Caveolin-1 Deficiency Dampens Toll-Like Receptor 4 Signaling through eNOS Activation

Caveolin-1 (Cav1), the scaffolding protein of caveolae, has been shown to play an important role in host defense and inflammation. However, the underlying molecular basis for these actions remains elusive. Here, using double mutant mice with genetic deletions of Cav1 and NOS3, we show that chronic endothelial nitric oxide synthase (eNOS) activation secondary to loss of Cav1 serves a crucial immunomodulatory function through tyrosine nitration-mediated impairment of interleukin-1 receptor associated kinase (IRAK)4, a signaling component required for nuclear factor-κB activation and innate immunity. We observed an eNOS-dependent decrease in the plasma concentration of pro-inflammatory cytokines and marked improvement of survival in Cav1^−/− mice following lipopolysaccharide challenge. Activation of eNOS secondary to loss of Cav1 resulted in decreased activation of nuclear factor-κB in response to lipopolysaccharide challenge, and thereby protected the animals from lipopolysaccharide-induced lung injury. IRAK4 was prominently nitrated in Cav1-deficient endothelial cells, whereas eNOS deletion in Cav1-deficient endothelial cells resulted in marked decrease of IRAK4 nitration and restored the inflammatory response after lipopolysaccharide challenge. Furthermore, in vitro nitration of IRAK4 resulted in impairment of the kinase activity. Thus, eNOS activation secondary to loss of Cav1 signals dampening of the innate immune response to lipopolysaccharide through IRAK4 nitration and the resultant impairment of kinase activity, and consequently mitigates inflammatory lung injury.Regulation of innate immunity, the primary line of defense against non-self, plays a key role in limiting disease.^1,2 The well-being of higher eukaryotes depends on the appropriate initiation and termination of the immune response.² Innate immune responses are comprised of phagocytosis of bacteria by macrophages and neutrophils, release of antimicrobial peptides, hydrolytic enzymes, and reactive oxygen intermediates by phagocytes, activation of complement system, and cytotoxic activity of natural killer cells against infected target cells.³ These rapid and nonspecific responses are elicited by surveillance of Gram-negative bacterial lipopolysaccharide (LPS), Gram-positive bacterial peptidoglycans and lipoteichoic acid-associated molecules, mycobacterial muramyl dipeptides, fungal glucans, and bacterial CpG-rich nucleotides.⁴ Interactions between pathogens and their hosts are initiated by the activation of pathogen recognition receptors, the toll-like receptors (TLR). As a potent activator of innate immunity, LPS activates TLR4 signaling, a critical event in the immune response to Gram-negative bacteria and in the etiology of endotoxic shock and acute lung injury.^5,6TLR4 binding by LPS recruits the adaptor molecule MyD88 through the TIR domain of TLR4 to initiate either MyD88-dependent or MyD88-independent pathways.^6,7 MyD88 recruits serine-threonine kinases interleukin(IL)-1R-associated kinase (IRAK)4 and IRAK1.^8,9 IRAK4 then phosphorylates IRAK1 resulting in recruitment of TRAF6 to the receptor complex¹⁰ and activation of transforming growth factor-β-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase family.¹¹ The activation of TAK1 leads to the activation of nuclear factor (NF)-κB, a regulator of immunity and inflammation,^12,13 which in return results in production of an array of pro-inflammatory cytokines, chemokines, and adhesive molecules, such as tumor necrosis factor (TNF)-α, macrophage inflammatory protein 1α (MIP-1α), and intercellular adhesion molecule (ICAM)-1.^2,14,15 The critical role of IRAK4 in TLR-mediated pathways was demonstrated in IRAK4 null mice and in humans with IRAK4 deficiency.^8,16,17 IRAK4 deficiency resulted in a severe impairment of TLR signaling.Nitric oxide (NO) plays an important role in host-defense and inflammation.^18,19,20 NO exerts its effect on innate immunity by direct antimicrobial activity and indirectly through reaction with reactive oxygen species and formation of antimicrobial metabolites such as peroxynitrite and nitrogen dioxide.¹⁸ The antimicrobial activity is mainly mediated by inducible NO synthase (iNOS)-derived NO. Endothelial NOS (eNOS)-derived NO blocks platelet and neutrophil activation, serves as a regulator of leukocyte recruitment, inhibits several features of mast cell-induced inflammation.^21,22 eNOS activity and NO release is mainly regulated by post-translational modifications by fatty acid and phosphorylation as well as protein–protein interaction with other effector molecules including heat shock protein 90 and caveolin-1.²³ Caveolin-1 (Cav1) is the scaffolding protein of caveolae in many non-muscle cell types including endothelial cells.²⁴ Cav1 binding to eNOS in the basal state suppresses eNOS activity, whereas on agonist activation, eNOS dissociates from Cav1 and synthesizes NO.²⁵ Plasma NO levels were markedly increased in Cav1^−/− mice.²⁶ Cav1-deficient mice exhibited multiple phenotypes: pulmonary hypertension, cardiomyopathy, pulmonary hypercellularity, and resistance to LPS-induced acute lung injury.^26,27,28,29 The pathology may be ascribed to the interaction of Cav1 with signaling molecules such as receptor and non-receptor tyrosine kinase receptors, G protein-coupled receptors, GTPases, and components of the mitogen-activated protein kinase, and eNOS.³⁰ Our recent study has shown that persistent eNOS activation secondary to Cav1 deficiency induces pulmonary hypertension through protein kinase G nitration and resultant impairment of its kinase activity.³¹ However, the precise pathophysiological role of Cav1-eNOS interaction in modulating innate immunity and LPS-induced inflammatory lung injury remains unclear because the approaches taken have used nonselective inhibitors. Here, using mice with genetic deletions of both Cav1 and NOS3 (DKO), we demonstrate the critical in vivo role of Cav1 in regulating the lung’s innate immune response to LPS by its ability to modulate the production of eNOS-derived NO. To our knowledge, we provide the first evidence that IRAK4 kinase activity is negatively regulated by tyrosine nitration. We showed that the decreased NF-κB activation and inflammatory lung injury seen in Cav1^−/− mice in response to LPS challenge was mediated by eNOS activation-dependent IRAK4 nitration.     

