Unraveling Verapamil's Multidimensional Role in Diabetes Therapy: From ß-Cell Regeneration to Cholecystokinin Induction in Zebrafish and MIN6 Cell-Line Models.

This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic ß-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 ß-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost ß-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resilience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for ß-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering ß-cell recovery post-metronidazole infliction. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing ß-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of ß-cell regeneration, thereby offering fresh avenues for diabetes management strategies aimed at preserving and augmenting ß-cell functionality.

Gut Dysbiosis Shaped by Cocoa Butter-Based Sucrose-Free HFD Leads to Steatohepatitis, and Insulin Resistance in Mice.

BACKGROUND: High-fat diets cause gut dysbiosis and promote triglyceride accumulation, obesity, gut permeability changes, inflammation, and insulin resistance. Both cocoa butter and fish oil are considered to be a part of healthy diets. However, their differential effects on gut microbiome perturbations in mice fed high concentrations of these fats, in the absence of sucrose, remains to be elucidated. The aim of the study was to test whether the sucrose-free cocoa butter-based high-fat diet (C-HFD) feeding in mice leads to gut dysbiosis that associates with a pathologic phenotype marked by hepatic steatosis, low-grade inflammation, perturbed glucose homeostasis, and insulin resistance, compared with control mice fed the fish oil based high-fat diet (F-HFD). RESULTS: C57BL/6 mice (5-6 mice/group) were fed two types of high fat diets (C-HFD and F-HFD) for 24 weeks. No significant difference was found in the liver weight or total body weight between the two groups. The 16S rRNA sequencing of gut bacterial samples displayed gut dysbiosis in C-HFD group, with differentially-altered microbial diversity or relative abundances. Bacteroidetes, Firmicutes, and Proteobacteria were highly abundant in C-HFD group, while the Verrucomicrobia, Saccharibacteria (TM7), Actinobacteria, and Tenericutes were more abundant in F-HFD group. Other taxa in C-HFD group included the Bacteroides, Odoribacter, Sutterella, Firmicutes bacterium (AF12), Anaeroplasma, Roseburia, and Parabacteroides distasonis. An increased Firmicutes/Bacteroidetes (F/B) ratio in C-HFD group, compared with F-HFD group, indicated the gut dysbiosis. These gut bacterial changes in C-HFD group had predicted associations with fatty liver disease and with lipogenic, inflammatory, glucose metabolic, and insulin signaling pathways. Consistent with its microbiome shift, the C-HFD group showed hepatic inflammation and steatosis, high fasting blood glucose, insulin resistance, increased hepatic de novo lipogenesis (Acetyl CoA carboxylases 1 (Acaca), Fatty acid synthase (Fasn), Stearoyl-CoA desaturase-1 (Scd1), Elongation of long-chain fatty acids family member 6 (Elovl6), Peroxisome proliferator-activated receptor-gamma (Pparg) and cholesterol synthesis (ß-(hydroxy ß-methylglutaryl-CoA reductase (Hmgcr). Non-significant differences were observed regarding fatty acid uptake (Cluster of differentiation 36 (CD36), Fatty acid binding protein-1 (Fabp1) and efflux (ATP-binding cassette G1 (Abcg1), Microsomal TG transfer protein (Mttp) in C-HFD group, compared with F-HFD group. The C-HFD group also displayed increased gene expression of inflammatory markers including Tumor necrosis factor alpha (Tnfa), C-C motif chemokine ligand 2 (Ccl2), and Interleukin-12 (Il12), as well as a tendency for liver fibrosis. CONCLUSION: These findings suggest that the sucrose-free C-HFD feeding in mice induces gut dysbiosis which associates with liver inflammation, steatosis, glucose intolerance and insulin resistance.

Differential effects of fish-oil and cocoa-butter based high-fat/high-sucrose diets on endocrine pancreas morphology and function in mice.

