Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1ß, IL-6, IL-12p40, IFN-ß) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.

Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response.

Introduction: The pathogenic bacterium Helicobacter pylori has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of H. pylori glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells. Methods: To test this hypothesis, human cell lines were challenged with wildtype H. pylori alongside an array of H. pylori glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion. Results: Our findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does. Discussion: The outcomes of this study demonstrate that H. pylori glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens.

[Inhibition of connexin 43-mediated hemichannel activity promotes odontoblast differentiation of human dental pulp cells induced by lipopolysaccharide].

PURPOSE: To investigate the role and mechanism of connexin 43（Cx43）in odontoblast differentiation of human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). METHODS: The maxillary first molar injury model of SD rats was established. The expression pattern of Cx43 in dental pulp repair after injury was detected by immunofluorescence(IF) staining. hDPCs was respectively stimulated with 0, 1, 10, 100 and 1 000 ng/mL LPS for 6 h to screen the optimal concentration, and then the expression of Cx43 was inhibited and overexpressed in hDPCs. Quantitative real-time PCR(qRT-PCR) and Western blot(WB) were used to detect the expression of Cx43 and dentin sialophosphoprotein (DSPP), dental matrix protein-1 (DMP-1), osterix (Osx) and extracellular signal-regulated kinase (ERK) activity. Furthermore, hDPCs were treated with specific Cx43 channel inhibitors to investigate the effect of Cx43-mediated channel activity in odontoblast differentiation of hDPCs, and to explore the role and mechanism of Cx43 in regulating odontoblast differentiation of hDPCs induced by LPS. Statistical analysis was performed with SPSS 26.0 software package. RESULTS: IF results showed that Cx43 was mainly expressed in the odontoblast layer in healthy dental pulp tissues. At 3-24 h after tooth injury, the expression of Cx43 decreased and then gradually increased to the normal level; from 3 days to 2 weeks after injury, the expression of Cx43 tended to be down-regulated which was in the odontoblast layer and pulp proper. The expression of DSPP mRNA was significantly up-regulated in the hDPCs stimulated with 10 ng/mL LPS for 6 h(P＜0.01). Inhibition of Cx43 significantly up-regulated the expression of DSPP, DMP-1 and Osx mRNA induced by LPS in hDPCs(P＜0.05), while overexpression of Cx43 obviously inhibited the expression of factors related to LPS-induced odontoblast differentiation(P＜0.01) and the fluorescence intensity of DSPP. 10 ng/mL LPS activated ERK signal in hDPCs, and overexpression of Cx43 significantly attenuated the activity of ERK signal induced by LPS(P＜0.01). Inhibition of Cx43-mediated hemichannel (HC) promoted mRNA expression of factors related to odontoblast differentiation in hDPCs and the activity of ERK signal induced by LPS(P＜0.05), while blocking Cx43-mediated gap junction channel (GJC) inhibited odontoblast differentiation. CONCLUSIONS: Cx43 participates in the regulation of dental pulp repair after injury, and its expression shows a downward trend as a whole. Inhibition of Cx43 or blocking of HC promotes LPS-induced ERK signal activity and odontoblast differentiation of hDPCs.

Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats. / è‰¾ç¸å¯¹è‚¥èƒ–å¤§é¼ è‚ é“å±éšœåŠŸèƒ½åŠTLR4/NF-κB p65信号通路的影响.

OBJECTIVES: To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. METHODS: Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. RESULTS: Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). CONCLUSIONS: Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.

Dual function of LapB (YciM) in regulating Escherichia coli lipopolysaccharide synthesis.

Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled-making LPS synthesis a promising target for developing new antibiotics. Escherichia coli adaptor protein LapB (YciM) plays an important role in regulating LPS synthesis by promoting degradation of LpxC, a deacetylase that catalyzes the first committed step in LPS synthesis. Under conditions where LPS is abundant, LapB recruits LpxC to the AAA+ protease FtsH for degradation. LapB achieves this by simultaneously interacting with FtsH through its transmembrane helix and LpxC through its cytoplasmic domain. Here, we describe a cryo-EM structure of the complex formed between LpxC and the cytoplasmic domain of LapB (LapBcyto). The structure reveals how LapB exploits both its tetratricopeptide repeat (TPR) motifs and rubredoxin domain to interact with LpxC. Through both in vitro and in vivo analysis, we show that mutations at the LapBcyto/LpxC interface prevent LpxC degradation. Unexpectedly, binding to LapBcyto also inhibits the enzymatic activity of LpxC through allosteric effects reminiscent of LpxC activation by MurA in Pseudomonas aeruginosa. Our findings argue that LapB regulates LPS synthesis in two steps: In the first step, LapB inhibits the activity of LpxC, and in the second step, it commits LpxC to degradation by FtsH.

Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis.

Oxidative stress-induced lipid accumulation is mediated by lipid droplets (LDs) homeostasis, which sequester vulnerable unsaturated triglycerides into LDs to prevent further peroxidation. Here we identify the upregulation of lipopolysaccharide-binding protein (LBP) and its trafficking through LDs as a mechanism for modulating LD homeostasis in response to oxidative stress. Our results suggest that LBP induces lipid accumulation by controlling lipid-redox homeostasis through its lipid-capture activity, sorting unsaturated triglycerides into LDs. N-acetyl-L-cysteine treatment reduces LBP-mediated triglycerides accumulation by phospholipid/triglycerides competition and Peroxiredoxin 4, a redox state sensor of LBP that regulates the shuttle of LBP from LDs. Furthermore, chronic stress upregulates LBP expression, leading to insulin resistance and obesity. Our findings contribute to the understanding of the role of LBP in regulating LD homeostasis and against cellular peroxidative injury. These insights could inform the development of redox-based therapies for alleviating oxidative stress-induced metabolic dysfunction.

Resveratrol prevents the release of neutrophil extracellular traps (NETs) by controlling hydrogen peroxide levels and nuclear elastase migration.

Neutrophil extracellular traps (NETs) are defense mechanisms that trap and kill microorganisms and degrade cytokines. However, excessive production, dysregulation of suppression mechanisms, or inefficient removal of NETs can contribute to increased inflammatory response and the development of pathological conditions. Therefore, research has focused on identifying drugs that inhibit or delay the NET release process. Since reactive oxygen species (ROS) play a significant role in NET release, we aimed to investigate whether resveratrol (RSV), with a wide range of biological and pharmacological properties, could modulate NET release in response to different stimuli. Thus, human neutrophils were pretreated with RSV and subsequently stimulated with PMA, LPS, IL-8, or Leishmania. Our findings revealed that RSV reduced the release of NETs in response to all tested stimuli. RSV decreased hydrogen peroxide levels in PMA- and LPS-stimulated neutrophils, inhibited myeloperoxidase activity, and altered the localization of neutrophil elastase. RSV inhibition of NET generation was not mediated through A2A or A2B adenosine receptors or PKA. Based on the observed effectiveness of RSV in inhibiting NET release, our study suggests that this flavonoid holds potential as a candidate for treating NETs involving pathologies.

Overexpression of Wnt5a promoted the protective effect of mesenchymal stem cells on Lipopolysaccharide-induced endothelial cell injury via activating PI3K/AKT signaling pathway.

BACKGROUND: Lung endothelial barrier injury plays an important role in the pathophysiology of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) therapy has shown promise in ARDS treatment and restoration of the impaired barrier function. It has been reported that Wnt5a shows protective effects on endothelial cells. Therefore, the study aimed to investigate whether overexpression of Wnt5a could promote the protective effects of MSCs on Lipopolysaccharide (LPS)-induced endothelial cell injury. METHODS: To evaluate the protective effects of MSCs overexpressing Wnt5a, we assessed the migration, proliferation, apoptosis, and angiogenic ability of endothelial cells. We assessed the transcription of protective cellular factors using qPCR and determined the molecular mechanism using Western blot analysis. RESULTS: Overexpression of Wnt5a upregulated the transcription of protective cellular factors in MSCs. Co-culture of MSCWnt5a promoted endothelial migration, proliferation and angiogenesis, and inhibited endothelial cell apoptosis through the PI3K/AKT pathway. CONCLUSIONS: Overexpression of Wnt5a promoted the therapeutic effect of MSCs on endothelial cell injury through the PI3K/AKT signaling. Our study provides a novel approach for utilizing genetically modified MSCs in the transplantation therapy for ARDS.

Role of gut-derived bacterial lipopolysaccharide and peripheral TLR4 in immobilization stress-induced itch aggravation in a mouse model of atopic dermatitis.

