wound closure and mineralized formation was reduced. The co-incubation with ascorbic acid significantly down-regulated the level of SA-β-gal staining. The reduction of senescence-associated gene expressions was observed. Ascorbic acid improved cell migration, mineralized nodule formation, and the expression of DSPP and BSP genes in LPS-treated hAPCs. LPS significantly promoted cellular senescence on hAPCs and diminished the cell function capacity. Co-presence of ascorbic acid could impede cellular senescence and possibly improve the regenerative capacity of LPS-induced senescent hAPCs in vitro. The data support the in vitro potential benefit of ascorbic acid on cellular senescence recovery of apical papilla cells.

Suppression of Neuroinflammation by Coffee Component Pyrocatechol via Inhibition of NF-κB in Microglia.

According to numerous studies, it has been epidemiologically suggested that habitual coffee intake seems to prevent the onset of neurodegenerative diseases. In this study, we hypothesized that coffee consumption suppresses neuroinflammation, which is closely related to the development of neurodegenerative diseases. Using microglial BV-2 cells, we first found that the inflammatory responses induced by lipopolysaccharide (LPS) stimulation was diminished by both coffee and decaffeinated coffee through the inhibition of an inflammation-related transcription factor, nuclear factor-κB (NF-κB). Pyrocatechol, a component of roasted coffee produced by the thermal decomposition of chlorogenic acid, also exhibited anti-inflammatory activity by inhibiting the LPS-induced activation of NF-κB. Finally, in an inflammation model using mice injected with LPS into the cerebrum, we observed that intake of pyrocatechol as well as coffee decoctions drastically suppressed the accumulation of microglia and the expression of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), CCL2, and CXCL1 in the inflammatory brain. These observations strongly encourage us to hypothesize that the anti-inflammatory activity of pyrocatechol as well as coffee decoction would be useful for the suppression of neurodegeneration and the prevention of the onsets of Alzheimer's (AD) and Perkinson's diseases (PD).

ALDH2 mitigates LPS-induced cardiac dysfunction, inflammation, and apoptosis through the cGAS/STING pathway

BackgroundSepsis is a severe syndrome of organ dysfunction that often leads to cardiac dysfunction and endangers life. The role of mitochondrial aldehyde dehydrogenase 2 (ALDH2) in LPS-induced myocardial injury is unclear. The purpose of this study was to assess the role of ALDH2 in lipopolysaccharide (LPS)-induced myocardial injury and the regulatory mechanism and to identify potential therapeutic strategies for treating this condition.MethodsAn in vivo model was established by 12 h of LPS (10 mg/kg, intraperitoneal injection) stimulation, and an in vitro model was generated by stimulating H9C2 cells with LPS (10 μg/ml) for 12 h. We then used the ALDH2 activator Alda-1 and the ALDH2 inhibitor daidzin to assess their effects on LPS-induced cardiac injury. Cardiac function in mice was evaluated by using cardiac ultrasound. We used various methods to evaluate inflammation, apoptosis, and oxidative stress, including ELISA, flow cytometry, JC-1 staining, Western blotting, and DCFH-DA staining. Additionally, we used a small interfering RNA (siRNA) to knock down cyclic GMP-AMP synthase (cGAS) to further investigate the relationship between ALDH2 and cGAS in LPS-induced cardiac injury.ResultsLPS-induced cardiac dysfunction and increased the levels of the cardiac injury markers creatine kinase-MB (CKMB) and lactate dehydrogenase (LDH) in vivo. This change was accompanied by an increase in reactive oxygen species (ROS) levels, which exacerbated the oxidative stress response and regulated apoptosis through cleaved caspase-3, BAX, BCL-2. The expression of inflammatory cytokines such as IL-6/IL-1β/TNF-α was also upregulated. However, these effects were reversed by pretreatment with Alda-1 via the inhibition of cGAS/stimulator of interferon genes (STING) signaling pathway. Interestingly, LPS, Alda-1 and daidzin altered the activity of ALDH2 but did not regulate its protein expression. Knocking down cGAS in H9C2 cardiomyocytes alleviated LPS-induced cardiac inflammation, apoptosis, and ROS production and weakened the synergistic effect of daidzin.ConclusionWe demonstrated that ALDH2 alleviated LPS-induced cardiac dysfunction, inflammation, and apoptosis through the cGAS/STING signaling pathway, thereby protecting against LPS-induced cardiac injury. This study identifies a novel therapeutic approach for treating sepsis-induced cardiomyopathy (SIC).

