Semaphorin 7A promotes endothelial permeability and inflammation via plexin C1 and integrin ß1 in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is a pediatric systemic vasculitis characterized by endothelial cell dysfunction. Semaphorin 7A (Sema7A) has been reported to regulate endothelial phenotypes associated with cardiovascular diseases, while its role in KD remains unknown. This study aims to investigate the effect of Sema7A on endothelial permeability and inflammatory response in KD conditions. METHODS: Blood samples were collected from 68 KD patients and 25 healthy children (HC). The levels of Sema7A and A Disintegrin and Metalloprotease 17 (ADAM17) in serum were measured by enzyme-linked immunosorbent assay (ELISA), and Sema7A expression in blood cells was analyzed by flow cytometry. Ex vivo monocytes were used for Sema7A shedding assays. In vitro human coronary artery endothelial cells (HCAECs) were cultured in KD sera and stimulated with Sema7A, and TNF-α, IL-1ß, IL-6, and IL-18 of HCAECs were measured by ELISA and qRT-PCR. HCAECs monolayer permeability was measured by FITC-dextran. RESULTS: The serum level of Sema7A was significantly higher in KD patients than in HC and correlated with disease severity. Monocytes were identified as one of the source of elevated serum Sema7A, which implicates a process of ADAM17-dependent shedding. Sera from KD patients induced upregulation of plexin C1 and integrin ß1 in HCAECs compared to sera from HC. Sema7A mediated the proinflammatory cytokine production of HCAECs in an integrin ß1-dependent manner, while both plexin C1 and integrin ß1 contributed to Sema7A-induced HCAEC hyperpermeability. CONCLUSIONS: Sema7A is involved in the progression of KD vasculitis by promoting endothelial permeability and inflammation through a plexin C1 and integrin ß1-dependent pathway. Sema7A may serve as a potential biomarker and therapeutic target in the prognosis and treatment of KD.

Protective role of forsythoside B in Kawasaki disease-induced cardiac injury: Inhibition of pyroptosis via the SIRT1-NF-κB-p65 signaling pathway.

Kawasaki disease (KD), an acute exanthematous febrile pediatric illness involving systemic non-specific inflammatory reactions in small- and medium-sized arteries, poses a significant risk of coronary artery and myocardial inflammatory injury. Developing new KD treatments with improved safety and fewer side-effects is highly desirable. Forsythoside B (FTS-B), extracted from the Forsythia suspensa plant, exerts anti-inflammatory activity by inhibiting NF-κB, which is regulated by SIRT1, the reduced expression of which is strongly associated with cardiovascular disease. However, it has yet to be established whether FTS-B influences KD-related inflammatory damage. In this study, we investigated the effects of FTS-B on inflammation in cellular and murine models of KD. Our findings revealed that KD is associated with cardiac dysfunction and inflammatory injury to myocardial and human coronary artery endothelial cells (HCAECs), resulting in a pyroptosis-feedback loop. Both cellular and KD models were characterized by reduced SIRT1 expression and increased NF-κB p65 expression. Contrastingly, the rates of pyroptosis in both murine model myocardial tissues and HCAECs were significantly alleviated in response to FTS-B treatment. Also in both models, we detected an increase of SIRT1 expression and a decrease in the expression of p65. Further examination of the protective mechanism of FTS-B using the SIRT1-specific inhibitor, EX 527, revealed that this inhibitor blocked the palliative effects of FTS-B on inflammatory injury-induced pyroptosis. These results highlight the potential utility of the SIRT1-NF-κB-p65 pathway as a therapeutic target for KD treatment and demonstrate that FTS-B can alleviate KD-induced cardiac and HCAEC inflammatory injury via inhibition of pyroptosis.

Inhibition of PDGFRß alleviates endothelial cell apoptotic injury caused by DRP-1 overexpression and mitochondria fusion failure after mitophagy.