Psychological stress and intestinal leakage are key factors in atopic dermatitis (AD) recurrence and exacerbation. Here, we demonstrate the mechanism underlying bacterial translocation across intestinal epithelial barrier damaged due to stress and further aggravation of trimellitic anhydride (TMA)-induced itch, which remain unclear, in AD mice. Immobilization (IMO) stress exacerbated scratching bouts and colon histological damage, and increased serum corticosterone and lipopolysaccharide (LPS). Orally administered fluorescein isothiocyanate (FITC)-dextran and surgically injected (into the colon) Cy5.5-conjugated LPS were detected in the serum and skin after IMO stress, respectively. The relative abundance of aerobic or facultative anaerobic bacteria was increased in the colon mucus layer, and Lactobacillus murinus, E. coli, Staphylococcus nepalensis, and several strains of Bacillus sp. were isolated from the spleens and mesenteric lymph nodes. Oral antibiotics or intestinal permeability blockers, such as lubiprostone (Lu), 2,4,6-triaminopyrimidine (TAP) and ML-7, inhibited IMO stress-associated itch; however, it was reinduced through intradermal or i.p. injection of LPS without IMO stress. I.p. injection of TAK-242 (resatorvid), a TLR4 inhibitor, abrogated IMO stress-associated itch, which was also confirmed in TLR4-KO mice. IMO stress alone did not cause itch in naïve mice. IMO stress-induced itch aggravation in TMA-treated AD mice might be attributed to the translocation of gut-derived bacterial cells and LPS, which activates peripheral TLR4 signaling.

Upregulated ATF1 Promotes Lipopolysaccharide Induced Inflammatory Response and Inhibits Osteogenic Differentiation of Human Periodontal Ligament Cells by Regulating NF-κB Pathway.

BACKGROUND: Periodontitis is a chronic inflammatory disease resulting from bacterial plaque infection. While the involvement of activating transcription factor 1 (ATF1) has been extensively explored in various human diseases, its specific role in periodontitis remains unclear. This study aims to elucidate the expression and biological function of ATF1 in the context of periodontitis. METHODS: Primary human periodontal ligament cells (hPDLCs) were procured from clinical samples and subsequently characterized. Following treatment with P. gingivalis lipopolysaccharide (LPS, 10 µg/mL), hPDLCs underwent transfection with either ATF1 vector or siRNA. The expression levels of ATF1 in LPS-treated hPDLCs or transfected cells were evaluated through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Inflammatory factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1ß), were quantified using Enzyme-linked Immunosorbent Assay (ELISA). The assessment of osteogenic proteins, such as runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG), as well as noncanonical nuclear factor-kappaB (NF-κB) pathway-related proteins (p65, p-p65, IkBα, p-IkBα), was conducted using western blot assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry assays were employed to detect cell viability. RESULTS: LPS induced an inflammatory response and hindered the osteogenic differentiation of hPDLCs (p < 0.05, p < 0.01). Furthermore, ATF1 silencing enhanced cell proliferation and suppressed apoptosis in LPS-stimulated hPDLCs (p < 0.05, p < 0.01). ATF1 silencing not only restrained the inflammatory response but also promoted the osteogenic differentiation of LPS-stimulated hPDLCs (p < 0.05, p < 0.01). Importantly, ATF1 silencing effectively blocked the LPS-induced activation of the NF-κB signaling pathway (p < 0.05, p < 0.01, p < 0.001). CONCLUSIONS: ATF1 emerges as a promising treatment option, inhibiting the osteogenic differentiation of hPDLCs and mitigating the inflammatory response by preventing the phosphorylation of the NF-κB signaling pathway.

Anti-inflammatory potential via the MAPK signaling pathway of Lactobacillus spp. isolated from canine feces.

