Increase In Expression Of Pro-inflammatory Cytokines Research Articles

SIRT1 alleviates Cd nephrotoxicity through NF-κB/p65 deacetylation–mediated pyroptosis in rat renal tubular epithelial cells

Cadmium (Cd) is a widely distributed environmental pollutant, primarily causing nephrotoxicity through renal proximal tubular cell impairment. Pyroptosis is an inflammation-related nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3)-dependent pathway for programmed cell death. We previously reported that inappropriate inflammation caused by Cd is a major contributor to kidney injury. Therefore, research on Cd-induced inflammatory response and pyroptosis may clarify the mechanisms underlying Cd-induced nephrotoxicity. In this study, we observed that Cd-induced nephrotoxicity is associated with NLRP3 inflammasome activation, leading to an increase in proinflammatory cytokine expression and secretion, as well as pyroptosis-related gene upregulation, both in primary rat proximal tubular (rPT) cells and kidney tissue from Cd-treated rats. In vitro, these effects were significantly abrogated through siRNA-based Nlrp3 silencing; thus, Cd may trigger pyroptosis through an NLRP3 inflammasome–dependent pathway. Moreover, Cd exposure considerably elevated reactive oxygen species (ROS) content. N-acetyl-l-cysteine, an ROS scavenger, mitigated Cd-induced NLRP3 inflammasome activation and subsequent pyroptosis. Mechanistically, Cd hindered the expression and deacetylase activity of SIRT1, eventually leading to a decline in SIRT1–p65 interactions, followed by an elevation in acetylated p65 levels. The administration of resveratrol (a SIRT1 agonist) or overexpression of Sirt1 counteracted Cd-induced RELA/p65/NLRP3 pathway activation considerably, leading to pyroptosis. This is the first study to reveal significant contributions of SIRT1-triggered p65 deacetylation to pyroptosis and its protective effects against Cd-induced chronic kidney injury. Our results may aid in developing potential therapeutic strategies for preventing Cd-induced pyroptosis through SIRT1-mediated p65 deacetylation.

Response of human peripheral blood monocyte-derived macrophages (PBMM) to demineralized and decellularized bovine bone graft substitutes.

The performance of apparently biocompatible implanted bovine bone grafts may be compromised by unresolved chronic inflammation, and poor graft incorporation leading to implant failure. Monitoring the intensity and duration of the inflammatory response caused by implanted bone grafts is crucial. In this study, the ability of demineralized (DMB) and decellularized (DCC) bovine bone substitutes in initiating inflammatory responses to peripheral blood monocyte-derived macrophages (PBMMs) was investigated. The response of PBMMs to bone substitutes was evaluated by using both direct and indirect cell culture, reactive oxygen species (ROS) generation, apoptosis, immunophenotyping, and cytokine production. Analysis of DMB and DCC substitutes using scanning electron microscope (SEM) showed a roughened surface with a size ranging between 500 and 750 μm. PBMMs treated with DMB demonstrated cell aggregation and clumping mimicking lipopolysaccharide (LPS) treated PBMMs and a higher proliferation ability (166.93%) compared to control (100%) and DCC treatments (115.64%; p<0.001) at 24h. This was associated with a significantly increased production of intracellular ROS in PBMMs exposed to DMB substitutes than control (3158.5 vs 1715.5; p<0.001) and DCC treatment (2117.5). The bone substitute exposure also caused an increase in percentage apoptosis which was significantly (p<0.0001) higher in both DMB (27.85) and DCC (29.2) treatment than control (19.383). A significant increase in proinflammatory cytokine expression (TNF-α: 3.4 folds; p<0.05) was observed in DMB substitute-treated PBMMs compared to control. Notably, IL-1β mRNA was significantly higher in DMB (21.75 folds; p<0.0001) than control and DCC (5.01 folds). In contrast, DCC substitutes exhibited immunoregulatory effects on PBMMs, as indicated by the expression for CD86, CD206, and HLDR surface markers mimicking IL-4 treatments. In conclusion, DMB excites a higher immunological response compared to DCC suggesting decellularization process of tissues dampen down inflammatory reactions when exposed to PBMM.