Introduction: A high-fat/high-sucrose diet leads to adverse metabolic changes that affect insulin sensitivity, function, and secretion. The source of fat in the diet might inhibit or increase this adverse effect. Fish oil and cocoa butter are a significant part of our diets. Yet comparisons of these commonly used fat sources with high sucrose on pancreas morphology and function are not made. This study investigated the comparative effects of a fish oil-based high-fat/high-sucrose diet (Fish-HFDS) versus a cocoa butter-based high-fat/high-sucrose diet (Cocoa-HFDS) on endocrine pancreas morphology and function in mice. Methods: C57BL/6 male mice (n=12) were randomly assigned to dietary intervention either Fish-HFDS (n=6) or Cocoa-HFDS (n=6) for 22 weeks. Intraperitoneal glucose and insulin tolerance tests (IP-GTT and IP-ITT) were performed after 20-21 weeks of dietary intervention. Plasma concentrations of c-peptide, insulin, glucagon, GLP-1, and leptin were measured by Milliplex kit. Pancreatic tissues were collected for immunohistochemistry to measure islet number and composition. Tissues were multi-labelled with antibodies against insulin and glucagon, also including expression on Pdx1-positive cells. Results and discussion: Fish-HFDS-fed mice showed significantly reduced food intake and body weight gain compared to Cocoa-HFDS-fed mice. Fish-HFDS group had lower fasting blood glucose concentration and area under the curve (AUC) for both GTT and ITT. Plasma c-peptide, insulin, glucagon, and GLP-1 concentrations were increased in the Fish-HFDS group. Interestingly, mice fed the Fish-HFDS diet displayed higher plasma leptin concentration. Histochemical analysis revealed a significant increase in endocrine pancreas ß-cells and islet numbers in mice fed Fish-HFDS compared to the Cocoa-HFDS group. Taken together, these findings suggest that in a high-fat/high-sucrose dietary setting, the source of the fat, especially fish oil, can ameliorate the effect of sucrose on glucose homeostasis and endocrine pancreas morphology and function.

Expression of Steroid Receptor RNA Activator 1 (SRA1) in the Adipose Tissue Is Associated with TLRs and IRFs in Diabesity.

Steroid receptor RNA activator gene (SRA1) emerges as a player in pathophysiological responses of adipose tissue (AT) in metabolic disorders such as obesity and type 2 diabetes (T2D). We previously showed association of the AT SRA1 expression with inflammatory cytokines/chemokines involved in metabolic derangement. However, the relationship between altered adipose expression of SRA1 and the innate immune Toll-like receptors (TLRs) as players in nutrient sensing and metabolic inflammation as well as their downstream signaling partners, including interferon regulatory factors (IRFs), remains elusive. Herein, we investigated the association of AT SRA1 expression with TLRs, IRFs, and other TLR-downstream signaling mediators in a cohort of 108 individuals, classified based on their body mass index (BMI) as persons with normal-weight (N = 12), overweight (N = 32), and obesity (N = 64), including 55 with and 53 without T2D. The gene expression of SRA1, TLRs-2,3,4,7,8,9,10 and their downstream signaling mediators including IRFs-3,4,5, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1), and nuclear factor-κB (NF-κB) were determined using qRT-PCR and SRA1 protein expression was determined by immunohistochemistry. AT SRA1 transcripts' expression was significantly correlated with TLRs-3,4,7, MyD88, NF-κB, and IRF5 expression in individuals with T2D, while it associated with TLR9 and TRAF6 expression in all individuals, with/without T2D. SRA1 expression associated with TLR2, IRAK1, and IRF3 expression only in individuals with obesity, regardless of diabetes status. Furthermore, TLR3/TLR7/IRAK1 and TLR3/TLR9 were identified as independent predictors of AT SRA1 expression in individuals with obesity and T2D, respectively. Overall, our data demonstrate a direct association between the AT SRA1 expression and the TLRs together with their downstream signaling partners and IRFs in individuals with obesity and/or T2D.

Adipose Tissue Steroid Receptor RNA Activator 1 (SRA1) Expression Is Associated with Obesity, Insulin Resistance, and Inflammation.