Two probiotic candidates, Lactobacillus reuteri C1 (C1) and Lactobacillus acidophilus C5 (C5), which were previously isolated from canines, were evaluated in the present study. L. reuteri and L. acidophilus have anti-oxidant, anti-inflammatory, immune-enhancing, and anti-cancer properties and exhibit various probiotic effects in humans and animals. The strains C1 and C5 demonstrated good tolerance to acid and bile salt exposure, exhibited effective adhesion to HT-29 cell monolayer, and displayed sensitivity to antibiotics, thus affirming their probiotic characteristics. Moreover, C1 and C5 exhibited the ability to downregulate the expression of inducible NO synthase (iNOS), an immunomodulatory factor, leading to a reduction in NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These strains also demonstrated potent anti-inflammatory effects in LPS-stimulated RAW 264.7 cells, achieved through the augmentation of anti-inflammatory cytokine IL-10 expression and the inhibition of pro-inflammatory cytokine IL-1ß expression. These anti-inflammatory effects of C1 and C5 were closely associated with the mitogen-activated protein kinase (MAPK) signaling pathway. The results of the present study suggest that the C1 and C5 probiotic candidates attenuate LPS-induced inflammation via the MAPK signaling pathway and the strains can be used as probiotics considering their anti-inflammatory potential.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

Vesicular Messages from Dental Biofilms for Neutrophils.

The encounter between dental biofilm and neutrophils in periodontitis remains elusive, although it apparently plays a crucial role in the periodontal pathology and constitutes a key topic of periodontology. Dental biofilm and neutrophils were isolated from orally healthy persons and patients with periodontitis. We investigated biofilm and its particle-shedding phenomenon with electron microscopy and nanoparticle tracking analysis (NTA); biofilm shedding-neutrophil interactions were examined ex vivo with epi-fluorescence microscopy. For this purpose, we used acellular dental biofilm shedding, purified lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA) as activators, and the interleukin 8 receptor beta (CXCR2) inhibitor and the anti-interleukin 8 receptor alpha (CXCR1) antibody as modulators. The shedding of acellular dental biofilms overwhelmingly consists of bacterial extracellular vesicles (BEVs). The latter induced the moderate formation of neutrophil extracellular traps (NETs) in orally healthy subjects and a strong formation in patients with periodontitis. A CXCR2 inhibitor and an anti-CXCR1 antibody had a minor effect on NET formation. Neutrophils from patients with periodontitis exhibited NET hyper-responsiveness. BEVs were stronger inducers of NET formation than purified LPS and PMA. A plateau of neutrophil responsiveness is reached above the age of 40 years, indicating the abrupt switch of maladaptive trained immunity (TI) into the activated modus. Our results suggest that dental biofilms consist of and disseminate immense amounts of outer membrane vesicles (OMVs), which initiate NET formation via a non-canonical cytosolic LPS/caspase-4/11/Gasdermin D pathway. This modus of NET formation is independent of neutrophil elastase (NE), myeloperoxidase (MPO), peptidylarginine deiminase 4 (PAD4), and toll-like receptors (TLR). In periodontitis, the hyper-responsiveness of neutrophils to BEVs and the increased NET formation appear to be a consequence of TI.

Propofol improves survival in a murine model of sepsis via inhibiting Rab5a-mediated intracellular trafficking of TLR4.

BACKGROUND: Propofol is a widely used anesthetic and sedative, which has been reported to exert an anti-inflammatory effect. TLR4 plays a critical role in coordinating the immuno-inflammatory response during sepsis. Whether propofol can act as an immunomodulator through regulating TLR4 is still unclear. Given its potential as a sepsis therapy, we investigated the mechanisms underlying the immunomodulatory activity of propofol. METHODS: The effects of propofol on TLR4 and Rab5a (a master regulator involved in intracellular trafficking of immune factors) were investigated in macrophage (from Rab5a-/- and WT mice) following treatment with lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) in vitro and in vivo, and peripheral blood monocyte from sepsis patients and healthy volunteers. RESULTS: We showed that propofol reduced membrane TLR4 expression on macrophages in vitro and in vivo. Rab5a participated in TLR4 intracellular trafficking and both Rab5a expression and the interaction between Rab5a and TLR4 were inhibited by propofol. We also showed Rab5a upregulation in peripheral blood monocytes of septic patients, accompanied by increased TLR4 expression on the cell surface. Propofol downregulated the expression of Rab5a and TLR4 in these cells. CONCLUSIONS: We demonstrated that Rab5a regulates intracellular trafficking of TLR4 and that propofol reduces membrane TLR4 expression on macrophages by targeting Rab5a. Our study not only reveals a novel mechanism for the immunomodulatory effect of propofol but also indicates that Rab5a may be a potential therapeutic target against sepsis.

A pattern recognition receptor interleukin-1 receptor is involved in reproductive immunity in Macrobrachium nipponense ovary.