REDD1-dependent GSK3β dephosphorylation promotes NF-κB activation and macrophage infiltration in the retina of diabetic mice

Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that the stress response protein regulated in development and DNA damage response 1 (REDD1) promotes diabetes-induced retinal inflammation by sustaining canonical activation of nuclear transcription factor κB (NF-κB). The studies here were designed to identify signaling events whereby REDD1 promotes NF-κB activation in the retina of diabetic mice. We observed increased REDD1 expression in the retina of mice after 16-weeks of streptozotocin (STZ)-induced diabetes and found that REDD1 was essential for diabetes to suppress inhibitory phosphorylation of GSK3β at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3β and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3β variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3β knockdown inhibited NF-κB activation and pro-inflammatory cytokine expression by preventing inhibitor of κB (IκB) kinase (IKK) complex autophosphorylation and IκB degradation. In both the retina of STZ-diabetic mice and in Müller cells exposed to hyperglycemic conditions, GSK3 inhibition reduced NF-κB activity and prevented an increase in pro-inflammatory cytokine expression. In contrast with STZ-diabetic mice receiving a vehicle control, macrophage infiltration was not observed in the retina of STZ-diabetic mice treated with GSK3 inhibitor. Collectively, the findings support a model wherein diabetes enhances REDD1-dependent activation of GSK3β to promote canonical NF-κB signaling and the development of retinal inflammation.

Fluoxetine and Curcumin Prevent the Alterations in Locomotor and Exploratory Activities and Social Interaction Elicited by Immunoinflammatory Activation in Zebrafish: Involvement of BDNF and Proinflammatory Cytokines.

The increase in proinflammatory cytokine expression causes behavioral changes consistent with sickness behavior, and this led to the suggestion that depression might be a psychoneuroimmunological phenomenon. Here, we evaluated the effects of the pretreatment with fluoxetine (10 mg/kg, i.p.) and curcumin (0.5 mg/kg, i.p.) on the immune response elicited by the inoculation of an Aeromonas hydrophila bacterin in zebrafish. Non-pretreated but A. hydrophila-inoculated and sham-inoculated groups of fish served as controls. The social preference, locomotor, exploratory activities, and cerebral expression of il1b, il6, tnfa, and bdnf mRNA were compared among the groups. Behavioral changes characteristic of sickness behavior and a significant increase in the expression of il1b and il6 cytokines were found in fish from the immunostimulated group. The behavioral alterations caused by the inflammatory process were different between males and females, which was coincident with the increased expression of cerebral BDNF. Fluoxetine and curcumin prevented the sickness behavior induced by A. hydrophila and the increased expression of proinflammatory cytokines. Our results point to the potential of zebrafish as a translational model in studies related to neuroinflammation and demonstrate for the first time the effects of fluoxetine and curcumin on zebrafish sickness behavior.

The Influence of Sex on Hippocampal Neurogenesis and Neurotrophic Responses on the Persistent Effects of Adolescent Intermittent Ethanol Exposure into Adulthood

In the United States, approximately 90% of alcohol consumed by adolescents is binge drinking. Binge-like ethanol exposure during adolescence promotes dysregulation of neurotrophic responses and neurogenesis in the hippocampus. These effects include changes in proliferation, regulation, differentiation, and maturation of neurons, and there is indication that such effects may be disproportionate between sexes. This study determined whether sex impacts neurotrophic responses and neurogenesis in adulthood after adolescent intermittent ethanol (AIE) exposure. To determine this, adolescent rats underwent AIE with ethanol (5 g/kg). In adulthood, animals were euthanized, and immunohistochemical techniques and ELISAs were utilized to determine AIE effects on sex-specific neurogenesis factors and neurotrophic markers, respectively. AIE exposure led to a significant decrease in neurogenesis in the dentate gyrus of the hippocampal formation indicated by reductions in the numbers of DCX+, SOX2+ and Ki-67+ cells in male and female AIE-exposed rats. Additionally, AIE increased markers for the pro-inflammatory cytokines, TNF-α and IL-1β, in the hippocampus into adulthood in male AIE-exposed rats only. No significant AIE-induced differences were observed in the anti-inflammatory cytokines, IL-10 and TGF-β, nor in the neurotrophic factors BDNF and GDNF. Altogether, our findings indicate that although AIE did not have a persistent effect on hippocampal neurotrophic levels, there was still a reduction in neurogenesis. The neurogenic impairment was not sex specific, but the neurogenic deficits in males may be attributed to an increase in pro-inflammatory cytokine expression. A persistent impairment in neurogenesis may have an impact on both behavioral maladaptations and neurodegeneration in adulthood.

Activation of GPR183 by 7α,25-Dihydroxycholesterol Induces Behavioral Hypersensitivity through Mitogen-Activated Protein Kinase and Nuclear Factor-κB.