Kawasaki disease (KD), described as "mucocutaneous lymph node syndrome", affects infants and toddlers. Patients with KD suffer from an inflammatory cascade leading to vasculitis with a predilection for coronary arteries. While the symptoms and pathogenesis of KD have received more and more attention, the precise mechanisms are still debated. Researches show that endothelial dysfunction process in KD leads to arterial damage and affect clinical outcome. In this study, we constructed a Candida albicans water soluble fraction (CAWS)-induced KD murine model and penetrated investigating the mechanisms behind endothelial dysfunction. CAWS-induced mice presented remarkably elevated vascular endothelial cell growth factor (VEGF) levels. Abundant expression of VEGF was documented in all vessels that showed edema from acute KD. It has been reported that Platelet-derived growth factor (PDGF) co-expression normalizes VEGF-induced aberrant angiogenesis. Hyperexpression of PDGFRß was induced in the thickened medial layer and vascular endothelium of KD mice. Masitinib (Mas) is an oral tyrosine kinase inhibitor of numerous targets, which can selectively target PDGFR signaling. We set out to explore whether Mas could regulate coronary pathology in KD. Mas administration significantly reduced the VEGF-induced endothelial cells migration. NOX4 was activated in vascular endothelial cells to produce more ROS. Mitochondrial dysregulated fission and mitophagy caused by DRP-1 overexpression precipitated the arterial endothelial cells injury. Here, mitophagy seemed to work as the driving force of DRP-1/Bak/BNIP3-dependent endothelial cells apoptosis. In summary, how mitophagy is regulated by DRP-1 under pathologic status is critical and complex, which may contribute to the development of specific therapeutic interventions in cardiovascular diseases patients, for example Masatinib, the inhibitor of PDGFRß. FACTS AND QUESTIONS: Kawasaki disease causing systemic vasculitis, affects infants and toddlers. Coronary artery injury remains the major causes of morbidity and mortality. DRP-1 overexpression induces DRP-1/Bak/BNIP3-dependent endothelial cells apoptosis. PDGFRß was high-expressed in the thickened medial layer of CAWS-induced KD mice. Inhibition of PDGFRß signaling alleviates arterial endothelial cells injury.

NFIL3 aggravates human coronary artery endothelial cell injury by promoting ITGAM transcription in Kawasaki disease.

OBJECTIVE: High expression of nuclear factor interleukin-3 (NFIL3) and integrin Alpha M (ITGAM) was found in serum samples from Kawasaki disease (KD) patients through bioinformatics analysis. Hence, this study aimed to explore the biological functions of NFIL3 and ITGAM in KD serum-stimulated human coronary artery endothelial cells (HCAECs). METHODS: The differentially-expressed genes in KD were analyzed through bioinformatics analysis. Serum samples were obtained from 18 KD patients and 18 healthy volunteers, followed by detection of NFIL3 and ITGAM levels in KD serum. After HCAECs were transfected with sh-NFIL3, sh-ITGAM, or sh-NFIL3 + oe-ITGAM and underwent 24-h KD serum stimulation, cell viability and apoptosis and the levels of inflammation-related factors were measured. The binding between NFIL3 and ITGAM was validated by dual-luciferase and chromatin immunoprecipitation (ChIP) assays. RESULTS: NFIL3 and ITGAM were up-regulated in serum from KD patients and KD serum-stimulated HCAECs. Down-regulation of NFIL3 or ITGAM inhibited KD serum-induced cell apoptosis and inflammatory response of HCAECs and promoted cell viability. Mechanistically, NFIL3 promoted ITGAM transcription level. Up-regulation of ITGAM reversed the improvement of NFIL3 down-regulation on KD serum-induced HCAEC injury. CONCLUSION: NFIL3 aggravated KD serum-induced HCAEC injury by promoting ITGAM transcription, which provided new insights into the treatment of KD.

Cathelicidin (LL-37) causes expression of inflammatory factors in coronary artery endothelial cells of Kawasaki disease by activating TLR4-NF-κB-NLRP3 signaling.

BACKGROUND: Kawasaki disease (KD) is a type of vasculitis with an unidentified etiology. Cathelicidin (LL-37) may be involved in the development of the KD process; therefore, further research to investigate the molecular mechanism of LL-37 involvement in KD is warranted. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, NLRP3, and LL-37 in the sera of healthy subjects, children with KD, and children with pneumonia. Subsequently, human recombinant LL-37 or/and toll-like receptors 4 (TLR4)-specific inhibitor TAK-242 stimulated human coronary artery endothelial cells (HCAECs), CCK-8 was used to detect cell proliferation, flow cytometry to detect apoptosis, transmission electron microscopy to observe cytoskeletal changes, Transwell to measure cell migration ability, ELISA to detect inflammatory factor levels, Western blot analysis to analyze protein levels of toll-like receptors 4 (TLR4) and NF-κB p-65, and quantitative real-time polymerase chain reaction (qRT-PCR) to determine LL-37, NLRP3 mRNA levels. RESULTS: In this study, we found that the level of LL-37 was highly expressed in the serum of children with KD, and after LL-37 stimulation, apoptosis was significantly increased in HCAECs, and the expression levels of TLR4, NLRP3 and inflammatory factors in cells were significantly enhanced. Intervention with the TLR4-specific inhibitor TAK-242 significantly alleviated the LL-37 effects on cellular inflammation, TLR4, NLRP3 promotion effect. CONCLUSIONS: Our data suggest that LL-37 induces an inflammatory response in KD coronary endothelial cells via TLR4-NF-κB-NLRP3, providing a potential target for the treatment of KD.

