Combination product of dermal matrix, human mesenchymal stem cells, and timolol promotes diabetic wound healing in mice.

Diabetic foot ulcers are a major health care concern with limited effective therapies. Mesenchymal stem cell (MSC)-based therapies are promising treatment options due to their beneficial effects of immunomodulation, angiogenesis, and other paracrine effects. We investigated whether a bioengineered scaffold device containing hypoxia-preconditioned, allogeneic human MSCs combined with the beta-adrenergic antagonist timolol could improve impaired wound healing in diabetic mice. Different iterations were tested to optimize the primary wound outcome, which was percent of wound epithelialization. MSC preconditioned in 1 µM timolol at 1% oxygen (hypoxia) seeded at a density of 2.5 × 105 cells/cm2 on Integra Matrix Wound Scaffold (MSC/T/H/S) applied to wounds and combined with daily topical timolol applications at 2.9 mM resulted in optimal wound epithelialization 65.6% (24.9% ± 13.0% with MSC/T/H/S vs 41.2% ± 20.1%, in control). Systemic absorption of timolol was below the HPLC limit of quantification, suggesting that with the 7-day treatment, accumulative steady-state timolol concentration is minimal. In the early inflammation stage of healing, the MSC/T/H/S treatment increased CCL2 expression, lowered the pro-inflammatory cytokines IL-1B and IL6 levels, decreased neutrophils by 44.8%, and shifted the macrophage ratio of M2/M1 to 1.9 in the wound, demonstrating an anti-inflammatory benefit. Importantly, expression of the endothelial marker CD31 was increased by 2.5-fold with this treatment. Overall, the combination device successfully improved wound healing and reduced the wound inflammatory response in the diabetic mouse model, suggesting that it could be translated to a therapy for patients with diabetic chronic wounds.

Crosstalk between adrenergic and toll-like receptors in human mesenchymal stem cells and keratinocytes: a recipe for impaired wound healing.

Previous studies demonstrate that skin wounds generate epinephrine (EPI) that can activate local adrenergic receptors (ARs), impairing healing. Bacterially derived activators of Toll-like receptors (TLRs) within the wound initiate inflammatory responses and can also impair healing. In this study, we examined the hypothesis that these two pathways crosstalk to one another, using EPI and macrophage-activating lipopeptide-2 (MALP2) to activate ARs and TLR2, respectively, in human bone marrow-derived mesenchymal stem cells (BM-MSCs) and neonatal keratinocytes (NHKs). BM-MSCs exposed to EPI significantly (p < .05) increased TLR2 message (sevenfold BM-MSCs), TLR2 protein (twofold), and myeloid differentiation factor 88 (MyD88) (fourfold). Conversely, activation of TLR2 by MALP2 in these cells increased ß2-AR message (twofold in BM-MSCs, 2.7-fold in NHKs), ß2-AR protein (2.5-fold), phosphorylation of ß-AR-activated kinase (p-BARK, twofold), and induced release of EPI from both cell types (twofold). Treating cells with EPI and MALP2 together, as would be encountered in a wound, increased ß2-AR and p-BARK protein expression (sixfold), impaired cell migration (BM-MSCs- 21%↓ and NHKs- 60%↓, p < .002), and resulted in a 10-fold (BM-MSCs) and 51-fold (NHKs) increase in release of IL-6 (p < .001) responses that were remarkably reduced by pretreatment with ß2-AR antagonists. In vivo, EPI-stressed animals exhibited impaired healing, with elevated levels of TLR2, MyD88, and IL-6 in the wounds (p < .05) relative to nonstressed controls. Thus, our data describe a recipe for decreasing cell migration and exacerbating inflammation via novel crosstalk between the adrenergic and Toll-like receptor pathways in BM-MSCs and NHKs.

Toll-like receptor expression and signaling in human diabetic wounds.

AIM: To examine the contribution of toll-like receptors (TLRs) expression and activation to the prolonged inflammation often seen in human diabetic wounds. METHODS: Debridement wound tissue was collected from diabetic patients with informed consent. Total RNA and protein were isolated and subjected to real-time polymerase chain reaction and Western blot analyses. RESULTS: TLR1, 2, 4, and 6 mRNA expressions were increased significantly in wounds of diabetic patients compared with non-diabetic wounds (P < 0.05). MyD88 protein expression was significantly increased in diabetic wounds compared to non-diabetic wounds. Interleukin-1beta, tumor necrosis factor-alpha concentration nuclear factor-kappa B activation, and thiobarbituric acid reactive substances were increased in diabetic wounds compared to non-diabetic wounds (P < 0.01). CONCLUSION: Collectively, our novel findings show that increased TLR expression, signaling, and activation may contribute to the hyper inflammation in the human diabetic wounds.