Emerging evidence implicates the G-protein coupled receptor (GPCR) GPR183 in the development of neuropathic pain. Further investigation of the signaling pathways downstream of GPR183 is needed to support the development of GPR183 antagonists as analgesics. In rodents, intrathecal injection of its ligand, 7α,25-dihydroxycholesterol (7α,25-OHC), causes time-dependent development of mechano-and cold- allodynia (behavioral hypersensitivity). These effects are blocked by the selective small molecule GPR183 antagonist, SAE-14. However, the molecular mechanisms engaged downstream of GPR183 in the spinal cord are not known. Here, we show that 7α,25-OHC-induced behavioral hypersensitivity is Gα i dependent, but not β-arrestin 2-dependent. Non-biased transcriptomic analyses of dorsal-horn spinal cord (DH-SC) tissues harvested at the time of peak hypersensitivity implicate potential contributions of mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB). In support, we found that the development of 7α,25-OHC/GPR183-induced mechano-allodynia was associated with significant activation of MAPKs (extracellular signal-regulated kinase [ERK], p38) and redox-sensitive transcription factors (NF-κB) and increased formation of inflammatory and neuroexcitatory cytokines. SAE-14 blocked these effects and behavioral hypersensitivity. Our findings provide novel mechanistic insight into how GPR183 signaling in the spinal cord produces hypersensitivity through MAPK and NF-κB activation. SIGNIFICANCE STATEMENT: Using a multi-disciplinary approach, we have characterized the molecular mechanisms underpinning 7α,25-OHC/GPR183-induced hypersensitivity in mice. Intrathecal injections of the GPR183 agonist 7α,25-OHC induce behavioral hypersensitivity, and these effects are blocked by the selective GPR183 antagonist SAE-14. We found that 7α,25-OHC-induced allodynia is dependent on MAPK and NF-κB signaling pathways and results in an increase in pro-inflammatory cytokine expression. This study provides a first insight into how GPR183 signaling in the spinal cord is pronociceptive.

Colonic MUC2 Mucin Is Stabilized By FCGBP To Resist Salmonella Enterica Invasion In Goblet Cells

MUC2 mucin produced by goblet cells form the colonic mucus bilayer as the first line of innate defense against pathogen invasion by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. Goblet cells also produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we elucidated the putative functions of FCGBP embedded in MUC2 mucin in response to Salmonella enterica adherence and invasion in human colonic goblet cells. We hypothesis that FCGBP is coordinately produced with MUC2 mucin to play an integral role in forming the protective mucus layer in innate host defense. The aims of the study are: 1) to determine if FCGBP alters the structural integrity of the mucus layer and 2) to quantify structural alterations of the mucus layer in S. enterica adherence and invasion of goblet cells.To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: Wildtype LS174T (WT) MUC2 mucus‐producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS). To determine if FCGBP MS led to loss in barrier function in the mucus layer, S. enterica adherence and invasion, and 0.2, 1, and 2 µM fluorescent bead penetrability inoculated on WT and FCGBP MS monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by S. enterica in WT and FCGBP MS goblet cells were analysed by RT‐PCR and Western blotting. Cell killing by S. enterica was determined by LDH assay. S. enterica induced pro‐inflammatory cytokines responses were analyzed by RT‐PCR and human focus 15‐plex cytokine and chemokine array.FCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetrability further towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of live S. enterica was significantly increased in FCGBP MS but not WT cells and induced robust MUC2 mRNA expression and mucus secretion in a time‐dependent manner. Similarly, S. enterica elicited high expression of pro‐inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by S. enterica that resulted in increased cell death as compared to WT cells.More S. enterica adhered, invaded, and killed FCGBP MS as compared to WT cells. Fluorescent beads penetrated the mucus layer closer to the cell surface in FCGBP MS cells. In WT goblet cells, FCGBP and MUC2 were coordinately upregulated in response to S. enterica. The concurrent increase in pro‐inflammatory cytokine expression in FCGBP MS cells in response to S. enterica suggests that bacteria directly interacted with the cell surface suggesting an impaired protective mucus layer. The overall trend for increased bacterial invasion, pro‐inflammatory response, and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity of the mucus layer.

