Investigations into the role of Toll-Like Receptors in the modulation of the inflammatory response during Osteoarthritis

Osteoarthritis (OA) is a multifactorial, painful and disabling disease that affects millions of people globally, with a largely unknown aetiology. OA remains undiagnosed until it becomes symptomatic with advanced structural alterations evident, thus joint replacement may be required. OA is now considered a whole-joint inflammatory disease, associated with synovitis of the fibroblast-like synoviocytes (FLS). FLS are sentinel cells that contribute to OA pathogenesis, through secretion of various catabolic and pro-inflammatory mediators, though the downstream stimuli which initiate and propagate the inflammatory pathway remain poorly defined. Activation of the innate immune Toll-Like Receptors (TLRs) leads to the induction of inflammatory mediators and cellular infiltration seen in most of the joint arthropathies, though the role of TLRs in OA is poorly understood. The aim of this research work was to characterise the role and functionality of TLRs in OA and to identify the key TLRs that modulate OA pathology. Interestingly, we found that TLR3, activated by dsRNA and endogenous alarm signals contained in the OA synovial fluid (SF), plays a key role in OA and this was confirmed by neutralisation of TLR3 expression which shifted the balance from pro-inflammatory to an anti-inflammatory cytokine milieu. Next using a proteomic approach, we found that prohibitin 1 (PHB1), an anti-proliferative molecule, was drastically down-regulated in FLS upon Poly(I:C) stimulation and this was validated through confocal and immunoblot analysis. Thus, PHB1 may be considered as a potential biomarker for tracing RNA borne synovial hyperplasia, indicative of synovitis which directly implies for OA severity and progression. Following proteomic analysis of grade-specific whole synovial tissue, suppression of key complement C3b in grade-2 OA, was evident. Furthermore, grade-specific OA-SF showed an ability to predominantly induce IFNβ in FLS and in HEK293-TLR3 cells in a TLR3 dependent manner. Neutralisation of TLR3 significantly inhibited IFNβ production, probably through regulation/blockade of downstream signalling cascades of OA-SF-induced persistent TLR3 activation. Further, luciferase reporter gene assays have suggested that, this effect may be mediated through the transcription factors IRF3 and IRF9, leading to sustained activation of IFNβ genes. Therefore, TLR3 blockade in FLS may inhibit OA-SF-induced activation of TLR3 and concomitant induction of IFNβ. Likewise, TLR3 blockade also inhibited RANTES production, primarily through blocking of NF-κB. Together, these data indicate that TLR3, expressed on the plasma membrane of FLS, may be a critical target for OA disease intervention. In conclusion, our data suggests, for the first time that, TLR3 hyper-activation plays a key role in perpetuating synovial inflammation in OA and suggests that therapeutic intervention of OA may be achieved through TLR3 blockade. Despite the significant advances in the understanding and management of OA, significant research must still be undertaken before clinicians can guarantee a quality of life for OA patients, which is free of the debilitating pain. We hope that our efforts would, at least in part, contribute to a better understanding of the pathogenic molecular mechanisms that drive this chronic inflammatory disease. The provision of better treatments will thereby improve the quality of life for patients whose lives are marred by OA and related inflammatory diseases

Nuclear Factor κB Subunits RelB and cRel Negatively Regulate Toll-like Receptor 3-mediated β-Interferon Production via Induction of Transcriptional Repressor Protein YY1

The induction of β-interferon (IFN-β) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-β requires the co-operative action of the transcription factors IRF-3/7, NF-κB, and ATF-2/c-Jun on the IFN-β promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-κB and promote its binding to the IFN-β promoter, recent studies have indicated that NF-κB is not essential for virus-induced expression of IFN-β. Herein, we examined the role of NF-κB in regulating IFN-β expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-κB pathway augments late phase expression of IFN-β expression in response to TLR3 stimulation. We show that the negative effect of NF-κB on IFN-β expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-β promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-κB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-β expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-β expression

