Upregulation Of IL-6 Research Articles

Gene expression profiling reveals host defense strategies for restricting Candida albicans invasion and gastritis to the limiting ridge of the murine stomach.

Candida albicans is a fungal constituent of the human gastrointestinal microbiota that can tolerate acidic environments like the stomach, where it can be associated with ulcers and chronic gastritis. In mice, C. albicans induces gastritis without concurrent intestinal inflammation, suggesting that the stomach is particularly prone to fungal infection. We previously showed that C. albicans invasion in the limiting ridge does not extend to or elicit an inflammatory response in the adjacent glandular region, indicating regionalized gastritis in the murine stomach. However, the molecular pathways involved in the host response to C. albicans specifically in the limiting ridge have not been investigated. Here, we found that gastric dysbiosis was associated with C. albicans limiting ridge colonization and gastritis. We isolated the limiting ridge and evaluated the expression of over 90 genes involved in mucosal responses. C. albicans infection triggered a type 3 immune response marked by elevated Il17a, Il17f, Il1b, Tnf, and Il36g, as well as an upregulation of Il12a, Il4, Il10, and l13. Chemokine gene induction (including Ccl2, Ccl3, Ccl4, Ccl1l, Cxcl1, Cxcl2, Cxcl9, and Cxcl10) coincided with an influx of neutrophils, monocytes/macrophages, and eosinophils. Hyphal invasion caused tissue damage, epithelial remodeling, and upregulation of genes linked to epithelium signaling and antimicrobial responses in the limiting ridge. Our findings support a need for continued exploration into the interactions between the immunological milieu, the host microbiota, and clinical interventions such as the use of antibiotics and immunotherapeutic agents and their collective impact on invasive candidiasis risk.

CSIG-23. CYTOMEGALOVIRUS-INFECTED HUMAN GLIOBLASTOMA CELL LINES SHOW AN AGGRESSIVE PHENOTYPE ASSOCIATED WITH UPREGULATION OF IL-6/STAT3 AND AKT SIGNALING

Abstract BACKGROUND Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) pathogenesis by affecting stemness, angiogenesis and immune pathways. Clinical trials targeting CMV in GBM patients have shown some early promise, and CMV seropositivity has been associated with poorer outcomes. However, the underlying mechanisms remain unclear. Here we investigated the effects of CMV infection on critical signaling pathways in GBM (IL6/STAT3, and Akt signaling) based on the hypothesis that CMV may contribute to poorer outcomes in GBM by increasing oncogenic signaling. MATERIALS AND METHODS Human U251 and LN229 GBM cell lines were infected with mCherry expressing human CMV TB40 strain at a multiplicity of infection (MOI) of 0.5. We performed basic phenotypic studies in vitro and investigated the status of IL6/STAT3, and Akt signaling by Western blotting. RESULTS Both LN229 and U251 cell lines were readily infectable by CMV. Infection of U251 cells was over 90% and in LN229 cells was approximately 60%. Interestingly expression of CMV was sustained over many passages in these cell lines. Infection was also verified by the presence of the CMV pp65 protein assessed by Western blotting. Measurement of cell proliferation indicated faster growth after CMV infection. Interrogation of key pro-oncogenic signaling pathways in GBM revealed a robust upregulation of IL6, as well as phospho(Tyr705)-STAT3 and phospho(Thr308)-Akt. This elevation of pro-oncogenic signaling pathways was observed in both cell lines compared with controls. CONCLUSIONS Here we show for the first time that infection of human GBM cell lines with CMV results in a more aggressive phenotype associated with upregulation of IL6/STAT3 signaling as well as a significant increase in phospho-Akt levels. We are currently studying sensitivity to standard therapies and additional molecular changes in these cells. These data support the hypothesis that CMV causes more aggressive disease and that CMV is a relevant therapeutic target in GBM.

Impact of Chondrocyte Inflammation on Glial Cell Activation: The Mediating Role of Nitric Oxide.

This study investigates how the inflammatory response of ATDC5 murine chondrogenic cells influences the activity of C6 (rat) and GL261 (mouse) glial cell lines. Prior research suggested nitric oxide (NO) involvement in cartilage-immune crosstalk. The current study explores whether NO, produced by inflamed chondrocytes, mediates signaling between chondrocytes and glial cells. Pre-challenged ATDC5 cells with 250 ng/ml of lipopolysaccharide (LPS) were cocultured with GL261 or C6 glioma cells for 24 h with a transwell culture system. Cell viability was assessed using MTT assay. Gene and protein expression were evaluated by qRT-PCR and WB, respectively. Real-time reverse transcription-polymerase chain reaction (RT-qPCR) indicated statistically significant upregulation of LCN2, IL-6, TNF-α, IL-1β, and GFAP in glial cells following 24-h coculture with challenged ATDC5 cells. Suppression of LPS-induced NO production by aminoguanidine decreased LPS-mediated LCN2 and IL-6 expression in glioma cells. We identified also the involvement of the ERK1/2 and AKT signaling pathways in the glial neuroinflammatory response. This study demonstrates, for the first time, that NO produced by inflamed murine chondrocytes mediated pro-inflammatory responses in glial cells via ERK1/2 and AKT signaling, highlighting a potential mechanism linking cartilage NO to neuroinflammation and chronic pain in osteoarthritis.