A198 COLONIC MUC2 MUCIN IS STABILIZED BY FCGBP TO RESIST SALMONELLA TYPHIMURIUM INVASION IN GOBLET CELLS

BackgroundThe colonic mucus bilayer is the first line of innate defense against pathogen invasion in the gut by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. While MUC2 mucin is the primary component of the mucus layer, it also contains several mucus associated proteins of which FCGBP is of significant interest due to its structural similarities to MUC2 with unknown functions. Here we elucidated the functions of FCGBP in MUC2 mucin in response to S. typhimurium ( St) adherence and invasion in human colonic goblet cells.AimsHypothesis: FCGBP is coordinately produced with MUC2 mucin and plays an important role in innate host defense. The specific aims are:1. To determine if FCGBP alters the structural integrity of the mucus layer2. To determine the role of FCGBP in St adherence and invasion of goblet cellsMethodsTo investigate whether FCGBP impaired mucus barrier functions, wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS) were used. To determine if FCGBP MS led to loss in barrier function, St adherence and invasion, and 0.2, 1, and 2 μM fluorescent bead penetration through the mucus monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by St were analysed by RT-PCR and Western blotting. St-induced pro-inflammatory cytokines responses were analyzed by RT-PCR and human focus 15-plex cytokine and chemokine array. Cell killing was enumerated by LDH assay.ResultsFCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration closer towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of St was significantly increased in FCGBP MS cells and induced robust MUC2 mRNA and protein expressions in a time-dependent manner. Similarly, St elicited robust expression of pro-inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by St that resulted in increased cell death as compared to WT cells.ConclusionsLoss of function by FCGBP MS, but not in WT cells, showed increased penetration of fluorescent beads through the mucus layer that resulted in increased St adherence, invasion, and cell death. In WT cells, FCGBP and MUC2 were coordinately upregulated in response to St. The concurrent increase in pro-inflammatory cytokine expression in FCGBP MS cells in response to St suggests that bacteria directly interacted with the cell surface indicative of an impaired mucus layer. The overall trend for increased bacterial invasion, pro-inflammatory response and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity and protective functions of the mucus layer.Funding AgenciesCIHR

Low Frequency of T Cell and Antibody Responses to Vaccination Against Sars-Cov-2 in Patients Post Allogeneic Stem Cell Transplantation in Comparison with Chronic Myeloid Malignancy Patients

Low Frequency of T Cell and Antibody Responses to Vaccination Against Sars-Cov-2 in Patients Post Allogeneic Stem Cell Transplantation in Comparison with Chronic Myeloid Malignancy Patients

Aged and young mice differentially respond to tape-stripping in epidermal gene expression.

Disruption of epidermal permeability barrier induces an increase in proinflammatory cytokine expression and release, stimulation of epidermal lipid and DNA synthesis, and expression of antimicrobial peptides. Although alterations in epidermal function in the aged skin are known, whether the epidermal transcriptomic responses to barrier disruption differ between aged and young mice remains unknown. Here, we performed RNA sequencing of the epidermis in 2-month- vs. 20-month-old mice following barrier disruption with repeated tape-stripping. At baseline condition, the epidermis of 20-month-old mice displayed an upregulation of inflammation-associated genes and down-regulation of epidermal structure- and development-related genes in comparison to 2-month-old mice. Barrier disruption upregulated expression levels of 327genes and downregulated 209genes in 2-month-old mice. In 20-month-old mice, the numbers of upregulated and down-regulated genes were 537 and 299, respectively. In comparison to young mice, the prominently upregulated genes in the 20-month-old mice were associated with the IL-17signalling pathway, while downregulated genes were mainly involved in the cytokine-cytokine receptor interaction pathway. These results indicate that inflammation-associated signalling pathways are upregulated, while epidermal structure- and development-related genes are downregulated in the epidermis of aged mice, with further aggravation following barrier disruption, suggesting the importance of improving epidermal function in the elderly.

Study on the Immune Escape Mechanism of Acute Myeloid Leukemia With DNMT3A Mutation.

DNA (cytosine-5)-methyltransferase 3A (DNMT3A)-mutated acute myeloid leukemia (AML) has a poor prognosis, but the exact mechanism is still unclear. Here, we aimed to explore the mechanism of immune escape in AML with DNMT3A mutation. We constructed a DNMT3A knockout clone and DNMT3A-R882H-mutated clones. RNA-seq results showed that transcription factors and macrophage inflammatory proteins were significantly downregulated in the DNMT3A mutant clones. KEGG enrichment and gene set enrichment analysis (GSEA) showed that a large number of genes were enriched in inflammatory immune-related pathways, such as the toll-like receptor signaling pathway. Therefore, we co-cultured AML cells with macrophages. The DNMT3A-mutated AML cells attenuated M1 macrophage polarization and resisted its killing effect in vitro and in vivo. In xenografts, the tumor volumes in the experimental group were significantly larger than those in the control group, and the proportion of M2 macrophages was significantly higher. After the co-culture, the increase in pro-inflammatory cytokine expression in the mutant cells was significantly lower than that in the control group, while that in immunosuppressive factors was not significantly different. In co-cultivated supernatants, the concentration of inflammatory factors in the experimental group was significantly lower than that in the control group, while that of immunosuppressive factors was significantly higher. Resistin significantly promoted the expression of inflammatory proteins in AML cells. It relieved the inhibitory effect of DNMT3A mutation, promoted the phenotypic recovery of the co-cultured macrophages, eliminated resistance, and regulated the immune microenvironment. Thus, resistin may serve as an ancillary drug for patients with DNMT3A-mutated AML.