Nuclear Factor κB Subunits RelB and cRel Negatively Regulate Toll-like Receptor 3-mediated β-Interferon Production via Induction of Transcriptional Repressor Protein YY1

The induction of β-interferon (IFN-β) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-β requires the co-operative action of the transcription factors IRF-3/7, NF-κB, and ATF-2/c-Jun on the IFN-β promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-κB and promote its binding to the IFN-β promoter, recent studies have indicated that NF-κB is not essential for virus-induced expression of IFN-β. Herein, we examined the role of NF-κB in regulating IFN-β expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-κB pathway augments late phase expression of IFN-β expression in response to TLR3 stimulation. We show that the negative effect of NF-κB on IFN-β expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-β promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-κB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-β expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-β expression

Investigations into the role of Toll-Like Receptors in the modulation of the inflammatory response during Osteoarthritis

Osteoarthritis (OA) is a multifactorial, painful and disabling disease that affects millions of people globally, with a largely unknown aetiology. OA remains undiagnosed until it becomes symptomatic with advanced structural alterations evident, thus joint replacement may be required. OA is now considered a whole-joint inflammatory disease, associated with synovitis of the fibroblast-like synoviocytes (FLS). FLS are sentinel cells that contribute to OA pathogenesis, through secretion of various catabolic and pro-inflammatory mediators, though the downstream stimuli which initiate and propagate the inflammatory pathway remain poorly defined. Activation of the innate immune Toll-Like Receptors (TLRs) leads to the induction of inflammatory mediators and cellular infiltration seen in most of the joint arthropathies, though the role of TLRs in OA is poorly understood. The aim of this research work was to characterise the role and functionality of TLRs in OA and to identify the key TLRs that modulate OA pathology. Interestingly, we found that TLR3, activated by dsRNA and endogenous alarm signals contained in the OA synovial fluid (SF), plays a key role in OA and this was confirmed by neutralisation of TLR3 expression which shifted the balance from pro-inflammatory to an anti-inflammatory cytokine milieu. Next using a proteomic approach, we found that prohibitin 1 (PHB1), an anti-proliferative molecule, was drastically down-regulated in FLS upon Poly(I:C) stimulation and this was validated through confocal and immunoblot analysis. Thus, PHB1 may be considered as a potential biomarker for tracing RNA borne synovial hyperplasia, indicative of synovitis which directly implies for OA severity and progression. Following proteomic analysis of grade-specific whole synovial tissue, suppression of key complement C3b in grade-2 OA, was evident. Furthermore, grade-specific OA-SF showed an ability to predominantly induce IFNβ in FLS and in HEK293-TLR3 cells in a TLR3 dependent manner. Neutralisation of TLR3 significantly inhibited IFNβ production, probably through regulation/blockade of downstream signalling cascades of OA-SF-induced persistent TLR3 activation. Further, luciferase reporter gene assays have suggested that, this effect may be mediated through the transcription factors IRF3 and IRF9, leading to sustained activation of IFNβ genes. Therefore, TLR3 blockade in FLS may inhibit OA-SF-induced activation of TLR3 and concomitant induction of IFNβ. Likewise, TLR3 blockade also inhibited RANTES production, primarily through blocking of NF-κB. Together, these data indicate that TLR3, expressed on the plasma membrane of FLS, may be a critical target for OA disease intervention. In conclusion, our data suggests, for the first time that, TLR3 hyper-activation plays a key role in perpetuating synovial inflammation in OA and suggests that therapeutic intervention of OA may be achieved through TLR3 blockade. Despite the significant advances in the understanding and management of OA, significant research must still be undertaken before clinicians can guarantee a quality of life for OA patients, which is free of the debilitating pain. We hope that our efforts would, at least in part, contribute to a better understanding of the pathogenic molecular mechanisms that drive this chronic inflammatory disease. The provision of better treatments will thereby improve the quality of life for patients whose lives are marred by OA and related inflammatory diseases