Runt-related transcription factor 1 (RUNX1) is a mediator of acute kidney injury.

Treatment for acute kidney injury (AKI) is suboptimal. A better understanding of the pathogenesis of AKI may lead to new therapeutic approaches. Kidney transcriptomics of folic acid-induced AKI (FA-AKI) in mice identified Runx1 as the most upregulated RUNX family gene. We then examined the expression of RUNX1 in FA-AKI, in bacterial lipopolysaccharide (LPS)-induced cytokine storm-AKI (CS-AKI), and in human AKI. In cultured mouse tubule cells, we explored the expression and role of RUNX1 in response to the cytokine TWEAK or LPS. A chemical inhibitor of RUNX1 (Ro5-3335) was used in animal models of AKI to test its potential as a therapeutic target. RUNX1 overexpression in FA-AKI was validated at the mRNA and protein levels and localized mainly to tubule cell nuclei. CS-AKI also upregulated kidney RUNX1. Increased tubule and interstitial RUNX1 expression were also observed in human AKI. In cultured mouse tubule cells, the pro-inflammatory cytokine TWEAK and LPS increased RUNX1 and IL-6 expression. Mechanistically, RUNX1 bound to the Il6 gene promoter and RUNX1 targeting with the chemical inhibitor Ro5-3335, or a specific small interfering RNA (siRNA), prevented the TWEAK- and LPS-induced upregulation of IL6 through a RUNX1/NFκB1 p50 pathway. In vivo, preventive Ro5-3335 improved kidney function and reduced inflammation in FA-AKI and CS-AKI. However, Ro5-3335 administration after the insult only improved kidney function in CS-AKI. Kidney transcriptomics identified inflammatory genes and transcription factor mRNAs such as Yap1 and Trp53 as key targets of Ro5-3335 in CS-AKI. In conclusion, RUNX1 contributes to AKI by driving the expression of genes involved in inflammation and represents a novel therapeutic target in AKI. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Acquired immunostimulatory phenotype of migratory CD103+ DCs promotes alloimmunity following corneal transplantation.

After transplantation, Th1-mediated immune rejection is the predominant cause of graft failure. Th1 cell sensitization occurs through complex and context-dependent interaction among antigen-presenting cell subsets, particularly CD11b+ DCs (DC2) and CD103+ DCs (DC1). This interaction necessitates further investigation in the context of transplant immunity. We used well-established preclinical models of corneal transplantation and identified distinct roles of migratory CD103+ DC1 in influencing the outcomes of the grafted tissue. In recipients with uninflamed corneal beds, migratory CD103+ DC1 demonstrate a tolerogenic phenotype that modulates the immunogenic capacity of CD11b+ DC2 primarily mediated by IL-10, suppressing alloreactive CD4+ Th1 cells via the PD-L1/PD-1 pathway and enhancing Treg-mediated tolerance via αvβ8 integrin-activated TGF-β1, thus facilitating graft survival. Conversely, in recipients with inflamed and vascularized corneal beds, IFN-γ produced by CD4+ Th1 cells induced migratory CD103+ DC1 to adopt an immunostimulatory phenotype, characterized by the downregulation of regulatory markers, including αvβ8 integrin and IL-10, and the upregulation of IL-12 and costimulatory molecules CD80/86, resulting in graft failure. The adoptive transfer of ex vivo induced tolerogenic CD103+ DC1 (iDC1) effectively inhibited Th1 polarization and preserved the tolerogenic phenotype of their physiological counterparts. Collectively, our findings underscore the essential role played by CD103+ DC1 in modulating host alloimmune responses.

RT-qPCR Analysis of Inflammatory & Apoptotic Factors-Related Gene Expression in ZIKV-Infected IFNAR1-/- Mice.

ZIKV was a mosquito-borne neglected tropical pathogen until it spread into the Pacific and South America, followed by large human outbreaks related to congenital abnormalities in neonates and neurological disorders in adults. The following study used the C57BL/6 IFNAR1 receptor knockout (IFN AR1-/-) mouse model to understand the role of selected cytokines and apoptotic factors in the pathogenicity of ZIKV strain PRVABC59. Mice infected with 102 particles of Zika viruses died until 9days post infection. The brain, spleen, and lung were collected from intramuscularly infected mice on day 6 post infection (pi) to quantify the mRNA expression of targeted cytokines and apoptosis-mediated factors by RT-qPCR. Upregulation of IL-6, IL-17α, IFN-α, and IFN-β were found in the brain and lung of infected mice. IFN-γ was also significantly upregulated in the infected brain and spleen. The collective findings from our study indicate that a strong immune response was developed against ZIKV PRVABC59 in the infected mice brain.