FCGBP stabilizes colonic MUC2 mucin structural integrity in innate host defense against Entamoeba histolytica

MUC2 mucin is the major component of the colonic mucus bilayer that serves as the first line of innate host defense against pathogens while supporting a healthy microbiota and regulating epithelial barrier function. Proteomic studies of colonic mucus have identified various mucus-associated proteins. One of the most abundant is FCGBP, similar to MUC2 mucin, but its interaction with MUC2 or function is not known. Here, we hypothesized that MUC2 mucin and FCGBP are coordinately produced and play an important role in innate host defense. The aims of the study are: 1) to determine if FCGBP alters the structural integrity of the mucus layer and 2) to determine the role of FCGBP in Eh infection. FCGBP mRNA and protein expression induced by Eh in WT and FCGBPCRISPR/Cas9 LS174T goblet cells were analysed by RT-PCR and Western blotting. To compare integrity of the mucus layer, fluorescent Eh and 1mM fluorescent beads were inoculated on WT and KO monolayers and adherent Eh and bead penetrability analyzed. To quantify MUC2 and FCGBP degradation by Eh, purified MUC2 and recombinant FCGBP were incubated with Eh proteases (SPs) and Western blotted using highly specific antibodies against various regions of the proteins. In response to live Eh, FCGBP and MUC2 mRNA and protein expressions were significantly increased in a time-dependent manner. Surprisingly, FCGBP KO cells elicited robust expression of pro-inflammatory cytokine mRNA and protein as compared to WT cells. More fluorescent Eh were attached to the mucus layer of FCGBP KO cells as compared to WT or MUC2 KO cells. Fluorescent beads penetrated further towards the epithelial cell surface in KO as compared to WT cells. Interestingly, while both MUC2 and FCGBP from purified polymeric mucins were degraded by Eh SPs, FCGBP cleavage occurred at a faster rate than MUC2. Degradation of FCGBP and MUC2 was mediated by EhCP-A5 cysteine proteinase using purified MUC2 and recombinant FCGBP. In WT goblet cells, FCGBP and MUC2 were upregulated temporally in response to Eh. The increase in pro-inflammatory cytokine expression in FCGBP KO cells in response to Eh suggests that Eh directly interacted with the cell surface suggesting an impaired protective mucus layer. In support of this, fluorescent beads penetrated the mucus layer close to the cell surface and more Eh wereattached to FCGBP KO mucus demonstrating that FCGBP was critical in providing structural integrity of the mucus layer. In response to Eh, FCGBP degradation was a prerequisite for MUC2 cleavage, providing direct evidence that FCGBP and MUC2 interactions conferred biophysical properties of the protective functions of the mucus gel.

A24 FCGBP MAINTAINS MUC2 MUCUS STRUCTURAL INTEGRITY BY STABILIZING THE MUCUS LAYER IN RESPONSE TO THE COLONIC PATHOGEN, ENTAMOEBA HISTOLYTICA

Abstract Background MUC2 mucin is the major component of the colonic mucus bilayer that serves as the first line of innate host defense against pathogens while supporting a healthy microbiota and regulating epithelial barrier function. Proteomic studies of colonic mucus have identified various mucus-associated proteins. One of the most abundant is FCGBP, similar to MUC2 mucin, but its interaction with MUC2 or function is not known. Here, we elucidated FCGBP functional role in stabilizing MUC2 mucus and in innate host defence against Entamoeba histolytica (Eh). Aims Hypothesis: MUC2 mucin and FCGBP are coordinately produced and play an important role in innate host defense. The specific aims are: 1. To determine if FCGBP alters the structural integrity of the mucus layer 2. To determine the role of FCGBP in Eh infection Methods FCGBP mRNA and protein expression induced by Eh, in WT and FCGBP CRISPR/Cas9 LS174T goblet cells were analysed by RT-PCR and Western blotting. To compare integrity of the mucus layer, fluorescent Eh and 1μM fluorescent beads were inoculated on WT and KO monolayers and adherent Eh and bead penetrability analyzed. To quantify MUC2 and FCGBP degradation by Eh, purified MUC2 and recombinant FCGBP were incubated with Eh proteases (SPs) and Western blotted using highly specific antibodies against various regions of the proteins. Results In response to live Eh, FCGBP and MUC2 mRNA and protein expressions were significantly increased in a time-dependent manner. Surprisingly, FCGBP KO cells elicited robust expression of pro-inflammatory cytokine mRNA and protein as compared to WT cells. More fluorescent Eh were attached to the mucus layer of FCGBP KO cells as compared to WT or MUC2 KO cells. Fluorescent beads penetrated further towards the epithelial cell surface in KO as compared to WT cells. Interestingly, while both MUC2 and FCGBP from purified polymeric mucins were degraded by Eh SPs, FCGBP cleavage occurred at a faster rate than MUC2. Degradation of FCGBP and MUC2 was mediated by EhCP-A5 cysteine proteinase using purified MUC2 and recombinant FCGBP. Conclusions In WT goblet cells, FCGBP and MUC2 were upregulated temporally in response to Eh. The increase in pro-inflammatory cytokine expression in FCGBP KO cells in response to Eh suggests that Eh directly interacted with the cell surface suggesting an impaired protective mucus layer. In support of this, fluorescent beads penetrated the mucus layer close to the cell surface and more Eh were attached to FCGBP KO mucus demonstrating that FCGBP was critical in providing structural integrity of the mucus layer. In response to Eh, FCGBP degradation was a prerequisite for MUC2 cleavage, providing direct evidence that FCGBP and MUC2 interactions conferred biophysical properties of the protective functions of the mucus gel. Funding Agencies CIHR

