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Background: MCT and MCTC types of human mast cells (MCs) are distinguished from one another on the basis of the protease compositions of their secretory granules, but their functional and developmental relationships have been uncertain. Objective: These studies better define the functional properties and developmental relationship of MCT and MCTC cells. Methods: Mast cells were dispersed from human skin and lung, purified with anti-Kit antibody, and separated into CD88+ and CD88− populations by cell sorting. These cells were evaluated by immunocytochemistry with antitryptase and antichymase mAbs; for chymase and tryptase mRNA by real-time RT-PCR; for conversion of MCT to MCTC cells during cell culture with recombinant human stem cell factor and recombinant human IL-6; and for degranulation and leukotriene C4 (LTC4) secretion when stimulated with anti-FcεRI, substance P, C5a, and compound 48/80. Results: Mature MCT and MCTC cells were separated from one another on the basis of selective expression of CD88, the C5aR, on MCTC cells. Lung MCT cells had negligible levels of chymase mRNA and retained their MCT phenotype in culture. Mature MCTC cells from skin and lung degranulated in response to FcεRI cross-linking, C5a, compound 48/80, and substance P. Lung MCTC cells released LTC4 on activation, but no LTC4 was detected when skin-derived MCTC cells were activated. MCT cells from lung degranulated and released LTC4 in response to anti-FcεRI and substance P, but not to C5a and compound 48/80. Conclusion: These observations functionally distinguish MCT from MCTC types of human mast cells and suggest important differences that may affect their participation in diseases such as asthma and urticaria. [Copyright &y& Elsevier]

We previously reported that rhIL-4 induced apoptosis and rhIL-6 mediated protection of human mast cells derived from cord blood mononuclear cells. Based on the result, we attempted to obtain the phenotypes and differentiation of CD3+ cells from cord blood by investigating their cell surface markers in the presence of rhSCF plus rhIL-4. The effect of co-cultured CD3+ cells on fetal liver mast cells (FLMCs) was also determined. Phenotypes from cord blood-derived cells were analyzed by flow cytometry and cell numbers were determined. Fetal liver mast cells were cultured with cord blood-derived cells (mainly CD3+) in the presence of rhSCF and/or rhIL-4 and were analyzed to determine cell number and expression of Kit+ and FcϵR1. The percentage of CD3+ cells from cord blood-derived cells on day 0 was about 41 ± 13.5%, following monocytes and granulocytes. CD3+ cells increased in number (1.5-fold) and purity (90%), whereas other cell types did not survive. More than 60% of CD3+ cells from cord blood at day 0 were CD4−CD8−. These double-negative cells dramatically decreased by 1 week of culture, while CD4+CD8+ cells increased in number and purity through 3 weeks of culture, and then decreased as greater numbers of single-positive T cells emerged. We also found that FcϵR expression on FLMC increased in the presence of rhIL-4, but was not affected by the T cells that developed from cord blood mononuclear cells. The results indicate that IL-4, a Th2 type cytokine, together with rhSCF, can induce T cell proliferations, differentiation, and maturation from cord blood progenitor cells. [Copyright &y& Elsevier]

Cholestenoic acid (CA) has been reported as an important biomarker of many severe diseases, but its physiological and pathological roles remain unclear. This study aimed to investigate the potential role of CA in hepatic lipid homeostasis. Enzyme kinetic studies revealed that CA specifically activates DNA methyltransferases 1 (DNMT1) at low concentration with EC 50 = 1.99 × 10 -6 M and inhibits the activity at higher concentration with IC 50 = 9.13 × 10 -6 M, and specifically inhibits DNMT3a, and DNMT3b activities with IC 50 = 8.41 × 10 -6 M and IC 50 = 4.89 × 10 -6 M, respectively. In a human hepatocyte in vitro model of high glucose (HG)-induced lipid accumulation, CA significantly increased demethylation of 5m CpG in the promoter regions of over 7,000 genes, particularly those involved in master signaling pathways such as calcium-AMPK and 0.0027 at 6 h. RNA sequencing analysis showed that the downregulated genes are affected by CA encoding key enzymes, such as PCSK9, MVK, and HMGCR, which are involved in cholesterol metabolism and steroid biosynthesis pathways. In addition, untargeted lipidomic analysis showed that CA significantly reduced neutral lipid levels by 60% in the cells cultured in high-glucose media. Administration of CA in mouse metabolic dysfunction-associated steatotic liver disease (MASLD) models significantly decreases lipid accumulation, suppresses the gene expression involved in lipid biosynthesis in liver tissues, and alleviates liver function. This study shows that CA as an endogenous epigenetic regulator decreases lipid accumulation via epigenetic regulation. The results indicate that CA can be considered a potential therapeutic target for the treatment of metabolic disorders. NEW & NOTEWORTHY To our knowledge, this study is the first to identify the mitochondrial monohydroxy bile acid cholestenoic acid (CA) as an endogenous epigenetic regulator that regulates lipid metabolism through epigenome modification in human hepatocytes. The methods used in this study are all big data analysis, and the results of each part show the global regulation of CA on human hepatocytes rather than narrow point effects.

