Mast cells (MCs) are long-lived immune cells, which were reported to play an important role in initiating innate and adaptive immune responses against various infections. MCs accumulate in high numbers in the stroma and at the invasion front of various human cancers, suggesting a possible contribution by MCs to tumour growth. Experimental studies using crosses of MC-deficient Kit-mutant mouse strains with mouse models of epithelial cancers have provided evidence for important MC tumour-promoting functions. However, the complex alterations of the immune system that characterize Kit-mutant mice in addition to their MC deficiency, limit the interpretation of these findings. Numerous key observations made in Kit mutant mice were not reproduced in novel, Kit-independent mouse models of MC deficiency. Thus, the impact of MCs on tumour biology remains unclear. The aim of this study is to clarify the contribution of MCs to the biology of Human papilloma virus (HPV)-induced skin cancer in a Kit-independent mouse model of MC deficiency. In K14-HPV16 transgenic mice, HPV oncogenes are constitutively expressed in the epidermis resulting in epidermal hyperplasia with 100% penetrance and squamous cell carcinoma in about 50% of the animals. A cross to a Kit-mutant line suggested that MCs are important tumour promoters in this model. We crossed K14-HPV16 mice to M5Cre R-DTA line, in which MCs are constitutively depleted with high efficiency and selectivity. Unexpectedly, the loss of MCs neither affected keratinocyte proliferation indices nor altered keratinocyte apoptosis at any stage of HPV-induced neoplasia. Furthermore, the loss of MCs did not result in any detectable changes in composition and gene expression of the inflammatory hematopoietic cell infiltrate in the tumour stroma. This shows that, contrary to current belief, MCs have no important function in orchestrating the tumour micro milieu. In keeping with this finding, MC deficiency resulted in no detectable difference in the incidence growth or grading of SSC in K14-HPV16 transgenic mice. Collectively, these results show that, despite their high density in HPV-induced neoplasia, MC have no role in cancerogenesis or neoplastic progression in the K14-HPV16 mouse model. Our findings also emphasize the importance of novel Kitindependent mouse models in the investigation of MC in vivo functions.

Mastocitoma é uma das principais neoplasias cutâneas em cães, caracterizada por uma multiplicação anormal de mastócitos, com comportamento biológico muito variável. Os principais fatores prognósticos incluem grau histopatológico, marcadores de proliferação, como índice mitótico, Ki67 e AgNOR, além de estadiamento clínico. Diversos estudos concentram-se na avaliação da relação da apoptose com a oncogênese e seu papel no prognóstico. Em condições fisiológicas, a apoptose ocorre na maturação e senescência celular, mantendo a homeostasia dos diferentes tecidos, removendo do organismo células que tenham sofrido alguma mutação. A genética da apoptose pode ser interrompida frente à ocorrência de mutações, levando à perda do controle na proliferação celular, o que resulta no desenvolvimento de uma neoplasia. O presente estudo avaliou a ocorrência de apoptose por meio de ensaio TUNEL em mastocitomas cutâneos caninos, com o objetivo de testar sua relação com as graduações histopatológicas e o valor prognóstico quanto à sobrevida pós-cirúrgica, assim como compará-lo à expressão imuno-histoquímica de caspase 3 e Ki67. Quarenta e quatro mastocitomas cutâneos caninos, provenientes de 36 cães, foram submetidos à avaliação histopatológica para graduação quanto à diferenciação tumoral, à análise imuno-histoquímica para avaliação das expressões de Ki67 e caspase 3. A marcação positiva para TUNEL não mostrou relação com grau histopatológico, nem foi um bom indicador para sobrevida ou mortalidade em função da doença. Apesar disso, houve correlação positiva entre os índices apoptóticos. / Mast cell tumor is a very common neoplasm in dogs and characterized by an abnormal proliferation of mast cells, with variable biological behavior. The main prognostic factors include histological grade, proliferation markers, such as mitotic index, Ki67 and AgNOR, and clinical staging. Several studies focus in the relation of apoptosis and oncogenesis and its role in prognostication. In physiological conditions, apoptosis occurs due to aging and cell senescence, maintaining the homeostasis of different tissues by removing mutated cells. The genetics of apoptosis can be interrupted by mutations, leading to loss of control in cellular proliferation, and resulting in cancer development. This study evaluated the occurrence of apoptosis by TUNEL assay in canine cutaneous mast cell tumors, compared it with histopathological grading and the immunohistochemical expressions of caspase-3 and Ki67, as well as tested its prognostic value for post-surgical survival. Forty-four canine cutaneous mast cell tumors, from 36 dogs were submitted to histopathologic and immunohistochemical analyses. Positive staining for TUNEL showed no relation with histological grade, and was not considered a good indicator for survival or mortality. Nevertheless, a positive correlation between the apoptotic indexes was found.