Differential expression of interleukin‐17 family cytokines in intact and complicated human atherosclerotic plaques

In addition to the classical TH1 and TH2 cytokines, members of the recently identified IL‐17 cytokine family play an important role in regulating cellular and humoral immune responses. At present nothing is known about the role of these cytokines in atherosclerosis. Expression of IL‐17A, ‐E and ‐F was investigated in atherosclerotic tissue by rtPCR and immunohistochemistry. IL‐17E and its receptor were further studied in cultured smooth muscle cells and endothelial cells, using rtPCR and western blot. rtPCR showed that IL‐17A, ‐E and ‐F were expressed in the majority of plaques under investigation. IL‐17A/F was expressed by mast cells in all stages of plaque development. IL‐17A/F⁺ neutrophils were always observed in complicated plaques, but hardly in intact lesions. IL‐17A/F⁺ Tcells (‘TH17’) were never observed. IL‐17E was expressed by smooth muscle cells and endothelial cells in both normal and atherosclerotic arteries, and in advanced plaques also extensively by mature B cells. Cultured smooth muscle cells and endothelial cells were found to express both IL‐17E and its functional receptor (IL‐17RB). The constitutive expression of IL‐17E by resident plaque cells, and the additional presence of IL‐17E⁺ B cells and IL‐17A/F⁺ neutrophils in advanced and complicated plaques indicates a complex contribution of IL‐17 family cytokines in human atherosclerosis, depending on the stage and activity of the disease. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Epstein Barr virus specific T-cells generated from unstable human atherosclerotic lesions: Implications for plaque inflammation

OBJECTIVE: T-cell activation is an essential feature of atherosclerotic plaque inflammation, which eventually may lead to plaque rupture. In this study, we investigated if EBV, a common herpes virus, is capable of stimulating atherosclerotic plaque derived T-cells and thus could contribute to atherosclerotic plaque inflammation. METHODS: Plaque derived T-cell cultures were established from symptomatic carotid atherosclerotic plaques of 19 patients. B-cells from the same patients were transformed with EBV to form lymphoblastoid cell lines (B-LCL) that served as antigen presenting cells. The proliferation of T-cells in the presence of autologous B-LCL was analyzed using 3H-thymidine incorporation. The presence of EBV in atherosclerotic material was analyzed by PCR. RESULTS: Of the 19 cell obtained T-cell cultures, 11 responded to EBV (58%, mean stimulation index: 10.1+/-3.1). PCR analysis showed that EBV DNA was present in 15 of the tissue samples (79%). All the specimens that contained EBV responding T-cells also contained EBV. EBV specific T-cells secreted granzymes, as indication of functional cytotoxic potential. CONCLUSIONS: EBV-specific cytotoxic T-cells and EBV DNA can be frequently observed in human atherosclerotic plaques. This suggests that a T-cell response against EBV could contribute to plaque inflammation, and thus to the onset of acute clinical symptoms.     

The oral implications of caustic soda ingestion in children

The morbidity related to caustic soda (sodium hydroxide) ingestion is well described in the literature. The majority of publications have concentrated on the effects to the trachea and gastrointestinal tract, with little reference to the oral and peri-oral areas. Accidental ingestion of sodium hydroxide-containing substances is fortunately rare; however the consequences can be devastating. Three cases of children who drank caustic substances are described. Treatment included fitting splints, injecting steroids, local surgical procedures, and the use of dynamic appliances to maintain mouth opening. Despite these interventions, all patients developed severe scarring, resulting in stenosis of the oral musculature and extra-articular ankylosis. There is perhaps a role for further investigation of early use of antiproliferative agents to prevent scarring, more aggressive surgery, and long-term physiotherapy appliance use. These patients require lifelong follow-up.     

A participatory program evaluation of a systems change program to improve access to information technology by people with disabilities

Purpose. To pilot-test and evaluate an innovative program providing information technology (IT) access to people with disabilities transitioning out of nursing homes into the community using a participatory approach.