Curcumin attenuates smoking and drinking activated NF-κB/IL-6 inflammatory signaling axis in cervical cancer

BackgroundHigh-risk strains of HPV are known to cause cervical cancer. Multiple clinical studies have emphasized that smoking and drinking are critical risk factors for cervical cancer and its high-grade precursors. In this study, we investigated if smoking and/or drinking augment the molecular mechanisms of cervical carcinogenesis and defined a potential therapeutic approach for their attenuation.MethodsThe impact of benzo[a]pyrene (B[a]P) and/or ethanol (EtOH) exposure on cervical cancer cells was assessed by measuring changes in their cell migration and invasion characteristics. Expression of HPV16 E6/E7, NF-κB, cytokines, and inflammation mediators was determined using qRT-PCR, immunoblotting, ELISA, luciferase reporter assay, and confocal microscopy. Herein, we used curcumin (Cur), and PLGA nanoparticle formulation of curcumin (PLGA-Cur) and determined effectiveness of free Cur and PLGA-Cur formulation on smoking and drinking activated NF-κB/IL-6 mediated inflammatory signaling pathways using in vitro cervical cancer models.ResultsTreatments with B[a]P and/or EtOH altered the expression of HPV16 E6/E7 oncogenes and EMT markers in cervical cancer cells; it also enhanced migration and invasion. In addition, B[a]P and/or EtOH exposure promoted inflammation pathways through TNF-α and NF-κB signaling, leading to IL-6 upregulation and activation of VEGF. The molecular effects caused by B[a]P and/or EtOH exposure were effectively attenuated by curcumin (Cur)/PLGA-Cur treatment.ConclusionsThese data suggest a molecular link between smoking, drinking, and HPV infectivity in cervical carcinogenesis. In addition, attenuation of these effects by treatment with Cur/PLGA-Cur treatment, implies the role of curcumin in cervical cancer prevention and treatment.

Protective Capacity of Helichrysum italicum Infusion Against Intestinal Barrier Disruption and Translocation of Salmonella Infantis.

Helichrysum italicum is a Mediterranean plant with well-known anti-inflammatory activity, but our previous whole transcriptome analysis has found that H. italicum infusion (HII) can also affect cytoskeletal rearrangement and tight junctions. The goal of the present study was to determine if HII improves the intestinal barrier (IB) dysfunction and by what mechanism. Caco-2 cells on Transwell inserts were used as a model of IB permeability. Heat-killed (HKB) or live Salmonella Infantis bacteria were used to induce IB integrity disruption upon three different testing conditions: pre-, co-, and post-treatment with 0.2 v/v% HII. Transepithelial electrical resistance values were used as an indicator of monolayer integrity before and after all treatments, and RT-PCR was used to assess the expression of tight junction proteins (TJPs) and inflammatory cytokines known to regulate intestinal permeability. We found that all three treatments with HII improved the HKB-induced integrity disruption and decreased the down-regulation of TJP1, OCLN, and CLDN1, with the greatest effect observed in the pre-treated cells. Treatment with HII also decreased the up-regulation of CLDN2, TNF-α, IL-1β, and IL-6. In addition, pre-treatment of Caco-2 cells with HII prevented translocation of S. Infantis but did not prevent adhesion and invasion. This study showed that HII can improve inflammation-disrupted IB function by indirect modulation of mRNA expression of TJPs, especially in a preventive manner.

Th1 and Th2 cytokine expression in hyperkeratotic chronic hand eczema and the role of Tofacitinib a oral JAK inhibitor.

Tissue cytokines in chronic hand eczema (CHE) can predict targeted therapy with novel drugs including JAK inhibitors. Our primary objective was to assess the tissue expression of cytokines of Th1 and Th2 cell lines in CHE patients and to study the efficacy of oral tofacitinib. We recruited patients presenting with recalcitrant CHE. Lesional and non-lesional tissue samples were assessed for Th1(IFN-γ, TNF-α) and Th2 related cytokines (IL-4, IL-13, IL-2,) using real time PCR. Tofacitinib 5mg twice daily was initiated with 4 weekly assessment and we also noted relapses post therapy.Of 21 refractory hyperkeratotic CHE patients, cytokine analysis was performed in 11 patients which showed upregulation of IL-4 [n = 5/11, 1.87-fold increase], TNF-α (n = 5/11, 5.13-fold) and IFN-γ (n = 6/11, 1.98-fold) as compared to uninvolved skin. All patients (100%) had used topical corticosteroids (TCS) and 4/21 (19%) had failed methotrexate and 2/21 (9.5%) had failed acitretin. Tofacitinib 5mg twice daily was given in 15/21 patients. The mean time to achieve hand eczema severity index 90 (HECSI 90) was 4 weeks (mean duration of treatment:5.8 months, n = 12). Side effects were observed in 4/12 (33.3%) patients and relapse was noted in 3/12 (25%) patients after a mean duration of 7 months after discontinuation of tofacitinib. Tofacitinib (pan-JAK inhibitor) showed an excellent response in refractory CHE patients with predominant tissue Th1/Th2 cells related cytokine expression.