Steroid receptor RNA activator 1 (SRA1) is involved in pathophysiological responses of adipose tissue (AT) in obesity. In vitro and animal studies have elucidated its role in meta-inflammation. Since SRA1 AT expression in obesity/type 2 diabetes (T2D) and the relationship with immune-metabolic signatures remains unclear, we assessed AT SRA1 expression and its association with immune-metabolic markers in individuals with obesity/T2D. For this, 55 non-diabetic and 53 T2D individuals classified as normal weight (NW; lean), overweight, and obese were recruited and fasting blood and subcutaneous fat biopsy samples were collected. Plasma metabolic markers were assessed using commercial kits and AT expression of SRA1 and selected immune markers using RT-qPCR. SRA1 expression was significantly higher in non-diabetic obese compared with NW individuals. SRA1 expression associated with BMI, PBF, serum insulin, and HOMA-IR in the total study population and people without diabetes. SRA1 associated with waist circumference in people without diabetes and NW participants, whereas it associated inversely with HbA1c in overweight participants. In most study subgroups AT SRA1 expression associated directly with CXCL9, CXCL10, CXCL11, TNF-α, TGF-ß, IL2RA, and IL18, but inversely with CCL19 and CCR2. TGF-ß/IL18 independently predicted the SRA1 expression in people without diabetes and in the total study population, while TNF-α/IL-2RA predicted SRA1 only in people with diabetes. TNF-α also predicted SRA1 in both NW and obese people regardless of the diabetes status. In conclusion, AT SRA1 expression is elevated in people with obesity which associates with typical immunometabolic markers of obesity/T2D, implying that SRA1 may have potential as a biomarker of metabolic derangements.

Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway.

Increased circulatory and adipose tissue expression of macrophage inflammatory protein (MIP)-1α (CC motif chemokine ligand-3/CCL3) and its association with inflammation in the state of obesity is well documented. Since obesity is associated with increases in both stearic acid and tumor necrosis factor α (TNF-α) in circulation, we investigated whether stearic acid and TNF-α together could regulate MIP-1α/CCL3 expression in human monocytic cells, and if so, which signaling pathways were involved in MIP-1α/CCL3 modulation. Monocytic cells were treated with stearic acid and TNF-α resulted in enhanced production of MIP-1α/CCL3 compared to stearic acid or TNF-α alone. To explore the underlying mechanisms, cooperative effect of stearic acid for MIP-α/CCL3 expression was reduced by TLR4 blocking, and unexpectedly we found that the synergistic production of MIP-α/CCL3 in MyD88 knockout (KO) cells was not suppressed. In contrast, this MIP-α/CCL3 expression was attenuated by inhibiting TBK1/IRF3 activity. Cells deficient in IRF3 did not show cooperative effect of stearate/TNF-α on MIP-1α/CCL3 production. Furthermore, activation of IRF3 by polyinosinic-polycytidylic acid (poly I:C) produced a cooperative effect with TNF-α for MIP-1α/CCL3 production that was comparable to stearic acid. Individuals with obesity show high IRF3 expression in monocytes as compared to lean individuals. Furthermore, elevated levels of MIP-1α/CCL3 positively correlate with TNF-α and CD163 in fat tissues from individuals with obesity. Taken together, this study provides a novel model for the pathologic role of stearic acid to produce MIP-1α/CCL3 in the presence of TNF-α associated with obesity settings.

The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways.