Hypothermia Attenuates Neurotoxic Microglial Activation via TRPV4.

Therapeutic hypothermia (TH) provides neuroprotection. However, the cellular mechanisms underlying the neuroprotective effects of TH are not fully elucidated. Regulation of microglial activation has the potential to treat a variety of nervous system diseases. Transient receptor potential vanilloid 4 (TRPV4), a nonselective cation channel, is activated by temperature stimulus at 27-35°C. Although it is speculated that TRPV4 is associated with the neuroprotective mechanisms of TH, the role of TRPV4 in the neuroprotective effects of TH is not well understood. In the present study, we investigated whether hypothermia attenuates microglial activation via TRPV4 channels. Cultured microglia were incubated under normothermic (37°C) or hypothermic (33.5°C) conditions following lipopolysaccharide (LPS) stimulation. Hypothermic conditions suppressed the expression of pro-inflammatory cytokines, inducible nitric oxide synthase, and the number of phagocytic microglia. AMP-activated protein kinase (AMPK)-NF-κB signaling was inhibited under hypothermic conditions. Furthermore, hypothermia reduced neuronal damage induced by LPS-treated microglial cells. Treatment with TRPV4 antagonist in normothermic culture replicated the suppressive effects of hypothermia on microglial activation and microglia-induced neuronal damage. In contrast, treatment with a TRPV4 agonist in hypothermic culture reversed the suppressive effect of hypothermia. These findings suggest that TH suppresses microglial activation and microglia-induced neuronal damage via the TRPV4-AMPK-NF-κB pathway. Although more validation is needed to consider differences according to age, sex, and specific central nervous system regions, our findings may offer a novel therapeutic approach to complement TH.

3-Hydroxy-3-(2-oxopropyl)indolin-2-one, a product of a human-derived Enterocloster strain, is an inhibitor of nitric oxide production.

When cultured anaerobically, Enterocloster sp. RD014215 was found to produce 1. Using nuclear magnetic resonance and mass spectroscopy, the planar structure of 1 was determined to be 3-hydroxy-3-(2-oxopropyl)indolin-2-one. The chirality of 1 was implied as S by comparing the optical rotation value of 1 with literature reports of the synthesized compounds. To our knowledge, this work represents the first discovery of the metabolite produced by Enterocloster strain. 1 exhibited inhibition of nitric oxide (NO) production, demonstrating a 50% inhibitory activity (IC50) of 34 µm for NO production by murine macrophage cells subjected to lipopolysaccharide stimulation.

Leukotriene B4 receptor 2 governs macrophage migration during tissue inflammation

Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies invitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation invivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.

Sex bias in celiac disease: XWAS and monocyte eQTLs in women identify TMEM187 as a functional candidate gene

BackgroundCeliac disease (CeD) is an immune-mediated disorder that develops in genetically predisposed individuals upon gluten consumption. HLA risk alleles explain 40% of the genetic component of CeD, so there have been continuing efforts to uncover non-HLA loci that can explain the remaining heritability. As in most autoimmune disorders, the prevalence of CeD is significantly higher in women. Here, we investigated the possible involvement of the X chromosome on the sex bias of CeD.MethodsWe performed a X chromosome-wide association study (XWAS) and a gene-based association study in women from the CeD Immunochip (7062 cases, 5446 controls). We also constructed a database of X chromosome cis-expression quantitative trait loci (eQTLs) in monocytes from unstimulated (n = 226) and lipopolysaccharide (LPS)-stimulated (n = 130) female donors and performed a Summary-data-based MR (SMR) analysis to integrate XWAS and eQTL information. We interrogated the expression of the potentially causal gene (TMEM187) in peripheral blood mononuclear cells (PBMCs) from celiac patients at onset, on a gluten-free diet, potential celiac patients and non-celiac controls.ResultsThe XWAS and gene-based analyses identified 13 SNPs and 25 genes, respectively, 22 of which had not been previously associated with CeD. The X chromosome cis-eQTL analysis found 18 genes with at least one cis-eQTL in naïve female monocytes and 8 genes in LPS-stimulated female monocytes, 2 of which were common to both situations and 6 were unique to LPS stimulation. SMR identified a potentially causal association of TMEM187 expression in naïve monocytes with CeD in women, regulated by CeD-associated, eQTL-SNPs rs7350355 and rs5945386. The CeD-risk alleles were correlated with lower TMEM187 expression. These results were replicated using eQTLs from LPS-stimulated monocytes. We observed higher levels of TMEM187 expression in PBMCs from female CeD patients at onset compared to female non-celiac controls, but not in male CeD individuals.ConclusionUsing X chromosome genotypes and gene expression data from female monocytes, SMR has identified TMEM187 as a potentially causal candidate in CeD. Further studies are needed to understand the implication of the X chromosome in the higher prevalence of CeD in women.