Neutrophil Extracellular Traps Formation and Citrullinated Histones 3 Levels in Patients with Kawasaki Disease.

Background: Kawasaki disease (KD) is a vasculitis associated with vascular injury and autoimmune response. Inflammatory factors stimulate neutrophils to produce web-like structures called neutrophil extracellular traps (NETs). Citrullinated histone 3 (H3Cit) is one of the main protein components of neutrophil extracellular traps involved in the process of NETosis. The levels of NETs and H3Cit in the KD are not known. Objective: To determine the changes in the levels of NETs and H3Cit in KD. Methods: Children with KD were recruited and divided into the acute KD and the sub-acute KD group according to the disease phase and whether intravenous immunoglobulin (IVIG) was used or not. Peripheral venous blood was taken before and after the IVIG administration and sent for the examination of NETs by flow cytometry. The level of H3Cit was measured by enzyme-linked immunosorbent assay (ELISA). Results: The counts of NETs in the acute KD group significantly increased compared with the healthy controls (p<0.01). The level of H3Cit was significantly higher in the acute KD group than in the healthy control subjects. Of note, both the counts of NETs and the level of H3Cit decreased in the KD patients treated with IVIG compared with the acute KD group (p<0.01). Conclusion: Acute KD is characterized by an increased formation of NETs and high levels of H3Cit. IVIG significantly inhibited NETs formation and also reduced the level of plasma H3Cit in children with KD.

MiR-223-3p affects the proliferation and apoptosis of HCAECs in Kawasaki disease by regulating the expression of FOXP3.

OBJECTIVE: Kawasaki disease (KD) can lead to permanent damage to coronary structures, the pathogenesis of which remains unknown. This experiment was designed to investigate whether miR-223-3p secreted in the serum of KD patients affects the proliferation and apoptosis of HCAECs in KD by regulating FOXP3. METHODS: Blood samples were collected in acute febrile phase of KD, after IVIG treatment, and from healthy controls. Transfected into HCAECs cells by synthetic FOXP3 siRNA/NC. A co-culture system was established between HCAECs cells transfected with FOXP3 siRNA/NC and THP1 cells added with three sera. RESULTS: Compared with the control group, the expressions of miR-223-3p, RORγt, and Th17 in serum of KD patients were significantly upregulated, and the expressions of TGF-ß1, FOXP3 and Treg were significantly downregulated. At the same time, the levels of IL-6, IL-17, and IL-23 were significantly increased, and the levels of IL-10 and FOXP3 were significantly decreased. After IVIG treatment, the patient's above results were reversed. The serum of KD patients increased the expression of miR-223-3p and inhibited the expression of FOXP3 in HCAECs cells. IVIG serum is the opposite. Overexpression of miR-223-3p also promoted the apoptosis of HCAECs. In addition, serum from KD patients promoted apoptosis, whereas serum after IVIG treatment inhibited apoptosis. KD patient serum downregulated the expression of FOXP3, Bcl2, TGF-ß1 and IL-10 in cells, and upregulated the expression of caspase3, Bax, IL-17, IL-6, and IL-23. The opposite results were obtained with IVIG-treated sera. CONCLUSION: miR-223-3p secreted in serum of KD patients can regulate the expression of FOXP3 and affect the proliferation, apoptosis, and inflammation of cells.

Single cell RNA-seq resolution revealed CCR1+/SELL+/XAF+ CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19.