Mouse mast cell chymases are granule-associated serine proteinases with chymotrypsin-like substrate specificities. cDNAs for two new chymases were isolated from a cDNA library constructec using mRNA from ABFTL-6 mouse mast cells by screening with a rat mast cell proteinase cDNA. The deduced amino acid sequence of mouse cymase 1 consists fo a 226 amino acid catalytic portion and a 21 amino acid preprosequence. Chymase 1 is unusual in that an Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine proteinase. Also, chymase 1 is expected to have a large positive charge (+13) at physiological pH. A partial cDNA for chymase 2 encodes 177 residues of the carboxy terminal portion of a second proteinase distinct from chymase 1. Chymase 2 cDNA contains highly conserved intron/exon junction, a high positive charge (+17) and a novel, second potential N-glycosylation site. Transcripts for both chymases are found in ABFTL-6 mast cells, but only chymase 2 mRNA is in mouse connective tissue mast cells. These data suggest that these chymases have distinct enzymatic properties and tissue-specific patterns of gene expression.

Indiana University-Purdue University Indianapolis (IUPUI) / Primary Sclerosing Cholangitis (PSC) is a rare and slow progressing cholangiopathy characterized by hepatic inflammation, fibrosis and ductular reaction with liver transplantation as the sole therapeutic option. PSC patients are at high risk of auto-immune comorbidities like irritable bowel disease (IBD), found in up to 80% of PSC patients (PSC-IBD). There are indications of genetic and environmental components for auto-immune development in IBD; however, its etiology remains unclear. Mast cells (MCs) infiltrate the liver and can become activated leading to degranulation and release of mediators, like histamine (HA), which result in increased intrahepatic bile duct mass, biliary senescence, hepatic inflammation, and hepatic stellate cell activation. Similarly, MCs infiltrate the intestine and increase inflammation which alters host-microbiome communication. MCs are necessary for successful liver regeneration and the combat of intestinal pathogens; however, chronic HA signaling exacerbates damage in cholangiopathies and IBD. Bile acid synthesis is tightly regulated by Farnesoid X Receptor (FXR) and its downstream mediator, fibroblast growth factor 15 (FGF15, -19 in humans). Cholangiocytes (i) are the target of cholangiopathies, (ii) modify and recycle bile acids through Apical Sodium Bile Acid Transporter (ASBT)-mediated cholehepatic shunting, which functions outside of enterohepatic circulation of bile acids and (iii) are capable of autocrine HA signaling. The complex relationship between hepatic and intestinal MC infiltration and bile acid signaling has not been established; therefore, identifying MC regulation of bile acid pool and FXR/FGF15 signaling pathway will provide insight into therapeutic treatment of PSC-IBD. Under the rationale that (i) cholestatic liver diseases are positively correlated with auto-immune comorbidities like IBD, (ii) during disease, MCs infiltrate the liver and intestine and release signaling factors like HA, and (iii) MCs express FXR and secrete FGF15/19; we propose the central hypothesis that MC activation regulates bile acid signaling and PSC progression through paracrine crosstalk with cholangiocytes in the liver and intestinal inflammation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20221号 / 医博第4180号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 中川 一路, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Indiana University-Purdue University Indianapolis (IUPUI) / Among the cytokines regulating immunity, interleukin 9 (IL-9) has gained considerable attention for its role in inflammation, immune tolerance, and tumor immunity. IL-9 has a broad array of functions