Differential transcriptomic host responses in the early phase of viral and bacterial infections in human lung tissue explants ex vivo

BackgroundThe first 24 h of infection represent a critical time window in interactions between pathogens and host tissue. However, it is not possible to study such early events in human lung during natural infection due to lack of clinical access to tissue this early in infection. We, therefore, applied RNA sequencing to ex vivo cultured human lung tissue explants (HLTE) from patients with emphysema to study global changes in small noncoding RNA, mRNA, and long noncoding RNA (lncRNA, lincRNA) populations during the first 24 h of infection with influenza A virus (IAV), Mycobacterium bovis Bacille Calmette-Guerin (BCG), and Pseudomonas aeruginosa.ResultsPseudomonas aeruginosa caused the strongest expression changes and was the only pathogen that notably affected expression of microRNA and PIWI-associated RNA. The major classes of long RNAs (> 100 nt) were represented similarly among the RNAs that were differentially expressed upon infection with the three pathogens (mRNA 77–82%; lncRNA 15–17%; pseudogenes 4–5%), but lnc-DDX60-1, RP11-202G18.1, and lnc-THOC3-2 were part of an RNA signature (additionally containing SNX10 and SLC8A1) specifically associated with IAV infection. IAV infection induced brisk interferon responses, CCL8 being the most strongly upregulated mRNA. Single-cell RNA sequencing identified airway epithelial cells and macrophages as the predominant IAV host cells, but inflammatory responses were also detected in cell types expressing few or no IAV transcripts. Combined analysis of bulk and single-cell RNAseq data identified a set of 6 mRNAs (IFI6, IFI44L, IRF7, ISG15, MX1, MX2) as the core transcriptomic response to IAV infection. The two bacterial pathogens induced qualitatively very similar changes in mRNA expression and predicted signaling pathways, but the magnitude of change was greater in P. aeruginosa infection. Upregulation of GJB2, VNN1, DUSP4, SerpinB7, and IL10, and downregulation of PKMYT1, S100A4, GGTA1P, and SLC22A31 were most strongly associated with bacterial infection.ConclusionsHuman lung tissue mounted substantially different transcriptomic responses to infection by IAV than by BCG and P. aeruginosa, whereas responses to these two divergent bacterial pathogens were surprisingly similar. This HLTE model should prove useful for RNA-directed pathogenesis research and tissue biomarker discovery during the early phase of infections, both at the tissue and single-cell level.

Evaluation of the immune response of peripheral blood mononuclear cells cultured on Ti6Al4V-ELI polished or etched surfaces.

While titanium and its alloys exhibit excellent biocompatibility and corrosion resistance, their polished surfaces can hinder fast and effective osseointegration and other biological processes, such as angiogenesis, due to their inert and hydrophobic properties. Despite being commonly used for orthopedic implants, research focuses on developing surface treatments to improve osseointegration, promoting cell adhesion and proliferation, as well as increasing protein adsorption capacity. This study explores a chemical treatment intended for titanium-based implants that enhances tissue integration without compromising the mechanical properties of the Ti6Al4V substrate. However, recognizing that inflammation contributes to nearly half of early implant failures, we assessed the impact of this treatment on T-cell viability, cytokine production, and phenotype. Ti6Al4V with extra low interstitial (ELI) content discs were treated with hydrofluoric acid followed by a controlled oxidation step in hydrogen peroxide that creates a complex surface topography with micro- and nano-texture and modifies the chemistry of the surface oxide layer. The acid etched surface contains an abundance of hydroxyl groups, crucial for promoting bone growth and apatite precipitation, while also enabling further functionalization with biomolecules. While cell viability remained high in both groups, untreated discs triggered an increase in Th2 cells and a decrease of the Th17 subset. Furthermore, peripheral blood mononuclear cells exposed to untreated discs displayed a rise in various pro-inflammatory and anti-inflammatory cytokines compared to the control and treated groups. Conversely, the treated discs showed a similar profile to the control, both in terms of immune cell subset frequencies and cytokine secretion. The dysregulation of the cytokine profile upon contact with untreated Ti6Al4V-ELI discs, namely upregulation of IL-2 could be responsible for the decrease in Th17 frequency, and thus might contribute to implant-associated bacterial infection. Interestingly, the chemical treatment restores the immune response to levels comparable to the control condition, suggesting the treatment's potential to mitigate inflammation by enhancing biocompatibility.