Investigations into the role of Toll-Like Receptors in the modulation of the inflammatory response during Osteoarthritis

Osteoarthritis (OA) is a multifactorial, painful and disabling disease that affects millions of people globally, with a largely unknown aetiology. OA remains undiagnosed until it becomes symptomatic with advanced structural alterations evident, thus joint replacement may be required. OA is now considered a whole-joint inflammatory disease, associated with synovitis of the fibroblast-like synoviocytes (FLS). FLS are sentinel cells that contribute to OA pathogenesis, through secretion of various catabolic and pro-inflammatory mediators, though the downstream stimuli which initiate and propagate the inflammatory pathway remain poorly defined. Activation of the innate immune Toll-Like Receptors (TLRs) leads to the induction of inflammatory mediators and cellular infiltration seen in most of the joint arthropathies, though the role of TLRs in OA is poorly understood. The aim of this research work was to characterise the role and functionality of TLRs in OA and to identify the key TLRs that modulate OA pathology. Interestingly, we found that TLR3, activated by dsRNA and endogenous alarm signals contained in the OA synovial fluid (SF), plays a key role in OA and this was confirmed by neutralisation of TLR3 expression which shifted the balance from pro-inflammatory to an anti-inflammatory cytokine milieu. Next using a proteomic approach, we found that prohibitin 1 (PHB1), an anti-proliferative molecule, was drastically down-regulated in FLS upon Poly(I:C) stimulation and this was validated through confocal and immunoblot analysis. Thus, PHB1 may be considered as a potential biomarker for tracing RNA borne synovial hyperplasia, indicative of synovitis which directly implies for OA severity and progression. Following proteomic analysis of grade-specific whole synovial tissue, suppression of key complement C3b in grade-2 OA, was evident. Furthermore, grade-specific OA-SF showed an ability to predominantly induce IFNβ in FLS and in HEK293-TLR3 cells in a TLR3 dependent manner. Neutralisation of TLR3 significantly inhibited IFNβ production, probably through regulation/blockade of downstream signalling cascades of OA-SF-induced persistent TLR3 activation. Further, luciferase reporter gene assays have suggested that, this effect may be mediated through the transcription factors IRF3 and IRF9, leading to sustained activation of IFNβ genes. Therefore, TLR3 blockade in FLS may inhibit OA-SF-induced activation of TLR3 and concomitant induction of IFNβ. Likewise, TLR3 blockade also inhibited RANTES production, primarily through blocking of NF-κB. Together, these data indicate that TLR3, expressed on the plasma membrane of FLS, may be a critical target for OA disease intervention. In conclusion, our data suggests, for the first time that, TLR3 hyper-activation plays a key role in perpetuating synovial inflammation in OA and suggests that therapeutic intervention of OA may be achieved through TLR3 blockade. Despite the significant advances in the understanding and management of OA, significant research must still be undertaken before clinicians can guarantee a quality of life for OA patients, which is free of the debilitating pain. We hope that our efforts would, at least in part, contribute to a better understanding of the pathogenic molecular mechanisms that drive this chronic inflammatory disease. The provision of better treatments will thereby improve the quality of life for patients whose lives are marred by OA and related inflammatory diseases

Investigations into the role of Toll-Like Receptors in the modulation of the inflammatory response during Osteoarthritis

