Trang T. Duong

TNF-α Is Necessary for Induction of Coronary Artery Inflammation and Aneurysm Formation in an Animal Model of Kawasaki Disease

Kawasaki disease is the most common cause of multisystem vasculitis in childhood. The resultant coronary artery lesions make Kawasaki disease the leading cause of acquired heart disease in children in the developed world. TNF-α is a pleiotropic inflammatory cytokine elevated during the acute phase of Kawasaki disease. In this study, we report rapid production of TNF-α in the peripheral immune system after disease induction in a murine model of Kawasaki disease. This immune response becomes site directed, with migration to the coronary arteries dependent on TNF-α-mediated events. Production of TNF-α in the heart is coincident with the presence of inflammatory infiltrate at the coronary arteries, which persists during development of aneurysms. More importantly, inflammation and elastin breakdown in the coronary vessels are completely eliminated in the absence of TNF-α effector functions. Mice treated with the TNF-α-blocking agent etanercept, as well as TNFRI knockout mice, are resistant to development of both coronary arteritis and coronary aneurysm formation. Taken together, TNF-α is necessary for the development of coronary artery lesions in an animal model of Kawasaki disease. These findings have important implications for potential new therapeutic interventions in children with Kawasaki disease.

Matrix metalloproteinase 9 activity leads to elastin breakdown in an animal model of Kawasaki disease.

OBJECTIVE Kawasaki disease (KD) is a multisystem vasculitis leading to damage in the coronary circulation and aneurysm formation. Because cardiac tissue from affected children is not available, investigation of the mechanisms responsible for coronary artery damage in KD requires use of a disease model. The present study was undertaken to examine, in an experimental model, the role of matrix metalloproteinase 9 (MMP-9) on coronary artery inflammation and vascular damage. METHODS C57BL/6 mice were injected with Lactobacillus casei cell wall extract to induce coronary arteritis. Hearts were isolated and assayed for MMP-9 protein expression and enzymatic activity by immunoblotting or confocal microscopy and zymography, respectively. MMP-9-deficient mice were used to examine the necessity of MMP-9 for disease development. RESULTS Localized inflammation at the coronary artery led to elastin breakdown and aneurysm formation. This occurred in the absence of smooth muscle cell apoptosis. Following disease induction, there was an increase in the amount and enzymatic activity of MMP-9, an elastolytic protease. MMP-9 was up-regulated by tumor necrosis factor alpha (TNFalpha) and produced primarily by vascular smooth muscle cells. In MMP-9-deficient animals, vascular inflammation continued to develop, but the incidence of elastin breakdown was significantly reduced. Elastin breakdown in the coronary artery was virtually eliminated by ablation of MMP-9. CONCLUSION These findings show that TNFalpha up-regulates expression of MMP-9, an important proteinase responsible for extracellular matrix breakdown, leading to coronary artery damage in this model of KD. These results have important implications regarding treatments for improving coronary outcome in affected children.

Assessment of sample collection and storage methods for multicenter immunologic research in children.

Multicenter studies involving both large and small centers separated by significant distances pose unique challenges to biological sample collection. The objective of this study was to evaluate protocols for determining inflammatory biomarkers that are cost and manpower efficient for handling blood destined for a sample repository. Tempus (Applied Biosystems) and Paxgene (Qiagen) blood collection systems were evaluated for RNA isolation. P100 tubes (BD), containing propriety stabilizers for preservation of plasma proteins, were evaluated for protein content and compared with plasma collected in conventional tubes. Blood for plasma separation was spiked with recombinant TNF-alpha and IL-2 prior to being processed and stored under various conditions. The Tempus RNA system produced a significantly greater yield of RNA at comparable quality when stored at 4 degrees C and shipped at ambient temperature than any other condition tested. The Tempus system was 20% less expensive and required approximately 40% less processing time thereby reducing costs. The P100 system preserved recombinant TNF-alpha in blood shipped at ambient temperature significantly better than conventionally collected plasma that was shipped on dry ice. There was no significant difference in IL-2 levels between the two collection methods and shipping temperatures. The Tempus RNA blood collection tubes and the P100 protein stabilization system provide the opportunity for reliable collection and ambient temperature transport of samples in multicenter studies. This cost-effective, standardized protocol for a large multicenter trial ensures the integrity of biological samples and maximizes study participation by both large and small centers.

