Shunsuke Kanno

Nod1 Ligands Induce Site-Specific Vascular Inflammation

Objective—The goal of this study was to investigate the effects of stimulants for a nucleotide-binding domain, leucine-rich repeat-containing (NLR) protein family on human artery endothelial cells and murine arteries. Methods and Results—Human coronary artery endothelial cells were challenged in vitro with microbial components that stimulate NLRs or Toll-like receptors. We found stimulatory effects of NLR and Toll-like receptor ligands on the adhesion molecule expression and cytokine secretion by human coronary artery endothelial cells. On the basis of these results, we examined the in vivo effects of these ligands in mice. Among them, FK565, 1 of the nucleotide-binding oligomerization domain (Nod)-1 ligands induced strong site-specific inflammation in the aortic root. Furthermore, coronary arteritis/valvulitis developed after direct oral administration or ad libitum drinking of FK565. The degree of the respective vascular inflammation was associated with persistent high expression of proinflammatory chemokine/cytokine and matrix metallopeptidase (Mmp) genes in each tissue in vivo by microarray analysis. Conclusion—This is the first coronary arteritis animal model induced by oral administration of a pure synthetic Nod1 ligand. The present study has demonstrated an unexpected role of Nod1 in the development of site-specific vascular inflammation, especially coronary arteritis. These findings might lead to the clarification of the pathogenesis and pathophysiology of coronary artery disease in humans.

Identification of Pathogenic Cardiac CD11c + Macrophages in Nod1-Mediated Acute Coronary Arteritis

Objective—Nod1 is an intracellular pattern recognition receptor for bacterial peptidoglycan fragments. We previously reported that a synthetic Nod1 ligand, FK565, induced acute coronary arteritis in mice similar to that of Kawasaki disease. However, the molecular mechanisms underlying this characteristic inflammation have remained elusive. Approach and Results—We found that CD11c+MHC class II+ cells accumulated in the heart of FK565-treated mice before arteritis development. Morphological features and gene expression signatures of the cardiac CD11c+MHC class II+ cells suggested that this population is closely related to macrophages, and thus, we designated them cardiac CD11c+ macrophages. Nod1 in nonhematopoietic cells, rather than hematopoietic cells, was required for the increase of cardiac CD11c+ macrophages and arteritis development. Among nonhematopoietic cells, cardiac endothelial cells produced a large amount of chemokines in response to FK565. Endothelial cell–specific blockade of Nod1 signaling suppressed FK565-induced expression of these chemokines, accumulation of cardiac CD11c+ macrophages, and subsequent coronary arteritis development. We also found that CCR2+Ly6Chi inflammatory monocytes in peripheral blood supplied precursors of cardiac CD11c+ macrophages. CCR2-deficient mice or pertussis toxin–treated mice exhibited decreased numbers of cardiac CD11c+ macrophages and reduced arteritis. Conclusions—These results suggest that Ly6Chi monocytes are recruited to FK565-activated endothelial cells to generate cardiac CD11c+ macrophages, which play a pivotal role in the pathogenesis of acute coronary arteritis.

Activation of an Innate Immune Receptor, Nod1, Accelerates Atherogenesis in Apoe−/− Mice

Atherosclerosis is essentially a vascular inflammatory process in the presence of an excess amount of lipid. We have recently reported that oral administration of a nucleotide-binding oligomerization domain (Nod)-1 ligand, FK565, induced vascular inflammation in vivo. No studies, however, have proven the association between Nod1 and atherosclerosis in vivo. To investigate a potential role of NOD1 in atherogenesis, we orally administered FK565 to apolipoprotein E knockout (Apoe−/−) mice for 4 wk intermittently and performed quantification of atherosclerotic lesions in aortic roots and aortas, immunohistochemical analyses, and microarray-based gene expression profiling of aortic roots. FK565 administration accelerated the development of atherosclerosis in Apoe−/− mice, and the effect was dependent on Nod1 in non–bone marrow origin cells by bone marrow transplantation experiments. Immunohistochemical studies revealed the increases in the accumulation of macrophages and CD3 T cells within the plaques in aortic roots. Gene expression analyses of aortic roots demonstrated a marked upregulation of the Ccl5 gene during early stage of atherogenesis, and the treatment with Ccl5 antagonist significantly inhibited the acceleration of atherosclerosis in FK565-administered Apoe−/− mice. Additionally, as compared with Apoe−/− mice, Apoe and Nod1 double-knockout mice showed reduced development of atherosclerotic lesions from the early stage as well as their delayed progression and a significant reduction in Ccl5 mRNA levels at 9 wk of age. Data in the present study show that the Nod1 signaling pathway in non–bone marrow-derived cells contributes to the development of atherosclerosis.