INTRODUCTION: The COVID-19 pandemic provide the opportunities to explore the numerous similarities in clinical symptoms with Kawasaki disease (KD), including severe vasculitis. Despite this, the underlying mechanisms of vascular injury in both KD and COVID-19 remain elusive. To identify these mechanisms, this study employs single-cell RNA sequencing to explore the molecular mechanisms of immune responses in vasculitis, and validate the results through in vitro experiments. METHOD: The single-cell RNA sequencing (scRNA-seq) analysis of peripheral blood mononuclear cells (PBMCs) was carried out to investigate the molecular mechanisms of immune responses in vasculitis in KD and COVID-19. The analysis was performed on PBMCs from six children diagnosed with complete KD, three age-matched KD healthy controls (KHC), six COVID-19 patients (COV), three influenza patients (FLU), and four healthy controls (CHC). The results from the scRNA-seq analysis were validated through flow cytometry and immunofluorescence experiments on additional human samples. Subsequently, monocyte adhesion assays, immunofluorescence, and quantitative polymerase chain reaction (qPCR) were used to analyze the damages to endothelial cells post-interaction with monocytes in HUVEC and THP1 cultures. RESULTS: The scRNA-seq analysis revealed the potential cellular types involved and the alterations in genetic transcriptions in the inflammatory responses. The findings indicated that while the immune cell compositions had been altered in KD and COV patients, and the ratio of CD14+ monocytes were both elevated in KD and COV. While the CD14+ monocytes share a large scale of same differentiated expressed geens between KD and COV. The differential activation of CD14 and CD16 monocytes was found to respond to both endothelial and epithelial dysfunctions. Furthermore, SELL+/CCR1+/XAF1+ CD14 monocytes were seen to enhance the adhesion and damage to endothelial cells. The results also showed that different types of B cells were involved in both KD and COV, while only the activation of T cells was recorded in KD. CONCLUSION: In conclusion, our study demonstrated the role of the innate immune response in the regulation of endothelial dysfunction in both KD and COVID-19. Additionally, our findings indicate that the adaptive immunity activation differs between KD and COVID-19. Our results demonstrate that monocytes in COVID-19 exhibit adhesion to both endothelial cells and alveolar epithelial cells, thus providing insight into the mechanisms and shared phenotypes between KD and COVID-19.

PROGRESSION OF VASCULAR FUNCTION AND BLOOD PRESSURE IN A MOUSE MODEL OF KAWASAKI DISEASE.

ABSTRACT: Kawasaki disease (KD) is a systemic vasculitis of childhood characterized by vascular damage in the acute stage, which can persist into the late stage. The vascular mechanisms in the cardiovascular risk of KD are not fully studied. We investigated the vascular function and blood pressure in a murine model of KD. We used the Candida albicans water-soluble (CAWS) fraction model. Mice were injected with 4 mg CAWS for 5 consecutive days and separated into three groups. Control, CAWS 7 days (C7), and CAWS 28 days (C28). Hearts and arteries were harvested for vascular characterization. Rat aortic smooth muscle cells were used to studies in vitro. C7 presented elevated inflammatory markers in the coronary area and abdominal aortas, whereas C28 showed severe vasculitis. No difference was found in blood pressure parameters. Vascular dysfunction characterized by higher contractility to norepinephrine in C7 and C28 in aortic rings was abolished by blocking nitric oxide (NO), reactive oxygen species, and cyclooxygenase (COX)-derived products. The CAWS complex increased COX2 expression in rat aortic smooth muscle cells, which was prevented by Toll-like receptor 4 antagonist. Our data indicate that the murine model of KD is associated with vascular dysfunction likely dependent on COX-derived products, oxidant properties, and NO bioavailability. Furthermore, vascular smooth muscle cell may present an important role in the genesis of vascular dysfunction and vasculitis via the Toll-like receptor 4 pathway. Finally, the CAWS model seems not to be appropriate to study KD-associated shock. More studies are necessary to understand whether vascular dysfunction and COXs are triggers for vasculitis.

Kawasaki disease: ubiquitin-specific protease 5 promotes endothelial inflammation via TNFα-mediated signaling.