Amelioration in wound healing in diabetic toll-like receptor-4 knockout mice.

Toll-like receptor-4 (TLR4) is a sentinel pathogen recognition receptor with a pivotal role in inflammation, tissue injury, diabetes and its complications. The aim of the study was to examine the contribution of TLR4 expression and activation to the prolonged inflammation observed in diabetic wounds. Diabetes was induced in male C57BL/6J and TLR4 knockout (KO) mice using streptozotocin (STZ) with matching non-diabetic mice as control. After 2weeks of persistent hyperglycemia in the mice, full-thickness excision wounds were made on the backs aseptically. Total RNA and protein were subjected to real-time PCR and western blot analyses. Wound sizes were measured using digital planimetry. TLR4 mRNA and protein expression increased significantly in wounds of diabetic mice compared with non-diabetic mice (P<0.05). IL-6, TNF-α concentration and nuclear factor-κB (NF-κB) activation were increased in diabetic wounds compared to non-diabetic wounds and knockout of TLR4 alleviates wound healing and decreases inflammation in diabetic TLR4 KO mice. Collectively, our findings show that increased TLR4 mRNA and protein expression and activation contribute to the prolonged inflammation in the diabetic wounds and that absence of TLR4 may result in decreased inflammation and improved wound healing.

Plant-based modulation of Toll-like receptors: an emerging therapeutic model.

Plant-based extracts present a large source of natural immune modulators, many of which have been used in traditional medicine for centuries. Recent research efforts have identified plant extracts as potential modulators of Toll-like receptors (TLRs), the first responders in immunological defenses in normal and disease conditions. This review aims to provide a comprehensive discussion of the modulatory effects of plant-based extracts on TLR expression, signaling, and activation. We organized the review by extraction solvent and plant part showing how they impacted the TLRs. The phytochemical components of the extracts discovered to enable these effects are diverse and vary based on the plant part. The role of the extraction solvent and differences between the different phytochemical components, such as phenolics and polysaccharides, are discussed. Plant extracts hold promising treatments for controlling inflammation and, conversely, for stimulating the immune response. Further research is needed to identify bioactive components of the extracts, mechanisms of their action, and in vivo pharmacological effects using appropriate disease models to ultimately adapt the findings for clinical use.

Toll-like receptors in wound healing: location, accessibility, and timing.

Toll-like receptors (TLRs) are considered to be the first responders in the defense against invading pathogens. TLR engagement by ligands triggers inflammatory responses in injury and trauma, and thus can impair or contribute to the healing process, depending on TLRs' expression pattern, cellular localization, signaling, and deployment of inflammatory responses. Understanding these attributes could improve therapeutic strategies for treating chronic wounds.

Toll-like receptors and diabetes: a therapeutic perspective.

Diabetes is a mutifactorial metabolic disorder that leads to a number of complications. Diabetes is estimated to affect 36 million people in the U.S.A., and the prevalence of diagnosed and undiagnosed diabetes is at 9.3% and continues to rise. Evidence from experimental animal models as well as humans has indicated that systemic inflammation plays a role in the pathophysiological processes of diabetes and is facilitated by innate immune responses. TLRs (Toll-like receptors) are key innate immune receptors that recognize conserved PAMPs (pathogen-associated molecular patterns), induce inflammatory responses essential for host defences and initiate an adaptive immune response. Although TLR expression is increased in a plethora of inflammatory disorders, the effects of metabolic aberrations on TLRs and their role in diabetes and its complications is still emerging. In the present paper, we provide a systematic review on how TLRs play a detrimental role in the pathogenic processes [increased blood sugar, NEFAs (non-esterified 'free' fatty acids), cytokines and ROS (reactive oxygen species)] that manifest diabetes. Furthermore, we will highlight some of the therapeutic strategies targeted at decreasing TLRs to abrogate inflammation in diabetes that may eventually result in decreased complications.