Osteoarthritis (OA) is a multifactorial, painful and disabling disease that affects millions of people globally, with a largely unknown aetiology. OA remains undiagnosed until it becomes symptomatic with advanced structural alterations evident, thus joint replacement may be required. OA is now considered a whole-joint inflammatory disease, associated with synovitis of the fibroblast-like synoviocytes (FLS). FLS are sentinel cells that contribute to OA pathogenesis, through secretion of various catabolic and pro-inflammatory mediators, though the downstream stimuli which initiate and propagate the inflammatory pathway remain poorly defined. Activation of the innate immune Toll-Like Receptors (TLRs) leads to the induction of inflammatory mediators and cellular infiltration seen in most of the joint arthropathies, though the role of TLRs in OA is poorly understood. The aim of this research work was to characterise the role and functionality of TLRs in OA and to identify the key TLRs that modulate OA pathology. Interestingly, we found that TLR3, activated by dsRNA and endogenous alarm signals contained in the OA synovial fluid (SF), plays a key role in OA and this was confirmed by neutralisation of TLR3 expression which shifted the balance from pro-inflammatory to an anti-inflammatory cytokine milieu. Next using a proteomic approach, we found that prohibitin 1 (PHB1), an anti-proliferative molecule, was drastically down-regulated in FLS upon Poly(I:C) stimulation and this was validated through confocal and immunoblot analysis. Thus, PHB1 may be considered as a potential biomarker for tracing RNA borne synovial hyperplasia, indicative of synovitis which directly implies for OA severity and progression. Following proteomic analysis of grade-specific whole synovial tissue, suppression of key complement C3b in grade-2 OA, was evident. Furthermore, grade-specific OA-SF showed an ability to predominantly induce IFNβ in FLS and in HEK293-TLR3 cells in a TLR3 dependent manner. Neutralisation of TLR3 significantly inhibited IFNβ production, probably through regulation/blockade of downstream signalling cascades of OA-SF-induced persistent TLR3 activation. Further, luciferase reporter gene assays have suggested that, this effect may be mediated through the transcription factors IRF3 and IRF9, leading to sustained activation of IFNβ genes. Therefore, TLR3 blockade in FLS may inhibit OA-SF-induced activation of TLR3 and concomitant induction of IFNβ. Likewise, TLR3 blockade also inhibited RANTES production, primarily through blocking of NF-κB. Together, these data indicate that TLR3, expressed on the plasma membrane of FLS, may be a critical target for OA disease intervention. In conclusion, our data suggests, for the first time that, TLR3 hyper-activation plays a key role in perpetuating synovial inflammation in OA and suggests that therapeutic intervention of OA may be achieved through TLR3 blockade. Despite the significant advances in the understanding and management of OA, significant research must still be undertaken before clinicians can guarantee a quality of life for OA patients, which is free of the debilitating pain. We hope that our efforts would, at least in part, contribute to a better understanding of the pathogenic molecular mechanisms that drive this chronic inflammatory disease. The provision of better treatments will thereby improve the quality of life for patients whose lives are marred by OA and related inflammatory diseases

Investigations into the role of Toll-Like Receptors in the modulation of the inflammatory response during Osteoarthritis