BACKGROUND: This study aimed to explore the functions of ubiquitin-specific protease 5 (USP5) in the endothelial inflammation of Kawasaki disease (KD). METHODS: USP5 expression levels in HCAECs were examined after stimulation with TNFα or KD sera. The inflammatory cytokine expression level and nuclear factor κB (NF-κB) signaling activation proteins were also investigated in HCAECs by using USP5 overexpression/knockdown lentivirus as well as its small molecule inhibitor vialinin A. RESULTS: USP5 expression level is upregulated in HCAECs after stimulation with KD sera. Similarly, the USP5 expression level is also increased in a time- and dose-dependent manner upon TNFα stimulation in HCAECs. Moreover, USP5 sustains proinflammatory cytokine production and NF-κB signaling activation, whereas USP5 knockdown causes the proinflammatory cytokine levels to decrease and suppress NF-κB signaling activation. Notably, the USP5 inhibitor vialinin A can suppress the expression of inflammatory genes induced by TNFα and IL-1ß in HCAECs. CONCLUSIONS: Our study identified USP5 as a positive regulator of TNFα production and its downstream signaling activation during the inflammatory responses in HCAECs, and demonstrated that its inhibitor vialinin A might serve as a candidate drug for KD therapy to prevent the excessive production of proinflammatory cytokines. IMPACT: USP5 is upregulated in human coronary artery endothelial cells (HCAECs) whether incubated with acute KD sera or TNFα in vitro. USP5 promotes proinflammatory cytokine expression by sustaining NF-κB signaling activation in HCAECs. The USP5 inhibitor vialinin A can suppress the expression levels of proinflammatory cytokines in HCAEC, thus providing a novel mechanism and intervention strategy in KD therapy.

Statins Show Anti-Atherosclerotic Effects by Improving Endothelial Cell Function in a Kawasaki Disease-like Vasculitis Mouse Model.

Kawasaki disease (KD) is an acute inflammatory syndrome of unknown etiology that is complicated by cardiovascular sequelae. Chronic inflammation (vasculitis) due to KD might cause vascular cellular senescence and vascular endothelial cell damage, and is a potential cause of atherosclerosis in young adults. This study examined the effect of KD and HMG-CoA inhibitors (statins) on vascular cellular senescence and vascular endothelial cells. Candida albicans water-soluble fraction (CAWS) was administered intraperitoneally to 5-week-old male apolipoprotein E-deficient (ApoE-) mice to induce KD-like vasculitis. The mice were then divided into three groups: control, CAWS, and CAWS+statin groups. Ten weeks after injection, the mice were sacrificed and whole aortic tissue specimens were collected. Endothelial nitric oxide synthase (eNOS) expression in the ascending aortic intima epithelium was evaluated using immunostaining. In addition, eNOS expression and levels of cellular senescence markers were measured in RNA and proteins extracted from whole aortic tissue. KD-like vasculitis impaired vascular endothelial cells that produce eNOS, which maintains vascular homeostasis, and promoted macrophage infiltration into the tissue. Statins also restored vascular endothelial cell function by promoting eNOS expression. Statins may be used to prevent secondary cardiovascular events during the chronic phase of KD.

The role of mitochondria in the pathogenesis of Kawasaki disease.

Kawasaki disease is a systemic vasculitis, especially of the coronary arteries, affecting children. Despite extensive research, much is still unknown about the principal driver behind the amplified inflammatory response. We propose mitochondria may play a critical role. Mitochondria serve as a central hub, influencing energy generation, cell proliferation, and bioenergetics. Regulation of these biological processes, however, comes at a price. Release of mitochondrial DNA into the cytoplasm acts as damage-associated molecular patterns, initiating the development of inflammation. As a source of reactive oxygen species, they facilitate activation of the NLRP3 inflammasome. Kawasaki disease involves many of these inflammatory pathways. Progressive mitochondrial dysfunction alters the activity of immune cells and may play a role in the pathogenesis of Kawasaki disease. Because they contain their own genome, mitochondria are susceptible to mutation which can propagate their dysfunction and immunostimulatory potential. Population-specific variants in mitochondrial DNA have also been linked to racial disparities in disease risk and treatment response. Our objective is to critically examine the current literature of mitochondria's role in coordinating proinflammatory signaling pathways, focusing on potential mitochondrial dysfunction in Kawasaki disease. No association between impaired mitochondrial function and Kawasaki disease exists, but we suggest a relationship between the two. We hypothesize a framework of mitochondrial determinants that may contribute to ethnic/racial disparities in the progression of Kawasaki disease.

[Recombinant human interleukin 35 (rhIL-35) alleviates the damage of coronary artery endothelial cells in Kawasaki disease by inhibiting NF-κB pathway].