8599 Development of an androgen receptor transgenic mouse that displays the complete attributes of Metabolic Syndrome

Abstract Disclosure: C. Alvarado: None. L.K. Beitel: None. M. Paliouras: None. M.A. Trifiro: None. The mouse androgen receptor (AR) shares over 90% homology with the human ortholog, but it lacks the CAG encoded poly-Q tract; instead, it contains a mixed glutamine/histidine tract. To study AR functionality in a more structured fashion, we created a knock-in mouse model that has only the equivalent of human poly-Q tract while maintaining the remaining mouse receptor sequence intact. Our humanized androgen receptor mouse (AR-19Q) displays a phenotype that is best described as Metabolic Syndrome (MetS). The mice gain excess weight, have hypertension, hyperglycemia, pancreatic islet cell abnormalities, and progressive non-alcoholic fatty liver disease (NAFLD). Features of MetS begin at approximately 4 months and progress till 12-14 months. Obesity: AR-19Q mice are significantly heavier, with a 35% gain of weight vs C57BL/6 mice. However, these mice are not hyperphagic at any time and eat identical quantities of chow. To assess obesity status, and found AR-19Q mice to have a significantly higher depositions of total white adipose visceral, lingual and subcutaneous vs age-matched controls. NAFLD: The progression of NAFLD is observed in AR-19Q mice. AR-19Q Histological analysis and immunohistochemistry reveal microvesicular steatosis, superseded by macrovesicular steatosis with hepatocyte balloon degeneration, scattered lobular inflammation (macrophages and lymphocytes) (6-9 months) and further superseded by perisinusoidal fibrosis (12 months). Biochemical blood analysis confirms. that there is a significant increase in alanine aminotransferase (ALT) levels, indicative of liver damage. With time classical NASH hepatitis and subsequent NASH fibrosis ensues; with several mice developing hepatocellular carcinoma (overall 20% of mice). Gene expression profiling AR-19Q mice for fatty liver genes, we observe an upregulation of inflammation genes Il-6, Il-10 and Tnf. In addition, increased Lpl (lipoprotein lipase) is indicative of increased blood triglyceride levels. On the other hand, down-regulation of G6pc (glucose-6-phosphatase) could be linked to glycogen storage disease. Down-regulation of Slc2a2 and Slc2a4, that encode GLUT2 and GLUT4, should have an influence on glucose levels, glucose homeostasis and insulin sensitivity, as insulin promotes glucose uptake through GLUT4. Type 2 Diabetes: Glucose intolerance is observed by 3-4 months followed by fasting hyperglycemia. Pancreases show advanced islet hyperplasia, concurrent with insulin resistance and type 2 diabetes. Pancreases of middle-age AR-19Q mice present with a significant islet hyperplasia, suggesting peripheral insulin resistance. We propose the use of the AR-19Q mice as a model to study the metabolic dysfunction/ deficiency leading to the MetS. The model will allow us to a better understanding into the mechanisms responsible for T2D and NAFLD-related disorders. Presentation: 6/2/2024

9285 Development of an androgen receptor transgenic mouse that displays the complete attributes of Metabolic Syndrome

Abstract Disclosure: C. Alvarado: None. L.K. Beitel: None. M. Paliouras: None. M.A. Trifiro: None. The mouse androgen receptor (AR) shares over 90% homology with the human ortholog, but it lacks the CAG encoded poly-Q tract; instead, it contains a mixed glutamine/histidine tract. To study AR functionality in a more structured fashion, we created a knock-in mouse model that has only the equivalent of human poly-Q tract while maintaining the remaining mouse receptor sequence intact. Our humanized androgen receptor mouse (AR-19Q) displays a phenotype that is best described as Metabolic Syndrome (MetS). The mice gain excess weight, have hypertension, hyperglycemia, pancreatic islet cell abnormalities, and progressive non-alcoholic fatty liver disease (NAFLD). Features of MetS begin at approximately 4 months and progress till 12-14 months. Obesity: AR-19Q mice are significantly heavier, with a 35% gain of weight vs C57BL/6 mice. However, these mice are not hyperphagic at any time and eat identical quantities of chow. To assess obesity status, and found AR-19Q mice to have a significantly higher depositions of total white adipose visceral, lingual and subcutaneous vs age-matched controls. NAFLD: The progression of NAFLD is observed in AR-19Q mice. AR-19Q Histological analysis and immunohistochemistry reveal microvesicular steatosis, superseded by macrovesicular steatosis with hepatocyte balloon degeneration, scattered lobular inflammation (macrophages and lymphocytes) (6-9 months) and further superseded by perisinusoidal fibrosis (12 months). Biochemical blood analysis confirms. that there is a significant increase in alanine aminotransferase (ALT) levels, indicative of liver damage. With time classical NASH hepatitis and subsequent NASH fibrosis ensues; with several mice developing hepatocellular carcinoma (overall 20% of mice). Gene expression profiling AR-19Q mice for fatty liver genes, we observe an upregulation of inflammation genes Il-6, Il-10 and Tnf. In addition, increased Lpl (lipoprotein lipase) is indicative of increased blood triglyceride levels. On the other hand, down-regulation of G6pc (glucose-6-phosphatase) could be linked to glycogen storage disease. Down-regulation of Slc2a2 and Slc2a4, that encode GLUT2 and GLUT4, should have an influence on glucose levels, glucose homeostasis and insulin sensitivity, as insulin promotes glucose uptake through GLUT4. Type 2 Diabetes: Glucose intolerance is observed by 3-4 months followed by fasting hyperglycemia. Pancreases show advanced islet hyperplasia, concurrent with insulin resistance and type 2 diabetes. Pancreases of middle-age AR-19Q mice present with a significant islet hyperplasia, suggesting peripheral insulin resistance. We propose the use of the AR-19Q mice as a model to study the metabolic dysfunction/ deficiency leading to the MetS. The model will allow us to a better understanding into the mechanisms responsible for T2D and NAFLD-related disorders. Presentation: 6/2/2024