Kawasaki Disease-Specific Molecules in the Sera Are Linked to Microbe-Associated Molecular Patterns in the Biofilms

Background Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. The innate immune system is involved in its pathophysiology at the acute phase. We have recently established a novel murine model of KD coronary arteritis by oral administration of a synthetic microbe-associated molecular pattern (MAMP). On the hypothesis that specific MAMPs exist in KD sera, we have searched them to identify KD-specific molecules and to assess the pathogenesis. Methods We performed liquid chromatography-mass spectrometry (LC-MS) analysis of fractionated serum samples from 117 patients with KD and 106 controls. Microbiological and LC-MS evaluation of biofilm samples were also performed. Results KD samples elicited proinflammatory cytokine responses from human coronary artery endothelial cells (HCAECs). By LC-MS analysis of KD serum samples collected at 3 different periods, we detected a variety of KD-specific molecules in the lipophilic fractions that showed distinct m/z and MS/MS fragmentation patterns in each cluster. Serum KD-specific molecules showed m/z and MS/MS fragmentation patterns almost identical to those of MAMPs obtained from the biofilms formed in vitro (common MAMPs from Bacillus cereus, Yersinia pseudotuberculosis and Staphylococcus aureus) at the 1st study period, and from the biofilms formed in vivo (common MAMPs from Bacillus cereus, Bacillus subtilis/Bacillus cereus/Yersinia pseudotuberculosis and Staphylococcus aureus) at the 2nd and 3rd periods. The biofilm extracts from Bacillus cereus, Bacillus subtilis, Yersinia pseudotuberculosis and Staphylococcus aureus also induced proinflammatory cytokines by HCAECs. By the experiments with IgG affinity chromatography, some of these serum KD-specific molecules bound to IgG. Conclusions We herein conclude that serum KD-specific molecules were mostly derived from biofilms and possessed molecular structures common to MAMPs from Bacillus cereus, Bacillus subtilis, Yersinia pseudotuberculosis and Staphylococcus aureus. Discovery of these KD-specific molecules might offer novel insight into the diagnosis and management of KD as well as its pathogenesis.

Memory B-Cell Pools Predict the Immune Response to Pneumococcal Conjugate Vaccine in Immunocompromised Children

BACKGROUND The immune responses to pneumococcal conjugate vaccine (PCV) are low in immunocompromised hosts. The effect of memory B cells on the immune response to PCV remains elusive. METHODS In this prospective study, 53 children who received 7-valent PCV were enrolled. Antipneumococcal immunoglobulin G (IgG) levels and opsonization index (OI) titers, along with lymphocyte subsets, were investigated in immunocompromised and immunocompetent hosts. Immunocompromised patients comprised 8 hematopoietic stem cell transplant recipients (group A) and 9 immunosuppressive therapy recipients (group B), and controls consisted of 14 children aged >1 year (group C) and 22 infants (group D). RESULTS Serotype-specific IgG concentrations and OIs in group A were lower than those in group C. These did not differ among groups B, C, and D. The rates of achieving immunity (defined as an IgG level of 1.0 µg/mL and an OI of 8) in group A were also lower than in group C. Despite the sustained numbers of total T cells and B cells, CD27(+) B-cell and CD4(+) T-cell counts in group A were lower than those in group C. In group B, the immunoglobulin D-expressing CD27(-) B-cell count was only lower than that in group C. CONCLUSIONS Circulating numbers of CD27(+) B cells, rather than CD4(+) T cells, may predict the effective PCV responses in immunocompromised children.

Evaluation of Teicoplanin Trough Values After the Recommended Loading Dose in Children With Associated Safety Analysis.