Reduced Population of CD36-/ABCA1+ Macrophages is Correlated with An Increase of Coronary Artery Disease Risk Markers in Type 2 Diabetes Mellitus

BACKGROUND: Cluster of differentiation (CD)36 and adenosine triphosphate-binding cassette transporter A1 (ABCA1) are 2 macrophages-expressed receptors that promote cholesterol uptake and efflux, in which their imbalance might be associated with the foam cell formation risk. Type 2 diabetes mellitus (T2DM) has been correlated with the increase of this plaque formation. Therefore, it is necessary to determine whether expression of CD36 and ABCA1 in macrophages are correlated with coronary artery disease (CAD) risk markers in T2DM cases.METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 13 diabetic patients and 11 healthy donors. Then, the PBMC-derived macrophages were cultured with supplement of oxidized low-density lipoprotein (ox-LDL) or lipopolysaccharide (LPS). Expression of CD36 and ABCA1 was measured using flowcytometry, meanwhile the supernatant concentration of interleukin (IL)-1b and IL-10 was measured by multiplex immunoassay.RESULTS: T2DM subjects more likely to have low proportion of CD36-ABCA+ macrophages compared to healthy donors (p=0.041) and it had negative correlation with glucose homeostasis and insulin resistance markers, including fasting blood glucose (FBG, r=-0.408, p=0.048), glycated hemoglobin (HbA1c, r=-0.380, p=0.049), triglyceride glucose index (r=-0,518, p=0.009), and high-sensitivity C-reactive protein (hs-CRP, r=-0.556, p=0.005). Moreover, it also had a negative correlation with atherogenic markers such as triglyceride (r=-0.417, p=0.043), triglyceride/HDL, and LDL/HDL, but had positive correlation with HDL (r=0.540, p=0.007). Most of T2DM subjects had high IL-1β/IL-10 ratio after ox-LDL and LPS stimulation (p=0.02 and p=0.05, respectively).CONCLUSION: Reduced proportion of CD36-ABCA1+ macrophages followed with high IL-1β/IL-10 can be a marker of CAD in T2DM.KEYWORDS: type 2 diabetes mellitus, coronary artery disease, macrophages, ABCA1, CD36

A Comparative Transcriptomic Study and Key Gene Targeting of Lamprey Gonadal Immune Response

ABSTRACT The mammalian testis and ovary possess special immunocompetence, which is central to provide protection against pathogens. However, the innate immune responses to immune challenges in lamprey gonads are poorly understood. In this study, we extracted RNA from testis and ovary tissues of lampreys at 0 hour, 8 hours and 17 days after lipopolysaccharides (LPS) stimulation and performed transcriptome sequencing. While the transcriptome profiles of the two tissues were different for the most part, genes LIP, LECT2, LAL2, GRN, ITLN, and C1q were found to be the most significantly up-regulated genes in both. Quantitative Real-time PCR (qRT-PCR) analysis confirmed that these genes were upregulated after stimulation. Furthermore, immunohistochemical staining showed that these genes in lamprey gonads are expressed in high quantities and have a specific distribution. Taken together, our results suggest that these genes could play an essential role in response of the gonads to LPS induction. This research establishes a basis for investigating the immune mechanism of vertebrate gonads and presents a fresh concept for gaining insight into the evolutionary development of jawless vertebrates.

TGF-β1 Decreases Microglia-Mediated Neuroinflammation and Lipid Droplet Accumulation in an In Vitro Stroke Model.