Free fatty acids in the presence of high glucose amplify monocyte inflammation via Toll-like receptors.

Type 2 diabetes (T2DM) is characterized by hyperglycemia, dyslipidemia, and increased inflammation. Previously, we showed that high glucose (HG) induces Toll-like receptor (TLR) expression, activity, and inflammation via NF-κB followed by cytokine release in vitro and in vivo. Here, we determined how HG-induced inflammation is affected by free fatty acids (FFA) in human monocytes. THP-1 monocytic cells, CD14(+) human monocytes, and transiently transfected HEK293 cells were exposed to various FFA (0-500 µM) and glucose (5-20 mM) for evaluation of TLR2, TLR4, NF-κB, IL-1ß, monocyte chemoattractant protein-1 (MCP-1), and superoxide release. In THP-1 cells, palmitate increased cellular TLR2 and TLR4 expression, generated reactive oxygen species (ROS), and increased NF-κB activity, IL-1ß, and MCP-1 release in a dose- and time-dependent manner. Similar data were observed with stearate and FFA mixture but not with oleate. Conversely, NADPH oxidase inhibitor treatment repressed glucose- and palmitate-stimulated ROS generation and NF-κB activity and decreased IL-1ß and MCP-1 expression. Silencing TLR2, TLR4, and p47phox with small inhibitory RNAs (siRNAs) significantly reduced superoxide release, NF-κB activity, IL-1ß, and MCP-1 secretion in HG and palmitate-treated THP-1 cells. Moreover, data from transient transfection experiments suggest that TLR6 is required for TLR2 and MD2 for TLR4 to augment inflammation in FFA- and glucose-exposed cells. These findings were confirmed with human monocytes. We conclude that FFA exacerbates HG-induced TLR expression and activity in monocytic cells with excess superoxide release, enhanced NF-κB activity, and induced proinflammatory factor release.

TLR2 expression and signaling-dependent inflammation impair wound healing in diabetic mice.

Toll-like receptor-2 (TLR2) is a pivotal pathogen recognition receptor that has a key role in inflammation, diabetes, and injury. Hyperglycemia, inflammation, and oxidative stress induce TLR2-myeloid differentiation factor-88 (MyD88) expression and signaling, and are major pathophysiological mechanisms in the impaired diabetic wound-healing process. The aim of the study was to examine the contribution of TLR2-MyD88 expression and signaling to the prolonged inflammation observed in diabetic wounds. Diabetes was induced in male C57BL/6J and TLR2(-/-) mice using streptozotocin (STZ) with matching nondiabetic mice as control. In addition, nonobese diabetic (NOD) mice were used to represent the spontaneous type 1 diabetes condition. After 2 weeks of persistent hyperglycemia in the mice, full-thickness excision wounds were made on the backs aseptically. Total RNA and protein were subjected to real-time PCR and western blot analyses. Wound sizes were measured using digital planimetry. TLR2 mRNA and protein expression increased significantly in wounds of C57BL/6J+STZ and NOD mice (P<0.05) compared with nondiabetic C57BL/6J mice. MyD88 expression, interleukin receptor-associated kinase-1 phosphorylation, and nuclear factor-κ B (NF-κB) activation were increased in diabetic wounds compared with nondiabetic wounds. Wounds of TLR2(-/-)+STZ mice showed less oxidative stress, decreased MyD88 signaling, NF-κB activation, and cytokine secretion. The wound closure was significant in TLR2(-/-)+ STZ mice compared with C57BL/6J+STZ mice. Collectively, our findings show that increased TLR2 mRNA and protein expression, signaling, and activation contribute to the prolonged inflammation in the diabetic wounds and that absence of TLR2 may result in decreased inflammation and improved wound healing.

C-reactive protein adversely alters the protein-protein interaction of the endothelial isoform of nitric oxide synthase.