The family of TIR domain-containing receptors includes numerous proteins involved in innate immunity. In this study, a member of this family was characterized from the ovary of the oriental river prawn Macrobrachium nipponense and identified as interleukin-1 receptor (MnIL-1R). Meanwhile, to elucidate the conservation of IL-1R, its orthologous were identified in several crustacean species as well. In addition, the expression pattern of MnIL-1R in various adult tissues and post different pathogen-associated molecular patterns (PAMPs) challenge in ovary was analyzed with qRT-PCR technology. Finally, the roles of MnIL-1R in the ovary were analyzed by RNAi technology. The main results are as follows: (1) MnIL-1R comprises a 1785 bp ORF encoding 594 amino acids and is structurally composed of five domains: a signal peptide, two immunoglobulin (IG) domains, a transmembrane region, and a TIR-2 domain; (2) the TIR domain showed a high conservation among analyzed crustacean species; (3) MnIL-1R is widely detected in all tested tissues including ovary; (4) MnIL-1R showed a positive response to challenges with LPS, PGN, and polyI:C in the ovary; (5) its IG domain showed strong binding ability to LPS and PGN, confirming its role as a pattern recognition receptor; (6) the expression patterns of several members of the Toll signaling pathway (Myd88, TRAF-6, Dorsal, and Relish) was similar to that of MnIL-1R after challenges with LPS, PGN, and polyI:C in the ovary; (7) the silencing of MnIL-1R resulted in down-regulation of theses gene' (Myd88, TRAF-6, Dorsal, and Relish) expression level in the ovary. These results suggest that MnIL-1R can activate the Toll signaling pathway in the ovary by directly recognizing LPS and PGN through its IG domain, thereby contributing to the immune response in the ovary of M. nipponense.

An LPS-induced TNF-α factor involved in immune response of oyster Crassostrea gigas by regulating haemocytes apoptosis.

LPS induced TNF-α Factor (LITAF) is a transcription factor widely involving in activation of Tumor Necrosis Factor (TNF) and other cytokines in the inflammatory response. In the present study, a homologue of LITAF with a conserved LITAF domain was identified from the Pacific oyster Crassostrea gigas. The transcripts of CgLITAF were detected in all examined tissues with highest expression in hepatopancrease. The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgLITAF protein in haemocytes. While the mRNA level of CgLITAF changed slightly after LPS stimulation. When the siRNA of CgLITAF was injected to inhibit its expression, the apoptotic level of haemocytes decreased observably after LPS stimulation. Consistently, the transcripts of CgTNF3 and CgTNF4 (LOC105343080, LOC105341146), the apoptotic-related molecules including CgBax, CgCytochrome c, CgCaspase9 and CgCaspase3, were significantly suppressed in the CgLITAF-RNAi oysters. While the mRNA expression level of CgBcl was enhanced significantly in the CgLITAF-RNAi oysters. These results indicated that CgLITAF promoted haemocyte apoptosis by regulating the expression of apoptotic-related factors, suggesting its important role in the immune response of oysters.

Differential tissue immune stimulation through immersion in bacterial and viral agonists in the Antarctic Notothenia rossii.

The genome evolution of Antarctic notothenioids has been modulated by their extreme environment over millennia and more recently by human-caused constraints such as overfishing and climate change. Here we investigated the characteristics of the immune system in Notothenia rossii and how it responds to 8 h immersion in viral (Poly I:C, polyinosinic: polycytidylic acid) and bacterial (LPS, lipopolysaccharide) proxies. Blood plasma antiprotease activity and haematocrit were reduced in Poly I:C-treated fish only, while plasma protein, lysozyme activity and cortisol were unchanged with both treatments. The skin and duodenum transcriptomes responded strongly to the treatments, unlike the liver and spleen which had a mild response. Furthermore, the skin transcriptome responded most to the bacterial proxy (cell adhesion, metabolism and immune response processes) and the duodenum (metabolism, response to stress, regulation of intracellular signal transduction, and immune system responses) to the viral proxy. The differential tissue response to the two proxy challenges is indicative of immune specialisation of the duodenum and the skin towards pathogens. NOD-like and C-type lectin receptors may be central in recognising LPS and Poly I:C. Other antimicrobial compounds such as iron and selenium-related genes are essential defence mechanisms to protect the host from sepsis. In conclusion, our study revealed a specific response of two immune barrier tissue, the skin and duodenum, in Notothenia rossii when exposed to pathogen proxies by immersion, and this may represent an adaptation to pathogen infective strategies.