Objective To investigate the protective effect and mechanism of recombinant human interleukin 35(rhIL-35) on coronary artery injury in Kawasaki disease (KD). Methods Human coronary artery endothelial cells (HCAECs) were cultured in vitro to establish KD vascular model. Tumor necrosis factor α(TNF-α) and the serum of KD patients stimulated HCAECs were used to mimic the local inflammatory lesions of KD. The cells were divided into control group, TNF-α and KD serum stimulation group, (25, 50) ng/mL rhIL-35 treatment group. Cell proliferation was detected by CCK-8 assay; mRNA levels of IL-1ß, IL-6, IL-17A and zonula occludens-1(ZO-1) of HCAECs were detected by real-time quantitative PCR; IL-35 expression in plasma and IL-1ß, IL-6 and IL-17A content in HCAEC supernatant were tested by ELISA; Western blot was performed to detect the expression of nuclear factor κB p65 (NF-κB p65) and ZO-1. Results TNF-α and KD serum inhibited the proliferation of HCAECs, while rhIL-35 significantly reversed the above effects. RhIL-35 significantly down-regulated the expression of IL-1ß, IL-6 and IL-17A after preconditioning HCAECs. Compared with TNF-α and KD serum stimulation group, rhIL-35 pretreated cells could significantly increase ZO-1 protein expression and inhibit NF-κB p65 expression. Conclusions rhIL-35 can alleviate the damage of KD coronary artery endothelial cells by inhibiting NF-κB pathway.

RNA Sequencing Reveals Beneficial Effects of Atorvastatin on Endothelial Cells in Acute Kawasaki Disease.

Background Damage to the coronary arteries during the acute phase of Kawasaki disease (KD) is linked to inflammatory cell infiltration, myointimal proliferation, and endothelial cell (EC) dysfunction. To understand the response of ECs to KD treatment, we studied the genome-wide transcriptional changes in cultured ECs incubated with KD sera before and after treatment with or without atorvastatin. Methods and Results RNA sequencing of human umbilical vein ECs incubated with pooled sera from patients with acute KD before or after treatment with intravenous immunoglobulin and infliximab revealed differentially expressed genes in interleukin-1, tumor necrosis factor-α, and inflammatory cell recruitment pathways. Subacute sera pooled from patients treated with intravenous immunoglobulin, infliximab, and atorvastatin uniquely induced expression of NOS3, Kruppel like factor (KLF2, and KLF4 (promotes EC homeostasis and angiogenesis) and ZFP36 ring finger protein (ZFP36) and suppressor of cytokine signaling 3 (SOCS3) (suppresses inflammation), and suppressed expression of TGFB2 and DKK1 (induces endothelial-mesenchymal transition) and sphingosine kinase 1 (SPHK1) and C-X-C motif chemokine ligand 8 (CXCL8) (induces inflammation). Conclusions These results suggest that atorvastatin treatment of patients with acute KD may improve EC health, reduce mediators of inflammation produced by ECs, and block KD-induced myofibroblast proliferation.

Melatonin alleviates vascular endothelial cell damage by regulating an autophagy-apoptosis axis in Kawasaki disease.

OBJECTIVES: Melatonin has been reported to be an appropriate candidate for mitigating various cardiovascular injuries, owing to its versatility. This study aimed to explore the role of melatonin in Kawasaki disease (KD)-associated vasculitis and its underlying mechanisms. MATERIAL AND METHODS: The role of melatonin was evaluated in human coronary artery endothelial cells (HCAECs), peripheral blood mononuclear cells from KD patients, human THP1 cell line in vitro, and a Candida albicans water-soluble fraction (CAWS)-induced KD mouse model in vivo. Cell proliferation assay, cell apoptosis assay, cell co-culture, RNA extraction, RNA sequencing, reverse transcription quantitative PCR, enzyme-linked immunosorbent assay (ELISA), transwell assay, western blot, dual-luciferase reporter assay, and autophagic flux assay were performed to investigate the function and regulatory mechanisms of melatonin in vitro, while haematoxylin and eosin staining, Verhoeff's van Gieson staining, ELISA, and immunohistochemical analysis were performed to detect the effect of melatonin in vivo. RESULTS: Melatonin suppressed cell apoptosis directly reduced the expression of endothelial cell damage markers in HCAECs, and alleviated vasculitis in the CAWS-induced KD mouse model. Mechanistically, melatonin promoted autophagy by activating the melatonin/ melatonin receptor (MT)/cAMP-response element binding protein (CREB) pathway and upregulating the expression of autophagy-related gene-3, thereby suppressing cell apoptosis in an autophagy-dependent manner. Additionally, melatonin decreased the production of pro-inflammatory cytokines in macrophages and indirectly reduced the immunopathological damage of HCAECs. CONCLUSIONS: This study revealed that melatonin protects vascular endothelial cells in KD, by suppressing cell apoptosis in an autophagy-dependent manner and reducing the immunopathological damage mediated by macrophages.