Chronic low-grade inflammation, also known as metabolic inflammation, is a hallmark of obesity and parallels with the presence of elevated circulatory levels of free fatty acids and inflammatory cytokines/chemokines. CCL4/MIP-1ß chemokine plays a key role in the adipose tissue monocyte recruitment. Increased circulatory levels of TNF-α, palmitate and CCL4 are co-expressed in obesity. We asked if the TNF-α/palmitate could interact cooperatively to augment the CCL4 production in human monocytic cells and macrophages. THP-1 cells/primary macrophages were co-treated with TNF-α/palmitate and CCL4 mRNA/protein expression was assessed using qRT-PCR/ELISA. TLR4 siRNA, a TLR4 receptor-blocking antibody, XBlue™-defMyD cells and pathway inhibitors were used to decipher the signaling mechanisms. We found that TNF-α/palmitate co-stimulation augmented the CCL4 expression in monocytic cells and macrophages compared to controls (p < 0.05). TLR4 suppression or neutralization abrogated the CCL4 expression in monocytic cells. Notably, CCL4 cooperative induction in monocytic cells was: (1) Markedly less in MyD88-deficient cells, (2) IRF3 independent, (3) clathrin dependent and (4) associated with the signaling mechanism involving ERK1/2, c-Jun, JNK and NF-κB. In conclusion, TNF-α/palmitate co-stimulation promotes the CCL4 expression in human monocytic cells through the mechanism involving a TLR4-MyD88 axis and MAPK/NF-κB pathways. These findings unravel a novel mechanism of the cooperative induction of CCL4 by TNF-α and palmitate which could be relevant to metabolic inflammation.

Oxidative Stress Induces Expression of the Toll-Like Receptors (TLRs) 2 and 4 in the Human Peripheral Blood Mononuclear Cells: Implications for Metabolic Inflammation.

BACKGROUND/AIMS: Innate immune toll-like receptors (TLRs) are emerging as nutrient sensors. Oxidative stress in the adipose tissue in obesity acts as a critical early trigger of altered pathophysiology. TLR2/TLR4 adipose upregulation has been associated with insulin resistance in humans; however, it remains unclear whether oxidative stress can modulate expression of TLR2/4 and related immune-metabolic regulators (IRF3/5) in immune cells. We, therefore, assessed their expression along with proinflammatory cytokines in the human PBMC following induction of oxidative stress. METHODS: PBMC were isolated from blood of healthy donors using Ficoll-Paque method and cells were treated with H2O2 to induce oxidative stress. ROS was measured by DCFH-DA assay. Target gene and protein expression was determined using real-time RT-PCR and flow cytometry/confocal microscopy, respectively. TLR2/4 expression by H2O2 in presence of ROS-inhibitors or leptin/LPS/fatty acids was also assessed. Expression of phosphorylated/total ERK1/2, c-Jun, p38, and NF-κB was determined by western blotting. The data (mean±SEM) were compared using unpaired student's t-test or ANOVA; all P-values <0.05 were considered significant. RESULTS: TLR2/4 mRNA/protein expression was elevated by oxidative stress in PBMC compared to controls (P<0.001). This induction was abrogated by apocynin/N-acetyl cysteine treatments (P<0.01). H2O2-induced TLR2/4 gene expression was further enhanced by leptin, LPS, oleate, or palmitate (P<0.05). Oxidative stress also promoted expression of IRF3/5 and proinflammatory cytokines including IFN-γ, IL-1ß, IL-6, TNF-α, and MCP-1/CCL2. This oxidative stress in PBMC involved MAPK/NF-κB dependent signaling. CONCLUSION: Taken together, oxidative stress upregulates expression of TLR2/4, IRF3/5 and signature proinflammatory cytokines in PBMC, involving MAPK/NF-κB dependent signaling, all of which may have implications for metabolic inflammation.

TNF-α Drives the CCL4 Expression in Human Monocytic Cells: Involvement of the SAPK/JNK and NF-κB Signaling Pathways.

