PI3K (phosphoinositide 3-kinase) alpha has been implicated in phagocytosis and fluid-phase pinocytosis in macrophages. The subtype-specific role of PI3K in these processes is poorly understood. To elucidate this issue, we made Raw 264.7 cells (a mouse leukaemic monocyte-macrophage cell line) deficient in each of the class-I PI3K catalytic subunits: p110alpha, p110beta, p110delta and p110gamma. Among these cells, only the p110alpha-deficient cells exhibited lower phagocytosis of opsonized and non-opsonized zymosan. The p110alpha-deficient cells also showed the impaired phagocytosis of IgG-opsonized erythrocytes and the impaired fluid-phase pinocytosis of dextran (molecular mass of 40 kDa). Receptor-mediated pinocytosis of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-labelled acetylated low-density lipoprotein and fluid-phase pinocytosis of Lucifer Yellow (molecular mass of 500 Da) were resistant to p110alpha depletion. None of these processes were impaired in cells lacking p110beta, p110delta or p110gamma, but were susceptible to a pan-PI3K inhibitor wortmannin. In cells deficient in the enzymes catalysing PtdIns(3,4,5)P3 breakdown [PTEN (phosphatase and tensin homologue deleted on chromosome 10) or SHIP-1 (Src-homology-2-domain-containing inositol phosphatase-1)], uptake of IgG-opsonized particles was enhanced. These results indicated that phagocytosis and fluid-phase pinocytosis of larger molecules are dependent on the lipid kinase activity of p110alpha, whereas pinocytosis via clathrin-coated and small non-coated vesicles may depend on subtypes of PI3Ks other than class I.

The complement system is a classic central player in innate immunity. Most pathogens activate both complement and the toll-like receptor (TLR) pathway. Therefore, to provide a more comprehensive understanding of innate immunity, it is important to understand the crosstalk between these two systems. Mouse macrophages produce IL-12 and IL-10 in response to TLR ligands such as LPS, CpG, Poly I:C and Malp2. The TLR-induced IL-12 production was decreased, while that of IL-10 was increased by concurrent stimulation with a complement fragment C5a. Pharmacological studies have suggested that C5a regulates TLR4-induced IL-12 production in a phosphoinositide 3-kinase (PI3K)-dependent mechanism. In the present study, however, we found that the C5a-mediated changes can be observed in macrophages from mice lacking PI3K p85α or PI3K p110γ. The result indicates that the C5a action is PI3K-independent; neither class IA nor class IB PI3K subtype is involved in this regulation. The actions of C5a were sensitive to pertussis toxin and PD98059, suggesting a role of G protein-mediated activation of the Erk1/2 pathway.

Fibrillin-1 (FBN1) is responsible for haploinsufficient and autosomal dominant Marfan syndrome. Even in the same Marfan pedigree, penetrance and expressivity in heterozygous individuals can differ and result in variable disease onset and severity. Thus, other factors in addition to mutations in FBN1 are likely to contribute to the disease. In this study, we examined the regulation of FBN1 in porcine Marfan syndrome model, focusing on DNA methylation patterns distinguishable as wild-type (WT) and FBN1 null (KO) alleles in heterozygous cells. Most importantly, the ratio of the transcriptionally active hypomethylated WT allele was altered during cellular passage and highly correlated with FBN1 mRNA level compared with that in the KO allele. Transcribed FBN1 RNA from the KO allele was abolished after splicing coupled with translational initiation, suggesting that the functional FBN1 mRNA levels were affected by DNA methylation of the WT allele.