Intravenous Immunoglobulin Therapy Restores the Quantity and Phenotype of Circulating Dendritic Cells and CD4+ T Cells in Children With Acute Kawasaki Disease.

Background: Intravenous immunoglobulin (IVIG) showed its therapeutic efficacy on Kawasaki disease (KD). However, the mechanisms by which it reduces systemic inflammation are not completely understood. Dendritic cells (DCs) and T cells play critical roles in the pathogenic processes of immune disorders. Assessing the quantity of DC subsets and T cells and identifying functional molecules present on these cells, which provide information about KD, in the peripheral blood may provide new insights into the mechanisms of immunoglobulin therapy. Methods: In total, 54 patients with KD and 27 age-matched healthy controls (HCs) were included in this study. The number, percentage, and phenotype of DC subsets and CD4+ T cells in peripheral blood were analyzed through flow cytometry. Results: Patients with KD exhibited fewer peripheral DC subsets and CD4+ T cells than HCs. Human leucocyte antigen-DR (HLA-DR) expression was reduced on CD1c+ myeloid DCs (CD1c+ mDCs), whereas that on plasmacytoid DCs (pDCs) did not change significantly. Both pDCs and CD1c+ mDCs displayed significantly reduced expression of co-stimulatory molecules, including CD40, CD86. pDCs and CD1c+ mDCs presented an immature or tolerant phenotype in acute stages of KD. Number of circulating pDC and CD1c+ mDC significantly inversely correlated with plasma interleukin-6 (IL-6) levels in KD patients pre-IVIG treatment. No significant differences were found concerning the DC subsets and CD4+ T cells in patients with KD with and without coronary artery lesions. Importantly, these altered quantity and phenotypes on DC subsets and CD4+ T cells were restored to a great extent post-IVIG treatment. T helper (Th) subsets including Th1 and Th2 among CD4+ T cells did not show alteration pre- and post-IVIG treatment, although the Th1-related cytokine IFN-γ level in plasma increased dramatically in patients with KD pre-IVIG treatment. Conclusions: pDCs and CD1c+ mDCs presented an immature or tolerant phenotype in acute stages of KD, IVIG treatment restored the quantity and functional molecules of DCs and CD4+ T cells to distinct levels in vivo, indicating the involvement of DCs and CD4+ T cells in the inflammation in KD. The findings provide insights into the immunomodulatory actions of IVIG in KD.

Human umbilical cord mesenchymal stem cells regulate CD54 and CD105 in vascular endothelial cells and suppress inflammation in Kawasaki disease.