BACKGROUND/AIMS: Increased circulatory levels of both TNF-α and CCL4/MIP-1ß are found in metabolic diseases. However, it is unclear whether TNF-α which is a signature proinflammatory cytokine involved in metabolic inflammation, can induce/promote the expression of CCL4. METHODS: THP-1 human monocytic cells and THP-1-derived macrophages were stimulated with TNF-α and LPS-treatment as a positive control. CCL4 mRNA/protein expression was measured using qRT-PCR/ELISA, respectively. Stress-activated protein kinases (SAPK)/ c-Jun N-terminal kinase (JNK) activity was determined using the assay kit. Mechanistic pathways were studied using anti-TNFR1/2 antibodies, pharmacological inhibitors, siRNAs, and NF-κB/AP-1 reporter-expressing THP-1-XBlue cells. Phosphorylation of signaling molecules was assessed by Western blotting. RESULTS: TNF-α induces CCL4 expression at mRNA and protein levels, in both THP-1 monocytic cells and macrophages (P<0.05). TNF-α-driven CCL4 production was markedly abrogated by pre-treatment with anti-TNFR1/2 neutralizing antibodies. TNF-α treatment induced phosphorylation of SAPK/JNK, c-Jun, and NF-κB. Genetic and/or pharmacologic inhibition of SAPK/JNK and NF-κB pathways suppressed the TNF-α-induced CCL4 expression (P<0.05). NF-κB/AP-1 activity was found to be significantly increased in TNFα-treated SEAP reporter-expressing monocytic cells. CONCLUSION: These data suggest that TNF-α drives CCL4 expression in THP-1 monocytic cells/macrophages via the activation of SAPK/JNK and NF-κB pathways. The findings may provide new insights into understanding the regulatory role of TNF-α in augmenting CCL4 expression during inflammatory conditions.

TLR4/MyD88 -mediated CCL2 production by lipopolysaccharide (endotoxin): Implications for metabolic inflammation.

BACKGROUND: Obese human and mice were reported to have higher circularity endotoxin (LPS) levels as compared to their lean counter parts. The current study was aimed to reveal the molecular mechanisms underlying the LPS mediated induction of CCL2 in human monocytes/macrophages. METHODS: Human monocytic cell line THP-1, THP-1 cells derived macrophages and primary macrophages were treated with LPS and TNF-α (positive control). CCL2 expression was determined with real-time RT-PCR and ELISA. THP-1-XBlue™ cells, THP-1-XBlue™-defMyD cells, TLR4 neutralization antibody, TLR4 siRNA and inhibitors for NF-kB and MAPK were used to study the signaling pathways. Phosphorylation of NF-kB and c-Jun was analyzed by ELISA. RESULTS: LPS upregulates CCL2 expression at both mRNA (THP-1: 23.40 ± .071 Fold, P < 0.0001; THP-1-derived macrophages: 103 ± 0.56 Fold, < 0.0001; Primary macrophages: 48 ± 1.41 Fold, P < 0.0005) and protein (THP1 monocytes:1048 ± 5.67 pg/ml, P < 0.0001; THP-1-derived macrophages; 2014 ± 2.12, P = 0.0001; Primary macrophages: 859.5 ± 3.54, P < 0.0001) levels in human monocytic cells/macrophages. Neutralization of TLR4 blocked LPS-induced CCL-2 secretion (P < 0.0001). Silencing of TLR4 by siRNA also significantly reduced LPS-induced CCL-2 production. Furthermore, MyD88-Knockout cells treated with LPS did not produce CCL-2. NF-kB and c-Jun phosphorylation was noted in LPS treated cells. CONCLUSION: Overall, our data reveal that LPS induces CCL-2 in monocytes/macrophages via TLR4/MyD88 signaling which leads to the activation of NF-kB/AP-1 transcription factors.

Palmitate Activates CCL4 Expression in Human Monocytic Cells via TLR4/MyD88 Dependent Activation of NF-κB/MAPK/ PI3K Signaling Systems.