Methods. Pre- and post-training data was collected on the 61 program participants to reflect three broad areas related to the IT training experience: performance; self-efficacy; importance, satisfaction and control. Additionally, semi-structured interviews were conducted with seven participants and five members of the program staff to explore environmental barriers to IT access for this group and the efficacy of the program in addressing these barriers. Data analyses followed a mixed methods approach incorporating both qualitative and quantitative techniques.

Results. Participants showed substantive changes in different spheres of IT use after completion of training. Post-training changes were significant particularly in areas related to self-efficacy, importance and satisfaction pertaining to use of IT. Qualitative findings substantiated the quantitative results and also revealed the numerous barriers to IT access that persons with disabilities continue to face within their communities.

Conclusion. Results indicate the feasibility, effectiveness and value of IT access to people with disabilities particularly those transitioning from institutional life to community living. Further action research aimed at increasing IT access for this group within local communities and neighborhoods is needed to address this issue at a broader societal level.     

Balance performance in three forms of multiple sclerosis

OBJECTIVES: To compare and document balance performance between patients with multiple sclerosis (MS) and healthy control subjects and balance performance among patients with different MS forms using a set of clinical balance tests. MATERIAL AND METHODS: Twenty eight primary progressive (PPMS), 34 secondary progressive (SPMS), and 62 relapsing remitting (RRMS), totalling 124 MS patients were included in the present study. Results from patients were compared with those of 31 healthy control subjects matching in age, gender, weight and height. Ashworth scale, mini-mental state examination and motricity index were used consecutively to evaluate spasticity, cognitive impairment and lower extremity muscle strength. Vision, sensation, proprioception, cerebellar and vestibular tests were also performed on the patients. The balance performance was evaluated using a set of clinical tests including steady stance tests (eyes in opened and closed positions, feet apart, feet together, stride stance, tandem stance and single stance), self-generated perturbations (functional reach, arm raise and step test), external perturbations, Tinetti-gait and 10 m gait time tests. RESULTS: There were no differences in age, sex, weight, height, sense impairment and lower extremity strength in patients with the three MS forms (p>0.05). No difference was found among patients with the three MS forms and the control subjects in the test of eyes closed with feet apart (p>0.05). The PPMS patients in all the balance tests except tests of eyes closed with feet apart and eyes opened with feet together, SPMS patients in all the balance tests except that of eyes closed with feet apart and RRMS patients in tandem stance, single leg stance, self-generated perturbations, external perturbations, Tinetti-gait and 10 m gait time tests had weaker balance than the control subjects (p<0.001). There were some differences between patients in the PPMS and SPMS groups in the eyes closed and feet apart test, between patients in the PPMS and RRMS groups in all the balance tests except eyes closed and feet apart and eyes opened and feet together tests and between patients in SPMS and RRMS group in all the balance tests except right and left arm raised tests (p<0.001). CONCLUSION: Balance in MS patients is impaired. The results of the present study show that there is more impairment in progressive MS forms than in RRMS. Meanwhile, patients with progressive MS are more likely to fall.     

Stability of CSF beta-amyloid(1-42) and tau levels by APOE genotype in Alzheimer patients

BACKGROUND: Cerebrospinal fluid (CSF) measures of beta-amyloid(1-42 )and tau differ between patients with Alzheimer's Disease (AD) and elderly normal controls. The effect of time and APOE genotype on these biomarkers continues to be elucidated. METHODS: We assessed CSF beta-amyloid(1-42) and tau in 20 mild-to-moderate AD patients, 11 APOE epsilon4+ and 9 APOE epsilon4-, over a mean time of 3.8 years (range 1-11.1 years). RESULTS: Over the period measured, CSF beta-amyloid(1-42) levels were lower in APOE epsilon4+ compared to APOE epsilon4- patients, and the levels decreased over time. Tau levels were stable over time and did not show an effect of APOE allele. CONCLUSIONS: While this is a limited clinical sample, the further decrease in CSF beta-amyloid(1-42 )(i.e., more abnormal) combined with the CSF tau stability over a mean period of almost 4 years suggests that beta-amyloid(1-42 )and tau maintain their potential usefulness as diagnostic biomarkers over time. These findings should be taken into account if CSF beta-amyloid(1-42) and tau are used as measures of treatment response.     

Importance of the immune system in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) are a heterogeneous group of tumors, mainly caused by exposure to cigarette smoke and/or alcohol. In recent years, a virally driven subset of cancers driven by human papillomavirus subtype 16 [HPV‐16]) has emerged. Our own data and data from other groups have demonstrated the favorable clinical outcome of HPV‐driven oropharyngeal tumors and in both HPV+ and HPV? cancers the importance of a high density of tumor‐associated lymphocytes for survival. These data underpin manipulation and activation of the patients' immune system by treatment, and as a result immunotherapy is rapidly taking its place in the management of HNSCC. Here we review the role the immune system in relation to HNSCC and consider the implications these have for HNSCC immunotherapy. Studies to quantify survival benefits and treatment‐associated toxicities are ongoing.