Background: This study evaluated whether the recommended teicoplanin loading dose (3 loading doses of 10 mg/kg every 12 hours) achieves a 15–30 &mgr;g/mL trough levels in 26 children (2−16 years). In addition, we examined the incidences of renal impairment and hepatic dysfunction in children treated with teicoplanin. Methods: This retrospective study was conducted between October 2008 and March 2014. Results: The percentage of patients with a trough level <10 and <15 &mgr;g/mL were 15.4% (4/26) and 46.2% (12/26), respectively. There were significant correlations between age and concentration/cumulative loading dose (C/D) ratio (P = 0.045), serum creatinine and C/D ratio (P < 0.001) and estimated glomerular filtration rate and C/D ratio (P = 0.005). Serum creatinine was significantly lower when trough levels were <15 &mgr;g/mL compared with ≥15 &mgr;g/mL. The incidences of renal impairment and hepatic dysfunction were 2.3% and 5.8%, respectively, with no significant difference between <20 and ≥20 &mgr;g/mL trough-level groups. Conclusions: The recommended loading dose may be insufficient to achieve 15–30 &mgr;g/mL in children with normal renal function. In addition, the target trough level ≥20 &mgr;g/mL for deep-seated infections seems to be safe in children.

Nontuberculous mycobacteria-associated hemophagocytic lymphohistiocytosis in MonoMAC syndrome

To the Editor: Hemophagocytic lymphohistiocytosis (HLH) has a wide spectrum of etiologies including both genetic defects and association with infection.1 Uncontrolled T-cell activation is the hallmark of HLH, but the T cell-independent pathway via activation of mononuclear phagocyte system has been implicated by several clinical reports and animal models.2,3 Monocytopenia andmycobacterial infection (MonoMAC) syndrome is caused byGATA2haploinsufficiency.4 Patientswith GATA2 deficiency suffer from nontuberculous mycobacterial (NTM) infection and myeloid malignancies. Hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for GATA2 deficiency.5–8 NTM-associated HLH has not been reported in GATA2deficient patients. Case report: A 14-year-old male was hospitalized because of prolonged fever and weight loss. He received the diagnosis of myelodysplastic syndrome (MDS) at age 11 years. Laboratory tests revealed leukocyte count, 1.58× 109/l (83% neutrophils, 16% lymphocytes, and 0%monocytes); hemoglobin, 9.2 g/dl; platelet count, 10.8 × 109/l; ferritin, 2,206 ng/ml; and soluble interleukin-2 receptor (sIL-2R), 2,349 U/ml. Flow cytometry of peripheral blood revealed CD3+ cells,

Calcineurin inhibitors exacerbate coronary arteritis via the MyD88 signalling pathway in a murine model of Kawasaki disease

Calcineurin inhibitors (CNIs) have been used off‐label for the treatment of refractory Kawasaki disease (KD). However, it remains unknown whether CNIs show protective effects against the development of coronary artery lesions in KD patients. To investigate the effects of CNIs on coronary arteries and the mechanisms of their actions on coronary arteritis in a mouse model of KD, we performed experiments with FK565, a ligand of nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1) in wild‐type, severe combined immunodeficiency (SCID), caspase‐associated recruitment domain 9 (CARD9)–/– and myeloid differentiation primary response gene 88 (MyD88)–/– mice. We also performed in‐vitro studies with vascular and monocytic cells and vascular tissues. A histopathological analysis showed that both cyclosporin A and tacrolimus exacerbated the NOD1‐mediated coronary arteritis in a dose‐dependent manner. Cyclosporin A induced the exacerbation of coronary arteritis in mice only in high doses, while tacrolimus exacerbated it within the therapeutic range in humans. Similar effects were obtained in SCID and CARD9–/– mice but not in MyD88–/– mice. CNIs enhanced the expression of adhesion molecules by endothelial cells and the cytokine secretion by monocytic cells in our KD model. These data indicated that both vascular and monocytic cells were involved in the exacerbation of coronary arteritis. Activation of MyD88‐dependent inflammatory signals in both vascular cells and macrophages appears to contribute to their adverse effects. Particular attention should be paid to the development of coronary artery lesions when using CNIs to treat refractory KD.