BACKGROUND/AIMS: Obesity is associated with adipose tissue inflammation which plays a key role in the development of insulin resistance and type 2 diabetes (T2D). Saturated free fatty acids (SFAs) levels are found to be elevated in obesity and T2D. Chemokines are known to have potent inflammatory functions in a wide range of biological processes linked to immunological disorders. Since CCL4 (Chemokine (C-C motif) ligand 4), also known as macrophage inflammatory protein-1ß (MIP-1ß), plays an important role in the migration of monocytes into the adipose tissue, we investigated the expression of CCL4 in monocytic cells/macrophages following activation with free fatty acid palmitate. METHODS: Human monocytic cell line THP-1 and macrophages derived from THP-1 and primary monocytes were stimulated with palmitate and LPS (positive control). CCL4 expression and secretion were measured with real time RT-PCR and ELISA respectively. Signaling pathways were identified by using THP-1-XBlueTM cells, THP-1-XBlueTM-defMyD cells, anti-TLR4 mAb and TLR4 siRNA. RESULTS: Palmitate induces CCL4 expression at both mRNA and protein levels in human monocytic cells. Palmitate-induced CCL4 production was markedly suppressed by neutralizing anti-TLR-4 antibody. Additionally, silencing of TLR4 by siRNA also significantly suppressed the palmitate-induced up-regulation of CCL4. MyD88-deficient cells did not express CCL4 in response to palmitate treatment. Inhibition of NF-kB and MAPK pathways suppressed the palmitate mediated induction of CCL4. Moreover, induction of CCL4 was blocked by PI3 Kinase inhibitors LY294002 and wortmannin. CONCLUSION: Collectively, our results show that palmitate induces CCL4 expression via activation of the TLR4-MyD88/NF-kB/MAPK/ PI3K signaling cascade. Thus, our findings suggest that the palmitate-induced CCL4 production might be an underlying mechanism of metabolic inflammation.

Increased Expression of the Innate Immune Receptor TLR10 in Obesity and Type-2 Diabetes: Association with ROS-Mediated Oxidative Stress.

BACKGROUND/AIMS: Metabolic diseases such as obesity and type-2 diabetes (T2D) are known to be associated with chronic low-grade inflammation called metabolic inflammation together with an oxidative stress milieu found in the expanding adipose tissue. The innate immune Toll-like receptors (TLR) such as TLR2 and TLR4 have emerged as key players in metabolic inflammation; nonetheless, TLR10 expression in the adipose tissue and its significance in obesity/T2D remain unclear. METHODS: TLR10 gene expression was determined in the adipose tissue samples from healthy non-diabetic and T2D individuals, 13 each, using real-time RT-PCR. TLR10 protein expression was determined by immunohistochemistry, confocal microscopy, and flow cytometry. Regarding in vitro studies, THP-1 cells, peripheral blood mononuclear cells (PBMC), or primary monocytes were treated with hydrogen peroxide (H2O2) for induction of reactive oxygen species (ROS)-mediated oxidative stress. Superoxide dismutase (SOD) activity was measured using a commercial kit. Data (mean±SEM) were compared using unpaired student's t-test and P<0.05 was considered significant. RESULTS: The adipose tissue TLR10 gene/protein expression was found to be significantly upregulated in obesity as well as T2D which correlated with body mass index (BMI). ROS-mediated oxidative stress induced high levels of TLR10 gene/protein expression in monocytic cells and PBMC. In these cells, oxidative stress induced a time-dependent increase in SOD activity. Pre-treatment of cells with anti-oxidants/ROS scavengers diminished the expression of TLR10. ROS-induced TLR10 expression involved the nuclear factor-kappaB (NF-κB)/mitogen activated protein kinase (MAPK) signaling as well as endoplasmic reticulum (ER) stress. H2O2-induced oxidative stress interacted synergistically with palmitate to trigger the expression of TLR10 which associated with enhanced expression of proinflammatory cytokines/chemokine. CONCLUSION: Oxidative stress induces the expression of TLR10 which may represent an immune marker for metabolic inflammation.

Plasma fetuin-A/α2-HS-glycoprotein correlates negatively with inflammatory cytokines, chemokines and activation biomarkers in individuals with type-2 diabetes.