Hypoxia triggers reactive microglial inflammation and lipid droplet (LD) accumulation under stroke conditions, although the mutual interactions between these two processes are insufficiently understood. Hence, the involvement of transforming growth factor (TGF)-β1 in inflammation and LD accumulation in cultured microglia exposed to hypoxia were analyzed herein. Primary microglia were exposed to oxygen-glucose deprivation (OGD) injury and lipopolysaccharide (LPS) stimulation. For analyzing the role of TGF-β1 patterns under such conditions, a TGF-β1 siRNA and an exogenous recombinant TGF-β1 protein were employed. Further studies applied Triacsin C, an inhibitor of LD formation, in order to directly assess the impact of LD formation on the modulation of inflammation. To assess mutual microglia-to-neuron interactions, a co-culture model of these cells was established. Upon OGD exposure, microglial TGF-β1 levels were significantly increased, whereas LPS stimulation yielded decreased levels. Elevating TGF-β1 expression proved highly effective in suppressing inflammation and reducing LD accumulation in microglia exposed to LPS. Conversely, inhibition of TGF-β1 led to the promotion of microglial cell inflammation and an increase in LD accumulation in microglia exposed to OGD. Employing the LD formation inhibitor Triacsin C, in turn, polarized microglia towards an anti-inflammatory phenotype. Such modulation of both microglial TGF-β1 and LD levels significantly affected the resistance of co-cultured neurons. This study provides novel insights by demonstrating that TGF-β1 plays a protective role against microglia-mediated neuroinflammation through the suppression of LD accumulation. These findings offer a fresh perspective on stroke treatment, suggesting the potential of targeting this pathway for therapeutic interventions.

Regulator of G protein signaling protein 6 alleviates acute lung injury by inhibiting inflammation and promoting cell self-renewal in mice

BackgroundAcute respiratory distress syndrome (ARDS) is a disease with high mortality and morbidity. Regulator of G protein signaling protein 6 (RGS6), identified as a tumor suppressor gene, has received increasing attention owing to its close relationship with oxidative stress and inflammation. However, the association between ARDS and RGS6 has not been reported.MethodsCongruously regulated G protein-coupled receptor (GPCR)-related genes and differentially expressed genes (DEGs) in an acute lung injury (ALI) model were identified, and functional enrichment analysis was conducted. In an in vivo study, the effects of RGS6 knockout were studied in a mouse model of ALI induced by lipopolysaccharide (LPS). HE staining, ELISA, and immunohistochemistry were used to evaluate pathological changes and the degree of inflammation. In vitro, qRT‒PCR, immunofluorescence staining, and western blotting were used to determine the dynamic changes in RGS6 expression in cells. The RGS6 overexpression plasmid was constructed for transfection. qRT‒PCR was used to assess proinflammatory factors transcription. Western blotting and flow cytometry were used to evaluate apoptosis and reactive oxygen species (ROS) production. Organoid culture was used to assess the stemness and self-renewal capacity of alveolar epithelial type II cells (AEC2s).ResultsA total of 110 congruously regulated genes (61 congruously upregulated and 49 congruously downregulated genes) were identified among GPCR-related genes and DEGs in the ALI model. RGS6 was downregulated in vivo and in vitro in the ALI model. RGS6 was expressed in the cytoplasm and accumulated in the nucleus after LPS stimulation. Compared with the control group, we found higher mortality, more pronounced body weight changes, more serious pulmonary edema and pathological damage, and more neutrophil infiltration in the RGS6 knockout group upon LPS stimulation in vivo. Moreover, AEC2s loss was significantly increased upon RGS6 knockout. Organoid culture assays showed slower alveolar organoid formation, fewer alveolar organoids, and impaired development of new structures after passaging upon RGS6 knockout. In addition, RGS6 overexpression decreased ROS production as well as proinflammatory factor transcription in macrophages and decreased apoptosis in epithelial cells.ConclusionsRGS6 plays a protective role in ALI not only in early inflammatory responses but also in endogenous lung stem cell regeneration.Graphical

Activation of toll-like receptor 4/nuclear factor-kappa B signaling by triggering a receptor expressed on myeloid cells 1 promotes alveolar macrophage M1 polarization and exacerbates septic acute lung injury.

Septic acute lung injury (ALI) is a life-threatening condition commonly occurring in the intensive care unit. Inflammation is considered as the basic pathological response of septic ALI. Triggering receptor expressed on myeloid cells 1 (TREM1) is a member of the immunoglobulin superfamily receptors that regulates the inflammatory response. However, the role of TREM1 in septic ALI has not yet been reported. Cell viability was tested using the MTT assay. TdT-mediated dUTP nick end labeling assay and flow cytometry were used for apoptosis. The level of protein was detected using western blot analysis. The levels of tumor necrosis factor-α and interleukin-1β were assessed using enzyme-linked immunosorbent assay. The lactate dehydrogenase content was assessed using the assay kit. Myeloperoxidase activity was determined using an assay. Histology of lung tissue was further analyzed through hematoxylin-eosin staining. We found that TREM1 knockdown by transfection with si-TREM1 inhibited lipopolysaccharide (LPS)-induced cell apoptosis of alveolar macrophage cell line MH-S. The LPS stimulation caused M1 polarization of MH-S cells, which could be reversed by TREM1 knockdown. In vivo assays proved that si-TREM1 injection improved lung injury and inflammation of cecal ligation and puncture-induced ALI in mice. In addition, TREM1 knockdown suppressed the activation of toll-like receptor 4/nuclear factor-kappa B signaling, implying the involvement of TLR4 in the effects of TREM1 in response to LPS stimulation. This study examined the proinflammatory role of TREM1 in septic ALI and its regulatory effect on alveolar macrophage polarization. These results suggest that TREM1 could potentially serve as a therapeutic target in the prevention and treatment of ALI.