The status of the GP130-STAT3 signaling pathway in humans with nonalcoholic fatty liver disease (NAFLD) and its relevance to disease pathogenesis are unknown. The expression of the gp130-STAT3 axis and gp130 cytokine receptors were studied in subjects with varying phenotypes of NAFLD including nonalcoholic steatohepatitis (NASH) and compared with lean and weight-matched controls without NAFLD. Gp130 and its downstream signaling element (Tyk2 and STAT3) expression were inhibited in obese controls whereas they were increased in NAFLD. IL-6 levels were increased in NASH and correlated with gp130 expression (P < 0.01). Palmitate inhibited gp130-STAT3 expression and signaling. IL-6 and palmitate inhibited hepatic insulin signaling via STAT3-dependent and independent mechanisms, respectively. STAT3 overexpression reversed palmitate-induced lipotoxicity by increasing autophagy (ATG7) and decreasing endoplasmic reticulum stress. These data demonstrate that the STAT3 pathway is activated in NAFLD and can worsen insulin resistance while protecting against other lipotoxic mechanisms of disease pathogenesis., (Copyright © 2015 the American Physiological Society.)

PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and γ-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling., (Copyright © 2014 the American Physiological Society.)

Human α- and β-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. β-Protryptase is sequentially processed in vitro by autocatalysis at R(-3) followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R(-3)Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. β-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q(-3)) and mutated β-protryptase (R(-3)Q) as well as wild-type β-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and β-protryptases from human mast cells to their mature enzymatically active products.

Background: Alpha and beta-tryptase levels in serum are clinical tools for the evaluation of systemic anaphylaxis and systemic mastocytosis. Basophils and mast cells are known to produce these proteins., Objective: The current study examines the effect of the alpha,beta-tryptase genotype on basophil tryptase levels and the type of tryptase stored in these cells., Methods: Tryptase extracted from purified peripheral blood basophils from 20 subjects was examined by using ELISAs measuring mature and total tryptase and by using an enzymatic assay with tosyl-Gly-Pro-Lys-p-nitroanilide. Tryptase genotypes (4:0, 3:1, and 2:2 beta/alpha ratios) were assessed by using a hot-stop PCR technique with alpha,beta-tryptase-specific primers. Total alpha,beta-tryptase mRNA was measured by means of competitive RT-PCR, and ratios of alpha to beta-tryptase mRNA were measured by means of hot-stop RT-PCR., Results: Tryptase in all but one of the basophil preparations was mature and enzymatically active. Tryptase quantities in basophils were less than 1% of those in tissue mast cells. Tryptase genotypes (beta/alpha) among the 20 donors were 4:0 in 7, 3:1 in 7, and 2:2 in 6. Tryptase protein and mRNA levels per basophil were not affected by the tryptase genotype., Conclusion: Basophils from healthy subjects contain modest amounts of mature and enzymatically active tryptase unaffected by the tryptase genotype.

Human cord blood-derived mast cells (HCMC) grown in medium with serum and recombinant human stem cell factor (rhSCF) with or without interleukin (IL)-6 are less mature than human skin mast cells (HSMC). We found that c-kit-positive HCMC cultured for 8-10 weeks with rhSCF in serum-free medium became sensitive to basic secretatogues and expressed the serine protease, chymase, which is preferentially expressed in HSMC. The HCMC release beta-hexosaminidase (beta-HEX) within 1 min of stimulation with compound 48/80 or substance P, and release was suppressed by pertussis toxin. Approximately 34% of the HCMC in the serum-free culture stained positively with chymase antibody. Chymase and c-kit levels, and responsiveness to basic secretagogues, increased substantially after an additional 2 weeks in a serum-free environment with rhIL-6 and rhSCF. Moreover, Fc(epsilon)RI-dependent activation of the HCMC resulted in induction of cytokines and cyclooxygenase-2. These results show that HCMC can differentiate into a phenotype morphologically and functionally similar to HSMC if exposed to SCF in serum-free medium.