Nontoxic dose of Phenethyl isothiocyanate ameliorates deoxynivalenol-induced cytotoxicity and inflammation in IPEC-J2 cells

The intestinal tract is a target for the deoxynivalenol (DON), which has adverse effects in animals and humans' health by affecting intestinal functions. Phenethyl isothiocyanate (PEITC) is an important degradation product of glucosinolates (GSLs), belonging to an anti-nutritional factor that affects the digestion and absorption of nutrients in the animals' intestinal. However, little attention has been paid to the interaction and its mechanism between DON and PEITC. Therefore, the purpose of this study was to assess the effects of PEITC on DON-induced cytotoxicity and inflammation, and explore the potential mechanisms in IPEC-J2 cells. Our results showed that DON exposure could decrease the cell viability and pro-inflammatory cytokine expression in IPEC-J2 cells in a dose-dependent manner. PEITC treatment at the concentrations of 1.25–5 μM had no significant effect on IPEC-J2 cells viability, but above 10 μM of PEITC treatment significantly reduced the cell viability. Interestingly, 1.25–5 μM of PEITC treatment could suppress 4 μM of DON-induced decrease in cell viability and increase in pro-inflammatory cytokine expression. Meanwhile, the protein ratios of p-p65/p-65 and p-IκBα/IκBα were markedly decreased in the groups treated with 1.25–5 μM PEITC compared to DON exposure alone. However, the protective effects of PEITC treatment were significantly blocked after pre-treatment with LPS, NF-κB activator, in IPEC-J2 cells. In conclusion, these findings indicated that the nontoxic dose of PEITC could alleviate DON-induced cytotoxicity and inflammation responses via suppressing the NF-κB signaling pathway in IPEC-J2 cells. Our results provide a new theoretical basis for the rational addition of rapeseed meal in animal feedstuff.

Long non-coding RNA SNHG29 regulates cell senescence via p53/p21 signaling in spontaneous preterm birth

IntroductionSpontaneous preterm birth affects>5–18% of pregnancies and causes infant morbidity and mortality. Long non-coding RNAs can regulate gene expression and have been associated with preterm birth. In this study, we investigated whether the long non-coding RNA SNHG29 was associated with spontaneous preterm birth. MethodsWe collected the placentas from women who underwent preterm/full-term birth with/without labor. We determined the levels of expression of SNHG29 in the placental tissues using quantitative real-time polymerase chain reaction. We generated a senescence model by treating HTR8/SVneo cells with 200 μM H2O2 for 2 h. The degree of senescence induced in cells depleted of or overexpressing SNHG29 was determined by measuring senescence-associated gene expression and β-galactosidase activity. ResultsSNHG29 was overexpressed in the placentas of women who delivered preterm with labor and in HTR8/SVneo cells treated with H2O2 (p < 0.05). The levels of mRNA of p53 and p21, protein levels of p53, phospho-p53, p21and phospho-p21, and β-galactosidase activity was decreased in HTR8/SVneo cells depleted of SNHG29, while the opposite trend was observed in HTR8/SVneo cells overexpressing SNHG29 (p < 0.05). We observed an increase in the expression of IL-8 and TNF-α in senescent HTR8/SVneo cells (p < 0.05). DiscussionSNHG29 was overexpressed in placentas from women who delivered preterm with labor compared to those in women who underwent preterm birth without labor and full-term birth with/without labor. High levels of SNHG29 enhanced senescence in vivo. The increase in pro-inflammatory cytokine expression and release by senescent cells may be pivotal to the pathophysiology of spontaneous preterm birth.