BACKGROUND: C-reactive protein (CRP) inhibits the activity of the endothelial isoform of nitric oxide synthase (eNOS) via uncoupling of the enzyme both in vitro and in vivo. eNOS activity appears to be related in part to its interaction with other cellular proteins, including heat shock protein 90 (Hsp90), caveolin-1, and porin. In this study, we examined the effect of CRP treatment of human aortic endothelial cells (HAECs) on eNOS interaction with caveolin-1, Hsp90, and porin. METHODS: We incubated HAECs with CRP (0, 12.5, and 25 mg/L) for 1, 6, or 24 h and assessed the interaction of these proteins with eNOS by immunoprecipitation and western blotting. RESULTS: CRP treatment (12.5 and 25 mg/L) of HAECs for 24 h significantly increased eNOS binding to caveolin-1 (40% and 54% increase, respectively; P < 0.05) and decreased binding to Hsp90 (33% and 66% decrease, respectively; P < 0.05). CRP (25 mg/L) also significantly decreased the binding of porin to eNOS (11% decrease, P < 0.05). Similar results were seen when HAECs were treated with CRP for 6 h. CONCLUSIONS: These negative protein-protein interactions of eNOS were able to partly explain the CRP-induced decreases in the activity of this critical enzyme, which caused endothelial dysfunction.

Diabetes is a proinflammatory state: a translational perspective.

The diabetic state confers an increased propensity to accelerated atherogenesis. Inflammation is pivotal in atherosclerosis; in addition to the established risk factors, inflammation appears to play a pivotal role in diabetes and its complications. Evidence for increased inflammation includes: increased levels of plasma C-reactive protein, the prototypic marker of inflammation; increased levels of plasminogen-activator inhibitor; increased monocyte superoxide and proinflammatory cytokine release (IL-1, IL-6 and TNF-alpha); increased monocyte adhesion to endothelium; increased NF-kappaB activity; and increased Toll-like receptor 2 and 4 expression and activity in diabetes. Thus, it appears that both Type 1 and Type 2 diabetes are proinflammatory states and that these could contribute to increased diabetic vasculopathies.

Increased toll-like receptor (TLR) activation and TLR ligands in recently diagnosed type 2 diabetic subjects.

OBJECTIVE: Individuals with type 2 diabetes have a myriad of metabolic aberrations including increased inflammation, increasing their cardiovascular risk. Toll-like receptors (TLRs) and their ligands play a key role in insulin resistance and atherosclerosis. However, there is a paucity of data examining the expression and activity of TLRs in type 2 diabetes. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression, their ligands, and signaling in monocytes of recently diagnosed type 2 diabetic patients. RESEARCH DESIGN AND METHODS: TLR mRNA, protein expression, TLR ligands, and TLR signaling were measured in freshly isolated monocytes from healthy human control subjects (n = 23) and type 2 diabetic subjects (n = 23) using real-time RT-PCR, Western blot, and flow cytometric assays. RESULTS: Type 2 diabetic subjects had significantly increased TLR2, TLR4 mRNA, and protein in monocytes compared with control subjects (P < 0.05). Increased TLR2 and TLR4 expression correlated with BMI, homeostasis model assessment-insulin resistance (HOMA-IR), glucose, A1C, N(epsilon)-(carboxymethyl) lysine (CML), and free fatty acid (FFA). Ligands of TLR2 and TLR4, namely, HSP60, HSP70, HMGB1, endotoxin, and hyaluronan levels, were elevated in type 2 diabetic subjects and positively correlated with TLR2 and TLR4. Type 2 diabetic subjects showed increased MyD88, phosphorylated IRAK-1, Trif, TICAM-1, IRF-3, and NF-kappaB p65 expression in monocytes compared with control subjects. Furthermore, TLR-MyD88-NF-kappaB signaling resulted in elevated levels of cytokines (P < 0.05), but increased interleukin (IL)-1beta, interferon (IFN)-gamma, and endotoxin were not significant when adjusted for BMI. CONCLUSIONS: In this comprehensive study, we make the novel observation that TLR2 and TLR4 expression and their ligands, signaling, and functional activation are increased in recently diagnosed type 2 diabetes and contribute to the proinflammatory state.

Pioglitazone inhibits Toll-like receptor expression and activity in human monocytes and db/db mice.