Intravenous immunoglobulin and salicylate differentially modulate pathogenic processes leading to vascular damage in a model of Kawasaki disease

OBJECTIVE Kawasaki disease (KD) is a multisystem vasculitis affecting children and is characterized by immune activation in the acute stage of disease. Systemic inflammation eventually subsides, although coronary arteritis persists, resulting in aneurysm formation. KD is the leading cause of acquired heart disease among children in North America. Accepted treatment guidelines include high-dose intravenous immunoglobulin (IVIG) and aspirin in the acute phase. Although this therapy is effective, the cellular and molecular mechanisms involved are not clear. The aim of this study was to examine the effect of IVIG and salicylate at each stage of disease development. METHODS Using a murine model of KD, we established and validated several in vitro techniques to reflect 3 key steps involved in disease pathogenesis, as follows: thymidine incorporation to evaluate T cell activation, enzyme-linked immunosorbent assay to measure tumor necrosis factor alpha (TNFalpha) production, and real-time polymerase chain reaction to examine TNFalpha-mediated expression of matrix metalloproteinase 9 (MMP-9). RESULTS At therapeutic concentrations, IVIG, but not salicylate, effectively reduced the immune response leading to TNFalpha expression. Unexpectedly, pharmacologic doses of salicylate were not able to inhibit TNFalpha production and in fact enhanced its production. Neither drug directly regulated MMP-9 expression but did so only indirectly via modulating TNFalpha. TNFalpha activity was a prerequisite for local expression of MMP-9 at the coronary artery. CONCLUSION Therapeutic concentrations of IVIG and salicylate differentially modulate the expression of TNFalpha and its downstream effects. Further dissection of the biologic effects of aspirin in acute KD is necessary for the rational design of therapy.

Presence of IFN-γ Does Not Indicate Its Necessity for Induction of Coronary Arteritis in an Animal Model of Kawasaki Disease

Kawasaki disease is the most common cause of vasculitis affecting children, and the leading cause of acquired heart disease in the developed world. To date, studies on the role of IFN-γ in the pathogenesis of Kawasaki disease have focused on peripheral production of IFN-γ, and have yielded conflicting results. Affected heart tissue is not available from children with Kawasaki disease. In this study, we use an animal model of Kawasaki disease, Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis, to examine the role of IFN-γ in the development of coronary artery lesions. We report the presence of IFN-γ, both at the mRNA and protein levels, in the affected vessels. Its biphasic expression, first at days 3–7 and again at days 28–42 post-LCWE injection, corresponds to the first appearance of inflammatory infiltrate in coronary arteries, and later to vascular wall disruption and aneurysm formation, respectively. Interestingly, ablation of IFN-γ expression did not dampen the inflammatory response, and IFN-γ-deficient lymphocytes proliferated more vigorously in response to LCWE than those of wild-type animals. Of more importance, the incidence of coronary arteritis was the same in IFN-γ-deficient and wild-type mice. Taken together, our findings demonstrate that IFN-γ regulates the immune response during development of coronary arteritis, but is not required for the induction of coronary artery disease.

The Biologic Basis of Clinical Heterogeneity in Juvenile Idiopathic Arthritis

Childhood arthritis encompasses a heterogeneous family of diseases. Significant variation in clinical presentation remains despite consensus‐driven diagnostic classifications. Developments in data analysis provide powerful tools for interrogating large heterogeneous data sets. We report a novel approach to integrating biologic and clinical data toward a new classification for childhood arthritis, using computational biology for data‐driven pattern recognition.

Elastolytic matrix metalloproteinases and coronary outcome in children with Kawasaki disease.