Burnout in the emergency department: Randomized controlled trial of an attention-based training program

BackgroundBurnout (encompassing emotional exhaustion, depersonalization and personal accomplishment) in healthcare professionals is a major issue worldwide. Emergency medicine physicians are particularly affected, potentially impacting on quality of care and attrition from the specialty. ObjectiveThe aim of this study was to apply an attention-based training (ABT) program to reduce burnout among emergency multidisciplinary team (MDT) members from a large urban hospital. Design, setting, participants and interventionsEmergency MDT members were randomized to either a no-treatment control or an intervention group. Intervention group participants engaged in a four session (4 h/session) ABT program over 7 weeks with a practice target of 20 min twice-daily. Practice adherence was measured using a smart phone application together with a wearable Charge 2 device. Main outcome measuresThe primary outcome was a change in burnout, comprising emotional exhaustion, depersonalization and personal achievement. The secondary outcomes were changes in other psychological and biometric parameters. ResultsThe ABT program resulted in a significant reduction (P < 0.05; T1 [one week before intervention] vs T3 [follow-up at two months after intervention]) in burnout, specifically, emotional exhaustion, with an effect size (probability of superiority) of 59%. Similar reductions were observed for stress (P < 0.05) and anxiety (P < 0.05). Furthermore, ABT group participants demonstrated significant improvements in heart rate variability, resting heart rate, sleep as well as an increase in pro-inflammatory cytokine expression. ConclusionThis study describes a positive impact of ABT on emergency department staff burnout compared to a no-treatment control group. Trial registrationClinicalTrials.gov identifier NCT02887300.

Pretreatment with a grape seed proanthocyanidin extract downregulates proinflammatory cytokine expression in airway epithelial cells infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) infections are associated with significant morbidity and mortality. Inflammation is mediated by cytokine secretion from RSV‑infected airway epithelial cells. Grape seed proanthocyanidin extract(GSPE) exhibits potent antioxidant capacity, as well as anti‑bacterial, anti‑viral, anti‑carcinogenic, anti‑inflammatory and anti‑allergic actions. However, few studies have explored the anti‑inflammatory effects of GSPE on airway epithelial cells infected with RSV. Airway epithelial A549 cells were pretreated with GSPE and its effects on cytokine production during RSV infection were investigated. A549 cells were infected with RSV, with or without GSPE pretreatment, and cultured for 24, 48 and 72h. The expression of interleukin (IL)‑1β, IL‑6 and IL‑8, were measured by reverse transcription‑quantitative polymerase chain reaction, ELISA and western blotting. RSV infection induced significant increases in proinflammatory cytokine expression. However, GSPE pretreatment decreased the mRNA and protein expression levels of IL‑1ß, IL‑6 and IL‑8. GSPE regulated the immune response by reducing the RSV‑induced transcription of proinflammatory cytokines in airway epithelial cells, suggesting that GSPE helps to prevent RSV‑induced airway disease.

Slc26a3 (DRA) Deficient Mice Display an Acidic Colonic pH‐microclimate, Develop a Strongly Altered Microbiome and Colonic Inflammation

BackgroundThe anion transporter Slc26a3 (DRA‐Down Regulated in Adenoma) is localized on the apical membrane of the colonic mucosa and is functionally involved in the absorption of luminal chloride (Cl−) in exchange for bicarbonate ions (HCO3−). Mutations in the dra gene result in congenital chloride diarrhoea (CLD) which is characterized by secretory diarrhoea, loss of Cl− in the stool, dehydration and metabolic alkalosis. These patients also have a higher risk of incidence of acute as well chronic intestinal inflammation (Wedenoja et al. Hum. Mut 2011). We previously reported low colonic HCO3− output rates and an increased susceptibility to Dextran Sodium Sulfate (DSS) damage in slc26a3−/− mice (Xiao, F. et al. Acta Phys. 2015).AimThe present study was undertaken to explore whether the slc26a3−/− mice develop spontaneous intestinal inflammation, and whether an altered microbiome composition may be the underlying molecular mechanism for inflammation.MethodsColonic surface pH was measured in vivo by two photon microscopy, the inflammatory state of the mucosa by Quantitative PCR (qPCR) and immunohistochemistry, the intestinal microbiome by 16S rRNA sequencing, and the effect of the microbiome of the slc26a3−/− colon on the development of inflammation in germ‐free mice by fecal crossing experiments.ResultsThe slc26a3−/− mice developed distal colonic inflammation evidenced by an increase in proinflammatory cytokine expression and the number of inflammatory cells in the colonic mucosa/submucosa at the time of sacrifice. The slc26a3−/− microbiome was significantly different from that of cohoused wild type (WT) littermates, with a strongly decreased diversity, and an increased percentage of several proinflammatory species. Transmission of this dysbiotic microbiome into germ free mice did not lead to a development of inflammation in the recipient mice. Instead, the species richness in the germ free mice gavaged with slc26a3−/− microbiome had increased, and the percentage of proinflammatory species decreased, in comparison to its donor microbiome.ConclusionsSlc26a3−/− mice display a very low colonic pH microclimate and develop spontaneous intestinal inflammation. The microbiome of slc26a3−/− colon is “dysbiotic” with strongly reduced diversity. However, transmission of this microbiome into germ‐free mice does not result in inflammation, but does result in microbiome normalization, suggesting that the reduced pH microclimate and reduced colonic transit time is necessary for the development of intestinal dysbiosis.Support or Funding InformationVolkswagen Foundation VW Vorab, DFG project SE460/17‐1 and 19‐1 and SFB621/C9This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellular and behavioral effects of lipopolysaccharide treatment are dependent upon neurokinin-1 receptor activation