BACKGROUND: Fetuin-A/AHSH is a novel hepatokine that acts as a vascular calcification inhibitor and as an endogenous TLR-4 ligand. Fetuin-A may act as a positive or negative acute phase protein (APP) in disease conditions. The relationship between circulatory fetuin-A and inflammatory biomarkers in type-2 diabetes (T2D) remains controversial. Therefore, the purpose of this study was to determine the plasma fetuin-A levels in 53 T2D (BMI = 29.7 ± 4.5 kg/m2) and 72 non-diabetic individuals (BMI = 28.2 ± 5.8 kg/m2) using premixed 38-plex MAP human cytokine/chemokine magnetic bead immunoassays and the data (mean ± SEM) were statistically analyzed to determine Pearson's correlation (r) between fetuin-A and detected analytes; P-values ≤0.05 were considered significant. RESULTS: The data show that plasma fetuin-A levels were comparable in both groups (P = 0.27) and in T2D individuals, fetuin-A associated negatively (P ≤ 0.05) with a large number of proinflammatory cytokines/chemokines and activation biomarkers including TNF-α, IFN-α2, IFN-γ, IL-1α, IL-1ß, IL-1RA, IL-3, IL-4, IL-7, IL-9, IL-12p40/p70, IL-15, CCL-2, CCL-4, CCL-11, CCL-22, CXCL-8, CX3CL-1, EFF-2, EGF, G-CSF, GM-CSF, GRO, sCD40L, and VEGF. In non-diabetics, fetuin-A also correlated positively with certain TH2 cytokines (IL-5, IL-13) and chemokines (CCL-3, CCL-5, CCL-7). Notably, in vitro fetuin-A production was significantly suppressed in HepG2 cells treated with TNF-α, IL-1ß, and IFN-γ which supported the clinical findings of a negative association between fetuin A and inflammatory mediators. CONCLUSIONS: The negative association between circulatory fetuin-A and systemic inflammatory mediators in T2D patients suggests that plasma fetuin-A may have predictive significance as a negative APP in metabolic disease.

Lack of the programmed death-1 receptor renders host susceptible to enteric microbial infection through impairing the production of the mucosal natural killer cell effector molecules.

The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.

GM-CSF produced by nonhematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa.

GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, as well as dendritic cell differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn's disease in humans and colitis in murine models has mainly been considered to reflect its activity on myeloid cells. We used GM-CSF-deficient (GM-CSF(-/-)) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS), at doses that resulted in little epithelial damage and mucosal ulceration in wild type mice, caused marked colon ulceration and delayed ulcer healing in GM-CSF(-/-) mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF(-/-) mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF(-/-) mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Nonhematopoietic cells, and not myeloid cells, produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury, as revealed by bone marrow chimera and dendritic cell-depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell-produced GM-CSF has a novel nonredundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium.

Chronic epithelial NF-κB activation accelerates APC loss and intestinal tumor initiation through iNOS up-regulation.

The role of NF-κB activation in tumor initiation has not been thoroughly investigated. We generated Ikkß(EE)(IEC) transgenic mice expressing constitutively active IκB kinase ß (IKKß) in intestinal epithelial cells (IECs). Despite absence of destructive colonic inflammation, Ikkß(EE)(IEC) mice developed intestinal tumors after a long latency. However, when crossed to mice with IEC-specific allelic deletion of the adenomatous polyposis coli (Apc) tumor suppressor locus, Ikkß(EE)(IEC) mice exhibited more ß-catenin(+) early lesions and visible small intestinal and colonic tumors relative to Apc(+/ΔIEC) mice, and their survival was severely compromised. IEC of Ikkß(EE)(IEC) mice expressed high amounts of inducible nitric oxide synthase (iNOS) and elevated DNA damage markers and contained more oxidative DNA lesions. Treatment of Ikkß(EE)(IEC)/Apc(+/ΔIEC) mice with an iNOS inhibitor decreased DNA damage markers and reduced early ß-catenin(+) lesions and tumor load. The results suggest that persistent NF-κB activation in IEC may accelerate loss of heterozygocity by enhancing nitrosative DNA damage.

Constitutive intestinal NF-κB does not trigger destructive inflammation unless accompanied by MAPK activation.