Comprehensive proteomic analysis reveals omega-3 fatty acids to counteract endotoxin-stimulated metabolic dysregulation in porcine enterocytes

Omega-3 polyunsaturated fatty acids (n-3 PUFA), such as the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are reported to beneficially affect the intestinal immunity. The biological pathways modulated by n-3 PUFA during an infection, at the level of intestinal epithelial barrier remain elusive. To address this gap, we investigated the proteomic changes induced by n-3 PUFA in porcine enterocyte cell line (IPEC-J2), in the presence and absence of lipopolysaccharide (LPS) stress conditions using shotgun proteomics analysis integrated with RNA-sequencing technology. A total of 33, 85, and 88 differentially abundant proteins (DAPs) were identified in cells exposed to n-3 PUFA (DHA:EPA), LPS, and n-3 PUFA treatment followed by LPS stimulation, respectively. Functional annotation and pathway analysis of DAPs revealed the modulation of central carbon metabolism, including the glycolysis/gluconeogenesis, pentose phosphate pathway, and oxidative phosphorylation processes. Specifically, LPS caused metabolic dysregulation in enterocytes, which was abated upon prior treatment with n-3 PUFA. Besides, n-3 PUFA supplementation facilitated enterocyte development and lipid homeostasis. Altogether, this work for the first time comprehensively described the biological pathways regulated by n-3 PUFA in enterocytes, particularly during endotoxin-stimulated metabolic dysregulation. Additionally, this study may provide nutritional biomarkers in monitoring the intestinal health of human and animals on n-3 PUFA-based diets.

Influence of organism stimulation with bacterial lipopolysaccharide on nitric oxide production and metabolism in rat heart on the background of metabolic syndrome

Aim. The aim of the study was to establish the changes in nitric oxide production and metabolism in rat heart during combined influence of organism stimulation with bacterial lipopolysaccharide (LPS) and modeling of metabolic syndrome (MetS).&#x0D; Materials and methods. The study was conducted on 24 mature male Wistar rats weighing 200–260 g. Experiment lasted 60 days. The animals were divided into 4 groups of 6 animals each: control group, MetS group, LPS stimulation group, LPS + MetS group. MetS was reproduced by using a 20 % fructose solution as the only source of drinking water. LPS of Salmonella typhi was administered at a dose of 0.4 μg/kg intraperitoneally. Animals from LPS + MetS group received a 20 % fructose solution as the only source of drinking water and were administered LPS. In 10 % tissue homogenate of rat heart we studied: total activity of NO-synthases (NOS), activity of constitutive (cNOS) and inducible (iNOS) isoforms, activity of nitrate (NaR) and nitrite (NiR) reductases, concentration of peroxynitrites (ONOO-), nitrites, nitrosothiols and hydrogen sulfide.&#x0D; Results. Combination of MetS and stimulation of organism with LPS led to increase in total NOS activity by 32.72 % compared to control group. Activity of cNOS did not change compared to control group. Activity of iNOS increased by 33.76 %. Arginase activity decreased by 23.53 %. NaR activity and NiR activity were increased by 86.67 % and by 149.29 %, respectively. Combination of MetS and stimulation of organism with LPS led to decrease in nitrite and nitrosothiols concentration by 38.73 % and by 54.79 %, respectively. Under these conditions concentration of ONOOelevated by 398.0 % compared to control group. Concentration of H S decreased by 27.56 %.&#x0D; Conclusions. Combination of metabolic syndrome and stimulation of organism with bacterial lipopolysaccharide leads to prevalence of peroxynitrite formation during increased nitric oxide production NO-synthase-dependent and nitrate-nitrite-NO pathways in rat heart.