BackgroundSeveral psychiatric conditions are affected by neuroinflammation and neuroimmune activation. The transcription factor nuclear factor kappa light-chain-enhancer of activated B cells (NFkB) plays a major role in inflammation and innate immunity. The neurokinin-1 receptor (NK1R) is the primary endogenous target of the neuroactive peptide substance P, and some data suggests that NK1R stimulation may influence NFkB activity. Both NK1R and NFkB have been shown to play a functional role in complex behaviors including stress responsivity, depression, and addiction. In this study, we test whether NFkB activity in the brain (stimulated by lipopolysaccharide administration) is dependent upon the NK1R.MethodsAdult male Wistar rats were treated systemically with the NK1R antagonist L822429 followed by administration of systemic lipopolysaccharide (LPS, a strong activator of NFkB). Hippocampal extracts were used to assess expression of proinflammatory cytokines and NFkB-DNA-binding potential. For behavioral studies, rats were trained to consume 1% (w/v) sucrose solution in a continuous access two-bottle choice model. After establishment of baseline, animals were treated with L822429 and LPS and sucrose preference was measured 12 h post-treatment.ResultsSystemic LPS treatment causes a significant increase in proinflammatory cytokine expression and NFkB-DNA-binding activity within the hippocampus. These increases are attenuated by systemic pretreatment with the NK1R antagonist L822429. Systemic LPS treatment also led to the development of anhedonic-like behavior, evidenced by decreased sucrose intake in the sucrose preference test. This behavior was significantly attenuated by systemic pretreatment with the NK1R antagonist L822429.ConclusionsSystemic LPS treatment induced significant increases in NFkB activity, evidenced by increased NFkB-DNA binding and by increased proinflammatory cytokine expression in the hippocampus. LPS also induced anhedonic-like behavior. Both the molecular and behavioral effects of LPS treatment were significantly attenuated by systemic NK1R antagonism, suggesting that NK1R stimulation lies upstream of NFkB activation following systemic LPS administration and is at least in part responsible for NFkB activation.

Hyperosmolarity-induced AQP5 upregulation promotes inflammation and cell death via JNK1/2 Activation in human corneal epithelial cells

Tear film hyperosmolarity and anterior ocular inflammation are two clinical signs that may be indicative of dry eye disease (DED). This condition can cause pathological and functional changes to the anterior ocular surface tissues. A contributing factor may be dysfunctional aquaporin 5 (AQP5) water channels as they are the AQP subtype that expressed in the corneal epithelium and contribute to fluid efflux needed for corneal function. We determined if described hyperosmolarity-induced increases in proinflammatory cytokine expression and cell death are mediated through AQP5 upregulation and JNK1/2 MAPK signaling activation in both primary human corneal epithelial cells (HCECs), and in a HCEC line. Real time RT-PCR identified rises in IL-1β, IL-6, IL-8, TNF-α, caspase-1, and AQP5 mRNA levels upon step increases in osmolarity up to 550 mOsm. Western blot analysis and the TUNEL assay identified corresponding rises in AQP5 and p-JNK1/2 protein expression and cell death respectively. JNK1/2 inhibition with SP600125, or siRNA AQP5 gene silencing reduced hypertonic-induced rises in proinflammatory cytokine expression and cell death. Taken together, hypertonicity-induced AQP5 upregulation leads to increases in proinflammatory cytokine expression and cell death through JNK1/2 MAPK activation. These results suggest that drug targeting AQP5 upregulation may be a therapeutic option in DED management.