DNA methylation in transcriptional regulatory regions is crucial for gene expression. The DNA methylation status of the edges of CpG islands, called CpG island shore, is involved in tissue/cell-type-specific gene expression. Haploinsufficiency diseases are caused by inheritance of one mutated null allele and are classified as autosomal dominant. However, in the same pedigree, phenotypic variances are observed despite the inheritance of the identical mutated null allele, including Fibrillin1 (FBN1), which is responsible for development of the haploinsufficient Marfan disease. In this study, we examined the relationship between gene expression and DNA methylation patterns of the FBN1 CpG island shore focusing on transcriptionally active hypomethylated alleles (Hypo-alleles). No difference in the DNA methylation level of FBN1 CpG island shore was observed in porcine fetal fibroblast (PFF) and the liver, whereas FBN1 expression was higher in PFF than in the liver. However, Hypo-allele ratio of the FBN1 CpG island shore in PFF was higher than that in the liver, indicating that Hypo-allele ratio of the FBN1 CpG island shore likely correlated with FBN1 expression level. In addition, oocyte-derived DNA hypermethylation in preimplantation embryos was erased until the blastocyst stage, and re-methylation of the FBN1 CpG island shore was observed with prolonged in vitro culture of blastocysts. These results suggest that the establishment of the DNA methylation pattern within the FBN1 CpG island shore occurs after the blastocyst stage, likely during organogenesis. In conclusion, Hypo-allele ratios of the FBN1 CpG island shore correlated with FBN1 expression levels in porcine tissues.

Background: The seasonal epidemic of Kawasaki disease (KD) in winter in Japan suggests that low vitamin D status may affect KD through the immune system. We aimed to evaluate the effect of vitamin D on the onset and clinical course of KD. Methods: We conducted a case-control study to compare 25-hydroxyvitamin D (25(OH)D) levels in KD patients admitted to our hospital between March 2018 and June 2021, with those in healthy controls from published Japanese data. In patients with KD, we evaluated the association of 25(OH)D levels with intravenous immunoglobulin resistance and coronary artery lesions. Results: We compared 290 controls and 86 age-group-adjusted patients with KD. The 25(OH)D levels in KD patients were lower than those in the controls (median: 17 vs. 29 ng/mL, P < 0.001). In winter, 25(OH)D levels in KD patients were lower than those in summer (median: 13 vs. 19 ng/mL). The adjusted odds ratios for the onset of KD were 4.9 (95% CI: 2.5-9.6) for vitamin D insufficiency (25(OH)D: 12-20 ng/mL) and 29.4 (95% CI: 12.5-78.2) for vitamin D deficiency (25(OH)D < 12 ng/mL). Among 110 KD patients, 25(OH)D levels at diagnosis of KD were not associated with intravenous immunoglobulin resistance or coronary artery lesions. Conclusions: The 25(OH)D levels in patients with KD were lower than those in the controls, especially in winter. Lower 25(OH)D levels in winter were associated with an increased risk of KD onset. It remains to be elucidated whether the observed association has a causal relationship.Key words 25-hydroxyvitamin D, coronary aneurysm, intravenous immunoglobulin, Kawasaki disease, vitamin D deficiency.Kawasaki disease (KD) is an acute systemic vasculitis of unknown cause that primarily affects infants and children. 1 Seasonal and regional epidemics have been reported and, in Japan, KD is most prevalent in winter, with a small peak in summer. 2 These epidemics have suggested that some infections trigger KD. 2 Vitamin D is an essential nutrient that is synthesized in the skin upon exposure to ultraviolet rays, in addition to absorption from food. 3 Vitamin D was initially reported to play an important role in bone metabolism and calcium homeostasis but recently, it has been recognized that it regulates innate and adaptive immunity. 3,4 Vitamin D status was defined based on serum 25-hydroxyvitamin D (25(OH)D) levels because of its relatively long half-life. 4 We hypothesized that lower 25(OH)D levels, especially in winter, predispose host immunity to exacerbate the onset and progression of KD. To test this hypothesis, we first conducted a case-control study comparing KD patients and previously reported healthy controls. Second, in patients with KD, we evaluated whether 25(OH)D levels at diagnosis were associated with the clinical course of KD, such as intravenous immunoglobulin (IVIG) resistance or coronary artery lesions (CALs). We have also considered the effect of breastfeeding as a possible confounding factor, which has been reported to be associated with a lower risk of KD and often leads to reduced ...