Kawasaki disease (KD) is a multisystem vasculitis that leads to coronary artery damage and aneurysm formation. Elastolytic matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of arterial aneurysms. To determine the relationship between circulating levels of elastolytic MMPs and development of coronary artery aneurysms in children with KD, we partnered studies done in affected children with an animal model of disease. In affected children, circulating protein levels and enzymatic activity of MMP-2 and MMP-9 did not have a statistically significant relationship with coronary artery outcome after adjusting for demographic characteristics, and clinical and laboratory features. Although matrix-degrading proteolytic activity was specific for affected mice and localized to inflamed coronary artery segments, the enzymatic activity in the systemic circulation of affected and control mice were not different. Similar to affected children, peripheral blood levels of MMP-9 enzymatic activity did not correlate with coronary artery disease in the animal model. Therefore, circulating levels of MMPs known to act locally may not be useful biomarkers of disease. This is especially relevant to enzymatic activity that is tightly regulated at multiple levels including the local tissue environment.

Inositol-Triphosphate 3-Kinase C Mediates Inflammasome Activation and Treatment Response in Kawasaki Disease

Kawasaki disease (KD) is a multisystem vasculitis that predominantly targets the coronary arteries in children. Phenotypic similarities between KD and recurrent fever syndromes point to the potential role of inflammasome activation in KD. Mutations in NLRP3 are associated with recurrent fever/autoinflammatory syndromes. We show that the KD-associated genetic polymorphism in inositol-triphosphate 3-kinase C (ITPKC) (rs28493229) has important functional consequences, governing ITPKC protein levels and thereby intracellular calcium, which in turn regulates NLRP3 expression and production of IL-1β and IL-18. Analysis of transcript abundance, protein levels, and cellular response profiles from matched, serial biospecimens from a cohort of genotyped KD subjects points to the critical role of ITPKC in mediating NLRP3 inflammasome activation. Treatment failure in those with the high-risk ITPKC genotype was associated with the highest basal and stimulated intracellular calcium levels and with increased cellular production of IL-1β and IL-18 and higher circulating levels of both cytokines. Mechanistic studies using Itpkc-deficient mice in a disease model support the genomic, cellular, and clinical findings in affected children. Our findings provide the mechanism behind the observed efficacy of rescue therapy with IL-1 blockade in recalcitrant KD, and we identify that regulation of calcium mobilization is fundamental to the underlying immunobiology in KD.

Inhibition of transforming growth factor β worsens elastin degradation in a murine model of Kawasaki disease.

Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) β increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-β would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-β signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-β by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-β suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-β, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.

The immune-stimulation capacity of liposome-treated red blood cells

Abstract Our in vivo studies on a rat model established safety of transfusing liposome-treated red blood cells (RBCs) but identified the potential for immune modulation as related to transfusion efficacy of liposome-treated RBCs. The aim of this study was at assessing the impact of liposome-induced membrane changes on the immune profile of liposome-treated RBCs by (a) evaluating their interaction with endothelial cells and monocytes; and (b) the resulting immune response derived from this interaction, in the form of cytokine release, adhesion molecules expression and phagocytosis. Unilamellar liposomes were synthesized to contain unsaturated phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC]:CHOL, 7:3 mol%). The human RBCs immune profile was assessed by incubating control and DOPC-treated RBCs with human umbilical vein endothelial cells (HUVECs) and monocytes. Cytokine release measured by Luminex technology, vascular cell adhesion molecule (VCAM)-1 and E-selectin on HUVECs measured by flow cytometry, and the erythrophagocytic activity of monocytes by monocyte monolayer assay (MMA) were determined. Fibroblast growth factor [FGF]-2 was the only cytokine released by HUVECs that remained increased after incubation with DOPC-treated RBCs compared to control throughout storage. The expression of both VCAM-1 (15.3 ± 5.6% versus 6.3 ± 0.9%, p = 0.008) and E-selectin (18.0 ± 6.3% versus 6.6 ± 0.7%, p = 0.004) by HUVECs were significantly increased after incubation with DOPC-treated RBCs at day 2 of storage. The MMA resulted in phagocytic indexes of zero for both control and DOPC-treated RBCs at day 2 and 42 of storage. The liposome treatment did not result in significant changes to the immune profile of stored DOPC-treated RBCs. These findings combined with previous in vivo results, make liposome treatment a potential candidate for application in RBC preservation and open the possibility for clinical use with other cell types.