The mitigating role of lysophospholipids in hepatic lipid metabolism and intestinal immunity in juvenile black seabream (Acanthopagrus schlegelii) fed a high-fat diet

An 8-week feeding experiment investigated the effects of lysophospholipids (LP) on growth performance, hepatic lipid metabolism, intestinal immunity and antioxidation in juvenile black seabream (Acanthopagrus schlegelii) (initial weight 4.60 ± 0.01 g) on a high-fat diet. Five experimental diets were established: normal fat diet (13 %, CON), high-fat diet (18 %, HFD) and HFD with different levels of LP (0.05 %, 0.1 %, and 0.2 %). The results indicated that dietary LP had no impact on the specific growth rate and survival rate of juvenile black seabream (P > 0.05), while 0.2 %LP supplementation reduced the intraperitoneal fat ratio (IPF) (P < 0.05). Dietary 0.1 % and 0.2 %LP supplementation reduced the total lipid content and the size and number of lipid droplets in the liver (P < 0.05). The diets supplemented with LP reduced the contents of TG and LDL-C in the serum (P < 0.05), without affecting T-CHO and HDL-C (P > 0.05). The expression of lipid transport genes including apoa, apob, fatp1, and fatp4, were markedly up-regulated with increased dietary LP supplementation (P < 0.05). Moreover, dietary 0.2 %LP supplementation notably increased mRNA expression levels of scarb1, ffar3, acsl5 and glp-1 in the intestine (P < 0.05). Dietary LP notably down-regulated the mRNA expression of genes related to the lipogenesis pathway including fas, aco and srebp-1c, while up-regulating mRNA expression of lipogenesis-related genes including cpt1α, lpl, and pparα (P < 0.05). Additionally, dietary 0.2 %LP supplementation notably increased total antioxidant capacity in the intestine and up-regulated expression levels of intestinal antioxidant genes, including mn sod, cu-zn sod, gpx, and cat (P < 0.05), fish fed diets with 0.2 %LP down-regulation of pro-inflammatory genes, including tnfα and nf-κb, and up-regulation of anti-inflammatory factors, tgfβ-1, il-10 and tight-junction protein genes including (oclna, cldni, cldn3, and tjp1b) in the intestine (P < 0.05). In conclusion, dietary LP improved intestinal fatty acid transport and liver lipolysis, alleviating intestinal oxidative damage and inflammatory reactions caused by HFD in black seabream.

Influence of titanium nanoparticles on cytotoxicity and inflammatory cytokines expression in gingival fibroblasts - An in vitro study

Background Despite the success of titanium (Ti) implants in dental rehabilitation, emerging evidence implicates the release of titanium oxide nanoparticles (TiO2NPs) from the implant surface as a potential contributor to the initiation of the pathogenic process leading to peri-implantitis and ultimately failure of the implants. However, a comprehensive investigation to elucidate the dose-dependent effects of TiO2NPs on cytotoxicity and inflammatory responses is lacking. The present study was undertaken to evaluate the effect of different concentrations of TiO2NPs on the cytotoxicity and inflammatory cytokine expression in Human Gingival Fibroblasts (HGFs). Methods HGFs were isolated from gingival tissue samples obtained from periodontally and systemically healthy subjects. Ti standard solution for ICP was diluted to create concentrations of (0.001 ppm, 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm, and 100 ppm) for cell culture media containing titanium. HGFs were then cultured in these varying concentrations for specific time periods (days 1, 3, 5, and 7) to assess cell viability. A cytotoxicity assay was performed to determine the levels and expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and anti-inflammatory cytokines (IL-10, IL-12, TGF-β) using qRT-PCR and ELISA techniques. Results Our findings demonstrate a concentration and duration-dependent decrease in HGF viability upon exposure to titanium nanoparticles. Notably, a 50% reduction in cell viability was observed at the highest concentration (100 ppm). qRT-PCR analysis revealed a significant upregulation of pro-inflammatory cytokines, particularly IL-1β and IL-6, in HGFs exposed to titanium. Interestingly, the expression of anti-inflammatory cytokines (IL-10, IL-12, and TGF-β) remained comparable or even equivalent compared to controls across different titanium concentrations. Conclusions The study revealed a concentration and duration-dependent influence on HGF viability and cytokine profile, suggesting potential cytotoxicity and modulation of the inflammatory response mediated by TiO2NPs. Further research is necessary to elucidate the underlying mechanisms and their implications for dental implant biocompatibility.