Toll-like receptors (TLRs) are key innate immune sensors of endogenous damage signals and play an important role in inflammatory diseases like diabetes and atherosclerosis. Pioglitazone (PIO), a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, has been reported to be an antiinflammatory agent. Thus, in the present study, we examined the antiinflammatory effects of PIO on TLR2 and TLR4 expression in human monocytes exposed to Pam3CSK4 (Pam; TLR2 ligand) and purified lipopolysaccharide (LPS; TLR4 ligand) using flow cytometry and real-time RT-PCR. Monocytes were isolated from healthy human volunteers and pretreated with PIO (1 microM) followed by Pam (170 ng/ml) and LPS (160 ng/ml) challenge. PIO significantly decreased Pam- and LPS-induced TLR2 (-56%) and TLR4 (-78%) expression (P < 0.05). In addition, PIO decreased TLR ligand-induced nuclear factor-kappaB activity (-63%), IL-1beta (-50%), IL-6 (-52%), monocyte chemoattractant protein-1(-83%), and TNF-alpha (-87%) compared with control. Next, PIO-treated db/db mice (n = 6/group) showed decreased TLR2 (-60%) and TLR4 (-45%) expression in peritoneal macrophages compared with vehicle control mice (P < 0.001) with associated decrease in MyD88-dependent signaling and nuclear factor-kappaB activation. Data suggest that Pam- and LPS-induced TLR2 and TLR4 expression are inhibited by PIO in human monocytes and db/db mice. Thus, we define a novel pathway by which PIO could induce antiinflammatory effects.

C-reactive protein stimulates superoxide anion release and tissue factor activity in vivo.

C-reactive protein (CRP), the prototypic marker of inflammation, is a cardiovascular risk marker and recent in vitro studies suggest that it may promote atherogenesis. CRP promotes oxidative stress in vitro and induces tissue factor (TF) release. However, there is a paucity of data examining the effects of CRP on oxidative stress and tissue factor procoagulant activity (PCA) in vivo. Thus, we tested the effects of CRP administration on superoxide anion release and tissue factor activity and examined mechanistic pathways using a rat sterile air pouch model. Intraperitoneal administration of CRP (20mg/kg body weight) compared to human serum albumin (HuSA) increased superoxide anion release and tissue factor activity from peritoneal macrophages in vivo (p<0.01). This was confirmed using intrapouch administration of CRP (25mug/mL) compared to HuSA. Pretreatment with reactive oxygen species (ROS) scavengers or protein kinase C (PKC) inhibitor significantly abrogated CRP-induced superoxide anion release and tissue factor activity. Pretreatment with extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) inhibitors, but not p38 mitogen-activated protein kinase (p38MAPK) significantly decreased CRP-induced superoxide anion release from macrophages in vivo. CRP-induced tissue factor activity in vivo was abrogated by pretreatment with inhibitors to p38MAPK, JNK and NFkappab (nuclear factor-kappab), but not ERK. Antibodies to Fc gamma receptors, CD32 and CD64 resulted in significant reduction in CRP-induced superoxide and tissue factor activity in vivo. Thus, CRP appears to induce oxidative stress in vivo by stimulating NADPH oxidase via PKC, ERK and JNK phosphorylation, and induces tissue factor PCA in vivo via upregulation of PKC, p38MAPK, JNK, ROS and NFkappab. CRP-induced ROS appears to precede tissue factor release. These effects are abrogated by blocking Fc gamma receptors, CD32 and CD64. This in vivo demonstration provides further evidence for a role for CRP in atherothrombosis.

Candesartan inhibits Toll-like receptor expression and activity both in vitro and in vivo.

INTRODUCTION: Toll-like receptors play an important role in the innate immune system and are found to be crucial in severe diseases like sepsis, atherosclerosis, and arthritis. TLR2 and TLR4 expression is upregulated in the inflammatory diseases. Angiotensin II in addition to stimulating vasoconstriction also induces an increase in ROS and a proinflammatory phenotype via AT(1)R. Angiotensin II type-1 receptor blocker (ARB), widely used as an antihypertensive drug, has been reported to also have anti-inflammatory effects. Thus, we investigated whether an ARB exerts anti-inflammatory effects via inhibiting TLR2 and TLR4 expression. METHODS AND RESULTS: Monocytes were isolated from healthy human volunteers and treated with the synthetic lipoprotein Pam3CSK4 or LPS in the absence or presence of candesartan. Pretreatment of human monocytes with candesartan significantly decreased Pam3CSK4 or LPS induced TLR2 and TLR4 expression of both mRNA and protein levels (P<0.05 vs. control) along with decrease in the activity of NF-kappaB and the expression of IL-1beta, IL-6, TNF-alpha, and MCP-1. Furthermore, candesartan treated mice show decreased TLR2 and TLR4 expression compared to vehicle control mice. CONCLUSION: Pam3CSK4 and LPS induced TLR2 and TLR4 expression at mRNA and protein levels are inhibited by candesartan both in vitro and in vivo. Thus, we define a novel pathway by which candesartan could induce anti-inflammatory effects.