Nuclear factor (NF)-κB, activated by IκB kinase (IKK), is a key regulator of inflammation, innate immunity, and tissue integrity. NF-κB and one of its main activators and transcriptional targets, tumor necrosis factor (TNF), are up-regulated in many inflammatory diseases that are accompanied by tissue destruction. The etiology of many inflammatory diseases is poorly understood, but often depends on genetic factors and environmental triggers that affect NF-κB and related pathways. It is unknown, however, whether persistent NF-κB activation is sufficient for driving symptomatic chronic inflammation and tissue damage. To address this question, we generated IKKß(EE)(IEC) mice, which express a constitutively active form of IKKß in intestinal epithelial cell (IECs). IKKß(EE)(IEC) mice exhibit NF-κB activation in IECs and express copious amounts of inflammatory chemokines, but only small amounts of TNF. Although IKKß(EE)(IEC) mice exhibit inflammatory cell infiltration in the lamina propria (LP) of their small intestine, they do not manifest tissue damage. Yet, upon challenge with relatively mild immune and microbial stimuli, IKKß(EE)(IEC) mice succumb to destructive acute inflammation accompanied by enterocyte apoptosis, intestinal barrier disruption, and bacterial translocation. Inflammation is driven by massive TNF production, which requires additional activation of p38 and extracellular-signal-regulated kinase mitogen-activated protein kinases (MAPKs).

Conventional dendritic cells regulate the outcome of colonic inflammation independently of T cells.

We explored the physiological role of conventional dendritic cells (cDCs) in acute colitis induced by a single cycle of dextran sodium sulfate administration. Depending on their mode of activation and independently of T cells, cDCs can enhance or attenuate the severity of dextran sodium sulfate-induced colitis. The latter beneficial effect was achieved, in part, by IFN-1 induced by Toll-like receptor 9-activated cDCs. IFN-1 inhibits colonic inflammation by regulating neutrophil and monocyte trafficking to the inflamed colon and restraining the inflammatory products of tissue macrophages. These data highlight a novel role of cDCs in the regulation of other innate immune cells and position them as major players in acute colonic inflammation.

Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-kappaB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IkappaBalpha degradation and activation of the NF-kappaB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IkappaB accumulates in the cytoplasm preventing NF-kappaB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-kappaB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

Transplanted embryonic stem cells successfully survive, proliferate, and migrate to damaged regions of the mouse brain.

An understanding of feasibility of implanting embryonic stem cells (ESCs), their behavior of migration in response to lesions induced in brain tissues, and the mechanism of their in vivo differentiation into neighboring neural cells is essential for developing and refining ESC transplantation strategies for repairing damages in the nervous system, as well as for understanding the molecular mechanism underlying neurogenesis. We hypothesized that damaged neural tissues offer a niche to which injected ESCs can migrate and differentiate into the neural cells. We inflicted damage in the murine (C57BL/6) brain by injecting phosphate-buffered saline into the left frontal and right caudal regions and confirmed neural damage by histochemistry. Enhanced yellow fluorescent protein-expressing ESCs were injected into the nondamaged left caudal portion of the brain. Using immunohistochemistry and fluorescent microscopy, we observed migration of ESCs from the injection site (left caudal) to the damaged site (right caudal and left frontal). Survival of the injected ESCs was confirmed by the real-time polymerase chain reaction analysis of stemness genes such as Oct4, Sox2, and FGF4. The portions of the damaged neural tissues containing ESCs demonstrated a fourfold increase in expression of these genes after 1 week of injection in comparison with the noninjected ESC murine brain, suggesting proliferation. An increased level of platelet-derived growth factor receptor demonstrated that ESCs responded to damaged neural tissues, migrated to the damaged site of the brain, and proliferated. These results demonstrate that undifferentiated ESCs migrate to the damaged regions of brain tissue, engraft, and proliferate. Thus, damaged brain tissue provides a niche that attracts ESCs to migrate and proliferate.