Kukoamine A alleviates nephrolithiasis by inhibiting endogenous oxalate synthesis via the IL-6/JAK/STAT3/DAO signaling pathway

BackgroundThe recurrent nature and socioeconomic burden of nephrolithiasis demand effective treatments. Delineating the crosstalk between inflammatory processes and the endogenous oxalate metabolism pathway, which underpins nephrolithiasis pathogenesis, is essential for advancing treatment strategies. PurposeWe aim to screen therapeutic Chinese herbal remedies and their bioactive constituents for kidney stone treatment using a fruit fly model, followed by efficacy and mechanism validation in a rodent nephrolithiasis model as well as in vitro human cell culture model. Study design and methodsWe developed a fruit fly model to screen for efficient traditional Chinese medicine herbs and their active compounds for kidney stone treatment. Candidate active compounds from efficient herbs were separated and identified by solid-phase chromatography coupled with LC-MS analysis. Fruit fly genetic tools were employed to manipulate the expression of related genes to explore the therapeutic mechanisms of the Lycii Cortex and kukoamine A (KuA). To confirm the therapeutic effects and mechanisms of KuA for mammalian nephrolithiasis, mouse model of glyoxylate-induced kidney stone and human renal tubular cells were used. The therapeutic role of kukoamine A in nephrolithiasis was evaluated by assessing tubular injury, crystal deposition, and adhesion. The level of expression and phosphorylation in cells and mice was assessed using RT-qPCR and western blot. ResultsOur findings indicate that Lycii Cortex potently inhibits calcium oxalate stone formation via activation of the JNK/Upd3/JAK/STAT signaling cascade, resulting in diminished endogenous oxalate synthesis by downregulating D-amino acid oxidase (DAO) gene expression, predominantly in fruit fly Malpighian tube stellate cells. KuA was identified as the principal bioactive constituent mediating these effects. Both mouse models and human cell assays confirmed KuA's efficacy in preventing calcium oxalate nephrolithiasis in mammals, through hepatic JAK/STAT3 pathway activation and upregulation of IL-6, culminating in reduced urinary crystal deposition. ConclusionOur research underscores the potential of kukoamine A as a lead compound in treating nephrolithiasis and reveals the interplay between the IL-6/JAK/STAT3 inflammatory pathway and endogenous oxalate metabolism in nephrolithiasis pathogenesis. Additionally, it highlights the utility of the fruit fly model as a powerful tool for deciphering the therapeutic mechanisms of traditional Chinese herbs.

Evaluation of attenuated Salmonella Typhimurium (STMΔznuABC) anticancer activity on canine mammary cancer-associated fibroblasts

Bacteria-mediated treatments gained increasing attention as alternative therapies against tumors. An attenuated mutant strain of Salmonella enterica serovar Typhimurium (STMΔznuABC) has recently been considered as a potential new anti-cancer strategy. However, it is unclear whether this activity is tumor-induced or species-specific, and no data are available regarding STMΔznuABC on canine mammary tumors (CMTs). This study aimed to investigate the ability of STMΔznuABC in modulating the response of CMTs, focusing on cancer-associated fibroblasts. Four CMT cell lines (CF33, TM51, TM52 TM53) were treated with STMΔznuABC. Then, antiproliferative activity (MTT assay), bacterial invasion, and CMT cell lines gene expression analysis (RT-qPCR) of genes involved in immune response and cancer aggressiveness were evaluated. STMΔznuABC penetrated in TM51, TM52, TM53, and CF33 cell lines, causing a significant reduction of cell viability. Moreover, the expression of several genes was significantly modulated in all CMT cell lines: STMΔznuABC infection determined a significant up-regulation of CXCL8, IL18, IL10, TLR4 and RAD51, while CD14, IL6, CXCR4, P53, PTEN, STAT5, TLR5 and TGFB were downregulated in TM53. In CF33, CXCL8 and P53 were upregulated, while MYD88, MD2, IL18, TLR4,5, TGFB were downregulated. In TM52, CXCL8, CD44 and MD2 were upregulated and PTEN was downregulated, while in TM51 CXCL8, CD44 and ErbB2 were downregulated. We demonstrated the anti-proliferative and immuno-modulatory activity of STMΔznuABC in CMTs, paving the way for potential new anti-cancer treatments.

Endothelial pyroptosis-driven microglial activation in choroid plexus mediates neuronal apoptosis in hemorrhagic stroke rats