High glucose induces toll-like receptor expression in human monocytes: mechanism of activation.

OBJECTIVE: Hyperglycemia-induced inflammation is central in diabetes complications, and monocytes are important in orchestrating these effects. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, there is a paucity of data examining the expression and activity of TLRs in hyperglycemic conditions. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression and mechanism of their induction in monocytic cells under high-glucose conditions. RESEARCH DESIGN AND METHODS: High glucose (15 mmol/l) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time- and dose-dependent manner (P < 0.05). High glucose increased TLR expression, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1, and nuclear factor-kappaB (NF-kappaB) p65-dependent activation in THP-1 cells. THP-1 cell data were further confirmed using freshly isolated monocytes from healthy human volunteers (n = 10). RESULTS: Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein (P < 0.05). Knocking down both TLR2 and TLR4 in the cells resulted in a 76% (P < 0.05) decrease in high-glucose-induced NF-kappaB activity, suggesting an additive effect. Furthermore, PKC-alpha knockdown decreased TLR2 by 61% (P < 0.05), whereas inhibition of PKC-delta decreased TLR4 under high glucose by 63% (P < 0.05). Small inhibitory RNA to p47Phox in THP-1 cells abrogated high-glucose-induced TLR2 and TLR4 expression. Additional studies revealed that PKC-alpha, PKC-delta, and p47Phox knockdown significantly abrogated high-glucose-induced NF-kappaB activation and inflammatory cytokine secretion. CONCLUSIONS: Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-alpha and PKC-delta, respectively, by stimulating NADPH oxidase in human monocytes.

Adiponectin decreases C-reactive protein synthesis and secretion from endothelial cells: evidence for an adipose tissue-vascular loop.

BACKGROUND AND OBJECTIVE: Inflammation is pivotal in atherosclerosis. C-reactive protein (CRP), in addition to being a cardiovascular risk marker, may also be proatherogenic. We have previously shown that in addition to the liver, human aortic endothelial cells (HAECs) synthesize and secrete CRP. Whereas CRP levels are increased in obesity, metabolic syndrome, and diabetes, levels of adiponectin are reduced in these conditions. We tested the hypothesis that adiponectin reduces CRP synthesis and secretion in HAECs under normoglycemic (5.5 mmol/L glucose) and hyperglycemic conditions (15 mmol/L glucose). METHODS AND RESULTS: Adiponectin dose-dependently reduced CRP mRNA and protein from HAECs. Adiponectin treatment of HAECs significantly decreased IkappaB phosphorylation and NFkappaB binding activity. There was no effect of adiponectin on STAT or C/EBP transcriptional activity. Adiponectin also activated AMP kinase resulting in decreased NFkappaB activity and decreased CRP mRNA and protein. These effects of adiponectin were mimicked by AICAR, an activator of AMPK, and reversed by inhibition of AMPK. Thus, adiponectin reduces CRP synthesis and secretion from HAECs under hyperglycemia via upregulation of AMP kinase and downregulation of NFkappaB. Similar findings were observed in rat primary hepatocytes. CONCLUSIONS: Thus, in obesity and diabetes, the hypoadiponectinemia could exacerbate the proinflammatory state by inducing CRP production.

Increased toll-like receptor (TLR) 2 and TLR4 expression in monocytes from patients with type 1 diabetes: further evidence of a proinflammatory state.