Synergistic effects of tetramethylpyrazine and astragaloside IV on spinal cord injury via alteration of astrocyte A1/A2 polarization through the Sirt1-NF-κB pathway.

OBJECTIVE: Reactive astrocytes are hallmarks of traumatic spinal cord injury (T-SCI) and are associated with neuropathic pain (NP). Mediating the functional phenotype of reactive astrocytes helps neural repair and ameliorates NP in T-SCI. Here, we aimed to explore the role of tetramethylpyrazine (TMPZ) and astragaloside IV (AGS-IV) in astrocyte polarization and the underlying molecular mechanism in T-SCI. METHODS: Primary cultured astrocytes from mice were treated with LPS or conditioned medium from "M1" polarized microglia (M1-CM), followed by TMPZ and/or AGS-IV administration. The expression levels of "A1" astrocyte-specific markers (including C3, GBP2, Serping1, iNOS), "A2" astrocyte-specific markers (including S100a10 and PTX3), Sirt1 and NF-κB were detected via western blotting. TNF-α and IL-1ß levels were detected via ELISA. RT-PCR was used to evaluate OIP5-AS1 and miR-34a expression. si-OIP5-AS1 or the Sirt1 inhibitor EX-527 was administered to astrocytes. A spinal cord injury (SCI) model was constructed in Sprague-Dawley (SD) rats. Alterations in astrocytic "A1/A2" polarization in the spinal cord tissues were evaluated. RESULTS: LPS and M1-CM induced "A1" polarization of primary astrocytes. TMPZ and ASG IV could substantially reduce the expression of "A1"-related biomarkers but enhance "A2"-related biomarkers. OIP5-AS1 and Sirt1 levels were reduced in "A1"-polarized astrocytes, while miR-34a and p-NF-κB p65 were elevated. TMPZ and ASG IV enhanced OIP5-AS1 and Sirt1 levels and, in contrast, attenuated the changes in miR-34a and p-NF-κB p65 levels. Notably, the TMPZ and ASG IV combination had stronger effects on astrocyte polarization than the single treatment with TMPZ or ASG IV. OIP5-AS1 knockdown and Sirt1 inhibition both reversed the regulatory effects of TMPZ and ASG IV in astrocytic polarization. According to the in vivo experiments, the expression of "A1"-associated markers was enhanced in the spinal cords of SCI rats. The TMPZ and ASG IV combination reduced astrocytic "A1" polarization and enhanced astrocytic "A2" polarization. The expression of lncRNA OIP5-AS1 and Sirt1 was enhanced by TMPZ and ASG IV, while that of miR-34a and p-NF-κB p65 was inhibited. CONCLUSION: The combination of TMPZ and ASG IV can ameliorate dysregulated astrocytic polarization induced by spinal cord injury by affecting the lncRNA OIP5-AS1-Sirt1-NF-κB pathway.

Roles of PD-L1 in human adipose-derived mesenchymal stem cells under inflammatory microenvironment.

Mesenchymal stem cells (MSCs) display unique homing and immunosuppression features which make them promising candidates for cell therapy in inflammatory disorders. It is known that C-X-C chemokine receptor type 4 (CXCR4, also known as CD184) is a critical receptor implicated in MSCs migration, and the protein programmed death ligand-1 (PD-L1) is involved in MSC's immunosuppression. However, it remains unclear how the molecular mechanisms regulate PD-L1 expression for migration and immunosuppression of MSCs under the inflammatory microenvironment. In this article, we used the human adipose-derived mesenchymal stem cells (hADMSCs) treated with lipopolysaccharide (LPS) as an in vitro inflammatory model to explore the roles of PD-L1 on the migration and immunosuppression of MSC. Our results demonstrate that in hADMSCs, LPS significantly increased PD-L1 expression, which mediated the migration of the LPS-treated hADMSCs via CXCR4. In addition, we found that the increased PD-L1 expression in the LPS-treated hADMSCs inhibited B cell proliferation and immunoglobulin G secretion through nuclear factor-κB. Our study suggests that the PD-L1 plays critical roles in the homing and immunosuppression of MSCs which are a promising cell therapy to treat inflammatory diseases.