Osteoarthritis (OA) is a multifactorial, painful and disabling disease that affects millions of people globally, with a largely unknown aetiology. OA remains undiagnosed until it becomes symptomatic with advanced structural alterations evident, thus joint replacement may be required. OA is now considered a whole-joint inflammatory disease, associated with synovitis of the fibroblast-like synoviocytes (FLS). FLS are sentinel cells that contribute to OA pathogenesis, through secretion of various catabolic and pro-inflammatory mediators, though the downstream stimuli which initiate and propagate the inflammatory pathway remain poorly defined. Activation of the innate immune Toll-Like Receptors (TLRs) leads to the induction of inflammatory mediators and cellular infiltration seen in most of the joint arthropathies, though the role of TLRs in OA is poorly understood. The aim of this research work was to characterise the role and functionality of TLRs in OA and to identify the key TLRs that modulate OA pathology. Interestingly, we found that TLR3, activated by dsRNA and endogenous alarm signals contained in the OA synovial fluid (SF), plays a key role in OA and this was confirmed by neutralisation of TLR3 expression which shifted the balance from pro-inflammatory to an anti-inflammatory cytokine milieu. Next using a proteomic approach, we found that prohibitin 1 (PHB1), an anti-proliferative molecule, was drastically down-regulated in FLS upon Poly(I:C) stimulation and this was validated through confocal and immunoblot analysis. Thus, PHB1 may be considered as a potential biomarker for tracing RNA borne synovial hyperplasia, indicative of synovitis which directly implies for OA severity and progression. Following proteomic analysis of grade-specific whole synovial tissue, suppression of key complement C3b in grade-2 OA, was evident. Furthermore, grade-specific OA-SF showed an ability to predominantly induce IFNβ in FLS and in HEK293-TLR3 cells in a TLR3 dependent manner. Neutralisation of TLR3 significantly inhibited IFNβ production, probably through regulation/blockade of downstream signalling cascades of OA-SF-induced persistent TLR3 activation. Further, luciferase reporter gene assays have suggested that, this effect may be mediated through the transcription factors IRF3 and IRF9, leading to sustained activation of IFNβ genes. Therefore, TLR3 blockade in FLS may inhibit OA-SF-induced activation of TLR3 and concomitant induction of IFNβ. Likewise, TLR3 blockade also inhibited RANTES production, primarily through blocking of NF-κB. Together, these data indicate that TLR3, expressed on the plasma membrane of FLS, may be a critical target for OA disease intervention. In conclusion, our data suggests, for the first time that, TLR3 hyper-activation plays a key role in perpetuating synovial inflammation in OA and suggests that therapeutic intervention of OA may be achieved through TLR3 blockade. Despite the significant advances in the understanding and management of OA, significant research must still be undertaken before clinicians can guarantee a quality of life for OA patients, which is free of the debilitating pain. We hope that our efforts would, at least in part, contribute to a better understanding of the pathogenic molecular mechanisms that drive this chronic inflammatory disease. The provision of better treatments will thereby improve the quality of life for patients whose lives are marred by OA and related inflammatory diseases

Analysis of Inflammatory Cytokines in Pre-Diabetic Subjects

Objective: Pre-diabetes is defined as either impaired fasting glucose ((IFG) between 5.6 and 6.9mmol/l) or impaired glucose tolerance (IGT) wherein fasting or post-prandial plasma glucose levels are above normal levels, but below that of clinical Type-2-Diabetes Mellitus (T2DM). Both IFG and IGT, risk factors for T2DM and macrovascular diseases, have previously been linked with inflammation. With this in mind, we sought to comparatively evaluate the levels of inflammatory markers in pre-diabetics relative to normal healthy individuals. Methods: We determined the levels of serum cytokines in a cohort of 9 patients with pre-diabetes and thirty four individuals with normal glucose control using a 96-Well Multi-array 7-plex assay or 1-plex IFN-β and Rantes 96-well plates (Meso Scale Discovery, Gaithersburg, Maryland, USA). Results: Our study demonstrated that the patient group with pre-diabetes exhibited a non-significant trend towards elevated IL-6, TNFα, IFN-β, IL-12, Rantes, IL-10 and IL-8 when compared with the healthy controls group. After adjustment for age, sex, BMI and WHR, in the study population, there was no difference in the levels of cytokines between the pre-diabetes and normal groups. Conclusion: Measurement of serum cytokine levels alone is unlikely to be a predictor of clinical disease activity in individuals with prediabetes

Nuclear Factor κB Subunits RelB and cRel Negatively Regulate Toll-like Receptor 3-mediated β-Interferon Production via Induction of Transcriptional Repressor Protein YY1

The induction of β-interferon (IFN-β) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-β requires the co-operative action of the transcription factors IRF-3/7, NF-κB, and ATF-2/c-Jun on the IFN-β promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-κB and promote its binding to the IFN-β promoter, recent studies have indicated that NF-κB is not essential for virus-induced expression of IFN-β. Herein, we examined the role of NF-κB in regulating IFN-β expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-κB pathway augments late phase expression of IFN-β expression in response to TLR3 stimulation. We show that the negative effect of NF-κB on IFN-β expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-β promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-κB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-β expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-β expression