CONTEXT: Type 1 diabetes (T1DM) is associated with increased cardiovascular mortality. It is a pro-inflammatory state as evidenced by increased circulating biomarkers and monocyte activity. The toll-like receptors (TLRs) are pattern recognition receptors, expressed abundantly on monocytes. TLR2 and TLR4 are important in atherosclerosis. However, there is a paucity of data examining TLR2 and TLR4 expression in T1DM and examining its contribution to the proinflammatory state. OBJECTIVE: Thus, we examined TLR2 and TLR4 expression in monocytes from T1DM patients compared with controls (n = 31 per group). SETTING: The study was performed at the University of California Davis Medical Center. PATIENTS: Healthy controls (n = 31) and T1DM patients (n = 31) were included in the study. RESULTS: TLR2 and TLR4 surface expression and mRNA were significantly increased in T1DM monocytes compared with controls. Downstream targets of TLR, nuclear factor kappaB, myeloid differentiation factor 88, Trif, and phosphorylated IL-1 receptor-associated kinase were significantly up-regulated in T1DM. Finally, the release of IL-1beta and TNF-alpha was significantly increased in monocytes from T1DM compared with controls and correlated with TLR2 and TLR4 expression (P < 0.005). In addition, TLR2 and TLR4 expression was significantly correlated to glycosylated hemoglobin, carboxymethyllysine, and nuclear factor kappaB (P < 0.02). CONCLUSION: Thus, we make the novel observation that TLR2 and TLR4 expression and signaling are increased in T1DM and contribute to the proinflammatory state.

High glucose induces IL-1beta expression in human monocytes: mechanistic insights.

Previously, IL-1beta secretion from Type 2 diabetic patients has been shown to be increased compared with controls. In this study, we aimed to delineate the mechanism of IL-1beta induction under high-glucose (HG) conditions in human monocytes. THP-1 cells cultured in normal glucose were treated with increasing concentrations of d-glucose (10-25 mM) for 6-72 h. IL-1beta and IL-1 receptor antagonist levels were measured by ELISA and Western blots, whereas mRNA was quantitated by RT-PCR. Specific inhibitors and small interfering RNAs of PKC, p38, ERK1/2, NF-kappaB, and NADPH oxidase were used to determine the mediators in parallel experiments under HG conditions. IL-1beta-secreted protein, cellular protein, and mRNA increase under HG conditions is time and dose dependent, with maximum increase at 15 mM (48 h; P < 0.05). IL-1 receptor antagonist release was time and dose dependent, similar to IL-1beta expression pattern; however, the molar ratio of IL-1beta to IL-1RA was increased. Data from inhibitor and small interfering RNA experiments indicate that IL-1beta release under HG is mediated by PKC-alpha, via phosphorylation of p38 MAPK, and ERK1/2 leading to NF-kappaB activation, resulting in increased mRNA and protein for IL-1beta. At the same time, it appears that NADPH oxidase via p47phox activates NF-kappaB, resulting in increased IL-1beta secretion. Data suggest that, under HG conditions, monocytes release significantly higher amounts of IL-1beta through multiple mechanisms, further compounding the disease progression. Targeting signaling pathways mediating IL-1beta release could result in the amelioration of inflammation and possibly diabetic vasculopathies.

The biological effects of CRP are not attributable to endotoxin contamination: evidence from TLR4 knockdown human aortic endothelial cells.

C-reactive protein (CRP) is the prototypic marker of inflammation and a strong predictor of cardiovascular events in humans. There are questions regarding the validity of the biological effects reported for CRP, in spite of adherence to rigorous control measures minimizing endotoxin [lipopolysaccharide (LPS)] contamination in these in vitro studies. In this study, we addressed the key question of endotoxin contamination in CRP preparations using Toll-like receptor 4 (TLR4) knockdown endothelial cells. Human aortic endothelial cells (HAECs) transfected with prevalidated TLR4 small interfering RNA (siRNA) and scrambled siRNA controls were challenged with pleural fluid-derived CRP or LPS for 12-16 h. Secreted interleukin-6 (IL-6), IL-1beta, IL-8, and plasminogen activator inhibitor-1 (PAI-1) levels and endothelial Nitric oxide synthase (eNOS) activity were determined. TLR4 knockdown in HAECs significantly decreased LPS-induced IL-1beta, IL-6, and IL-8, whereas the stimulatory effects of CRP were similar in both scrambled control and TLR4 knockdown cells. Furthermore, CRP significantly stimulated PAI-1 levels in both control and TLR4-transfected cells and inhibited eNOS activity, whereas LPS effects were negated in TLR4-transfected cells. The data presented cogently demonstrate and further confirm that the biological effects of CRP on HAECs are independent of LPS and thus are attributable to native protein per se. This is the first study to positively authenticate the significance of earlier in vitro reports on CRP biological effects.