BackgroundSpontaneous intracerebral hemorrhage (ICH) is associated with alarmingly high rates of disability and mortality, and current therapeutic options are suboptimal. A critical component of ICH pathology is the initiation of a robust inflammatory response, often termed “cytokine storm,” which amplifies the secondary brain injury following the initial hemorrhagic insult. The precise sources and consequences of this cytokine-driven inflammation are not fully elucidated, necessitating further investigation. MethodsTo address this knowledge gap, our study conducted a comprehensive cytokine profiling using Luminex® assays, assessing 23 key cytokines. We then employed single-cell RNA sequencing and spatial transcriptomics at three critical time points post-ICH: the hyperacute, acute, and subacute phases. Integrating these multimodal analyses allowed us to identify the cellular origins of cytokines and elucidate their mechanisms of action. ResultsLuminex® cytokine assays revealed a significant upregulation of IL-6 and IL-1β levels at the 24-h post-ICH time point. Through the integration of scRNA-seq and spatial transcriptomics in the hemorrhagic hemisphere of rats, we observed a pronounced activation of cytokine-related signaling pathways within the choroid plexus. Initially, immune cell presence was sparse, but it surged 24 h post-ICH, particularly in the choroid plexus, indicating a substantial shift in the immune microenvironment. We traced the source of IL-1β and IL-6 to endothelial cells, establishing a link to pyroptosis. Endothelial pyroptosis post-ICH induced the production of IL-1β and IL-6, which activated microglial polarization characterized by elevated expression of Msr1, Lcn2, and Spp1 via the NF-κB pathway in the choroid plexus. Furthermore, we identified neuronal populations undergoing apoptosis, mediated by the Lcn2-SLC22A17 pathway in response to IL-1β and IL-6 signaling. Notably, the inhibition of pyroptosis using VX-765 significantly mitigated neurological impairments. ConclusionsOur study provides evidence that endothelial pyroptosis, characterized by the release of IL-1β and IL-6, triggers microglial polarization through NF-κB pathway activation, ultimately leading to microglia-mediated neuronal apoptosis in the choroid plexus post-ICH. These findings suggest that targeted therapeutic strategies aimed at mitigating endothelial cell pyroptosis and neutralizing inflammatory cytokines may offer neuroprotection for both microglia and neurons, presenting a promising avenue for ICH treatment.

The rs6967330 minor allele in CDHR3 is a significant risk factor for severe acute exacerbations in chronic rhinosinusitis

Acute exacerbations of chronic rhinosinusitis (AECRS) are commonly triggered by rhinovirus (RV) infections with secondary bacterial infections. Risk factors for AECRS are not well understood. To determine if carriers of the minor allele rs6967330 (AA/AG) in the Cadherin related family member 3 (CDHR3) gene have an increased risk for RV infections in AECRS in vivo and identify CDHR3 genotype-dependent host responses to RV infection in differentiated nasal airway liquid interface (ALI) cultures ex vivo. We performed a prospective year-long study of adult subjects with chronic rhinosinusitis (CRS) by rs6967330 genotype (AA/AG, n=16; GG, n=38). We contacted subjects every 2 weeks, and if they reported AECRS clinical data were collected. ALI cultures of adults with CRS (AG/AA,n=19; GG,n=19) were challenged with RV-A and RV-C. We measured viral copy numbers at 4- and 48-hours post-infection and RNA transcriptomes and cytokines at 48 hours post infection. Subjects with the minor allele had significantly higher rates of RV and bacterial infections than those with the major allele. ALI minor allele cultures had higher viral copy numbers of RV-A and RV-C after 48 hours compared to the major allele. Differentially expressed genes (DEG) and pathways identified an upregulation of IL-10 and IL4/13 pathways and a significant downregulation of toll like receptor (TLR) pathways in the minor allele cultures after RV-A and RV-C infection. Unsupervised hierarchical analysis of all DEGs suggest that allergic rhinitis had an additive effect on this response. The rs6967330 minor allele is associated with increased RV-A and RV-C replication, downregulation of TLR-mediated responses and increased T2-type and cytokine and chemokine responses during RV infection.

Enhanced Third Degree Burn Wound Healing by Hypoxic Mesenchymal Stem Cells' Secretome: IL-10 Upregulation and TNF-α/PGE2 Suppression

Background: Severe third-degree burns pose a notable obstacle in clinical settings, frequently leading to hindered healing of wounds and the development of scars. In this research, we explored how the secretome of hypoxic mesenchymal stem cells (HS-MSCs) could be improved the healing of burn wounds. Methods: In this study, we used 20 male Wistar rats that induced third degree wound by metal 80 exposure for 10 s. These rats divided into 4 groups: Healthy, control, HS-MSCs 100 µL, and HS-MSCs 200 µL groups. We injected the HS-MSCs, which carry various beneficial molecules like IL-10, TGF-β, PDGF, and VEGF, directly into the skin in a third-degree burn rat model on day 1 after burn induction, and harvest on day 14. Results: Our research discovered that using HS-MSCs resulted in a dosage-related rise in IL-10, an anti-inflammatory cytokine, alongside a notable decrease in TNF-α and PGE2, pro-inflammatory cytokines. These effects quickened the healing process by improving re-epithelialization and minimizing scar formation. Conclusions: Our findings indicate that HS-MSCs show promise as a treatment for severe burns by potentially controlling inflammation and fostering a regenerative healing process. More investigation is needed to understand the exact mechanisms and to explore how HS-MSCs could be applied in clinical treatments. HIGHLIGHTS HS-MSCs suppress inflammation and promote regenerative healing in severe burns HS-MSCs accelerated re-epithelialization and reduced scarring GRAPHICAL ABSTRACT