OBJECTIVE: The objective was to evaluate the expression levels of CD31+CD54+ and CD31+CD105+ endothelial microparticles (EMPs) before and after intravenous immunoglobulin (IVIG) treatment of Kawasaki disease (KD). To explore the role of human umbilical cord mesenchymal stem cells (hucMSCs) in inhibiting endothelial inflammation in KD, the effects of hucMSCs on the expression of CD54 and CD105 in endothelial cells in KD were analyzed in vivo and in vitro. METHODS: The concentrations of IL-1ß and VEGF in the peripheral blood of KD or healthy children were detected, and the distributions of CD31+CD54+ and CD31+CD105+ EMPs in platelet-poor plasma (PPP) were analyzed by flow cytometry. Human umbilical vein endothelial cells (HUVECs) were first cocultured with the patients' peripheral blood mononuclear cells (PBMCs). Next, HUVECs were cocultured with hucMSCs after stimulation with inactivated serum from patients. Cell proliferation and migration activities were assessed, and the expression of CD54, CD105 and IL-1ß was analyzed. In an in vivo study, hucMSCs were transplanted into KD mice. The locations and expression levels of CD54, CD105 and IL-1ß in the heart tissues of mice were analyzed. RESULTS: The levels of IL-1ß and CD31+CD54+ EMPs were significantly higher before IVIG treatment and 2 weeks after treatment in KD patients (P < 0.01). However, the levels of VEGF and CD31+CD105+ EMPs increased significantly in KD only after IVIG treatment (P < 0.01). KD-inactivated serum stimulation combined with cocultivation of PBMCs can activate inflammation in HUVECs, leading to reduced cell proliferation and migration activities. Cocultivation also increased the expression of CD54 and decreased the expression of CD105 (P < 0.001). Cocultivation with hucMSCs can reverse these changes. Additionally, hucMSC transplantation downregulated the expression of IL-1ß and CD54 and significantly upregulated the expression of CD105 in KD mice. CONCLUSION: The expression levels of CD31+CD54+ and CD31+CD105+ EMPs showed inconsistent changes at different KD statuses, providing potential markers for clinical application. HucMSCs suppress inflammation and regulate the expression levels of CD54 and CD105 in vascular endothelial cells in KD, possibly providing a new basis for stem cell therapy for KD.

Leucine-rich alpha-2-glycoprotein 1 and angiotensinogen as diagnostic biomarkers for Kawasaki disease.

OBJECTIVE: Kawasaki disease (KD) is a systemic vasculitis in childhood that can lead to coronary artery lesions (CALs). Although early diagnosis and treatment is important for preventing KD patients from development of CALs, diagnosis depends on the clinical features of KD. We studied the usefulness of leucine-rich alpha-2-glycoprotein 1 (LRG1) and angiotensinogen (AGT), previously reported as KD-related proteins, for KD diagnosis and estimation of intravenous immunoglobulin (IVIG) efficacy. METHODS: We undertook a prospective cohort study with patients having two or more KD symptoms in multiple centers in Japan, between July 2017 and February 2019. RESULTS: Two hundred forty-two patients were included. In multivariable analysis, one unit increase in LRG1 was associated with higher odds of KD diagnosis (Odds ratio [OR] 1.02 [95% confidence interval (CI) 1.001-1.03]). Double-positivity for AGT (≥ 26 µg/mL) and LRG1 (≥ 123.5 µg/mL) was an independent biomarker for KD diagnosis in both the total cohort and the subgroup of patients with two to four KD symptoms (OR 5.01 [95% CI 1.86-13.50] and 3.71 [95% CI 1.23-11.16], respectively). There was no association between LRG1/AGT and IVIG efficacy. CONCLUSION: Double-positivity for LRG1 and AGT is an biomarker for KD diagnosis, especially useful in diagnosing incomplete KD from non-KD. Future studies with larger cohorts should seek to determine whether LRG1 and AGT are valuable as definitive data referred at the diagnosis of KD and for estimating the risk of CALs.

NLRP3 Inflammasome Mediates Immune-Stromal Interactions in Vasculitis.

[Figure: see text].

The autoimmune signature of hyperinflammatory multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) manifests as a severe and uncontrolled inflammatory response with multiorgan involvement, occurring weeks after SARS-CoV-2 infection. Here, we utilized proteomics, RNA sequencing, autoantibody arrays, and B cell receptor (BCR) repertoire analysis to characterize MIS-C immunopathogenesis and identify factors contributing to severe manifestations and intensive care unit admission. Inflammation markers, humoral immune responses, neutrophil activation, and complement and coagulation pathways were highly enriched in MIS-C patient serum, with a more hyperinflammatory profile in severe than in mild MIS-C cases. We identified a strong autoimmune signature in MIS-C, with autoantibodies targeted to both ubiquitously expressed and tissue-specific antigens, suggesting autoantigen release and excessive antigenic drive may result from systemic tissue damage. We further identified a cluster of patients with enhanced neutrophil responses as well as high anti-Spike IgG and autoantibody titers. BCR sequencing of these patients identified a strong imprint of antigenic drive with substantial BCR sequence connectivity and usage of autoimmunity-associated immunoglobulin heavy chain variable region (IGHV) genes. This cluster was linked to a TRBV11-2 expanded T cell receptor (TCR) repertoire, consistent with previous studies indicating a superantigen-driven pathogenic process. Overall, we identify a combination of pathogenic pathways that culminate in MIS-C and may inform treatment.