and acts on multiple cell types to regulate immune responses. IL-9 receptor is expressed on both non-hematopoietic cells and hematopoietic cells in the innate and adaptive immune system. IL-9 demonstrates a remarkable degree of tissue-specific functionality that varies by tissue site and the context of the inflammatory milieu. In this dissertation, we investigate the biological activities of IL-9 and identify distinct IL-9-responsive cell type in the immune pathogenesis of disease models including allergic airway disease, lung cancer, and urinary tract infection. When examining airway hyperreactivity, we found IL-9-dependent mast cell function was critical. Using adoptive transfer models and newly generated mice with an inactivation of the Il9 gene restricted to T cells generated by CD4-cre/LoxP-mediated targeting, we demonstrate that T cell secreted IL-9 promotes mast cell progenitor proliferation and CCR2-dependent mast cell migration during allergic airway inflammation. In IL-9-mediated pro-tumor responses, interstitial macrophages, but not mast cells, respond to T cell IL-9 to enhance B16 metastatic tumor growth. In the context of urinary tract infection, IL-9 contributes to protection against E. coli bladder infection potentially by enhancing CCL20 production in epithelial cells to recruit macrophages and neutrophils. Altogether, IL-9 can exert cell type-specific effects that identify its roles in immunity and disease. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial, and where it has the potential to complicate clinical outcomes.

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No / Nerve growth factor (NGF) has long been recognized as an important mast cell (MC) growth factor. To explore whether other neurotrophins (NTs) of the NGF family, which are widely expressed in mouse skin, affect the numbers and/or functions of MCs we examined the effects of NT-3 on neonatal skin MCs. We demonstrate that TrkC, the high affinity NT-3 receptor, is expressed by virtually all neonatal skin MCs in C57BL/6 mice, which indicates that MCs can respond to NT-3. Skin of neonatal and early postnatal NT-3-overexpressing mice (promoter: K14) displayed significantly and up to twofold increased numbers of MCs during the first 20 days after birth, as compared to wild-type mice. To check whether this increase in MC numbers in NT-3 transgenic mice reflects a higher rate of proliferation, we performed immunohistochemistry, which revealed that only 1-2% of all skin MCs both in NT-3-overexpressing and in wild-type controls showed Ki-67-positive nuclei, suggesting that the observed differences in the number of MCs do not reflect a higher rate of MC proliferation. Additionally, we show that the effect of NT-3 on the number of MCs is most likely to be stem cell factor (SCF)-independent, because NT-3 significantly downregulates secretion of SCF-protein in cultured dermal fibroblasts, as assessed by enzyme-linked immunosorbent assay. Numbers of skin MCs in neonatal TrkC-deficient mice were found to be modestly reduced, as compared to wild-type mice, indicating that NT-3 can modulate the number of MCs directly via TrkC, although TrkC does not seem to be essential for the number of basal MCs. To further analyze the effects of NT-3 on MCs, we stimulated skin organ culture of early postnatal C57BL/6 mouse skin with 5-50 ng/ml NT-3, which induced a significant increase in MC degranulation, as visualized by Giemsa staining. However, stimulation of isolated neonatal dermal skin MCs with NT-3 in vitro failed to result in MC activation, as measured by serotonin release. Our data suggest a role for NT-3 in the maturation of MCs, such as a TrkC-mediated stimulation of the differentiation of pre-existing, less mature MCs and/or by enhancing the migration of circulating MC precursors into the skin.