Tissue Homeostatic Control by CSF-dependent Macrophage-Stromal Cell Interactions

描述(申请人提供)：所有组织都含有间质和实质细胞以及组织驻留的巨噬细胞，这对组织的动态平衡、修复和免疫至关重要。巨噬细胞集落刺激因子是巨噬细胞生长、分化和发育的关键细胞因子，它的缺失导致CSF1op/OP小鼠出现大量的巨噬细胞缺陷。虽然已知基质细胞分泌的CSF1在各种细胞因子和激素的作用下上调，但对于它如何通过与巨噬细胞的相互作用进行局部调节却知之甚少。巨噬细胞是MCSF的主要消费者。除了巨噬细胞和浸润性乳腺癌细胞之间可能存在的EGF-CSF1旁分泌正反馈回路外，对肿瘤细胞如何操纵MCSF的合成知之甚少。这种反馈环不能解释组织微环境中巨噬细胞与基质细胞的组织特异性比率的明显稳定性，也不能解释对巨噬细胞功能或巨噬细胞产生的生长因子的适应性生理需求如何影响局部MCSF的产生设定点。我们假设巨噬细胞产生一个信号，向基质细胞报告它们对MCSF的需求，基质细胞通过诱导CSF1转录，通过反馈抑制抑制巨噬细胞信号的产生来响应这个信号，并假设存在一个相互的负反馈环，报告基质细胞对巨噬细胞功能或生长因子的需求。与我们的假设一致的是，我们在小鼠胚胎成纤维细胞-巨噬细胞共培养系统中的初步数据表明，基质细胞CSF1的表达可能显著地被未知的细胞间信号所诱导，而条件培养液转移实验表明，这种CSF1诱导信号可能是由可溶性因子介导的。此外，关于肿瘤产生的单核细胞集落刺激因子和肿瘤相关巨噬细胞()单核细胞集落刺激因子需求之间的功能不匹配如何导致肿瘤病理，我们知之甚少。虽然已知一些肿瘤能够成功地过表达CSF1来招募TAMs，但我们假设肿瘤未能充分调节局部MCSF的产生，以满足的要求，导致坏死、缺氧、促炎和肿瘤细胞死亡的增加。在这个项目中，我们结合了体外巨噬细胞和基质细胞共培养系统，转录分析，以及生物物理和生化方法来分离分子信号。此外，我们在肝脏特异的CSF1条件基因敲除模型上建立了CSF1减毒的肿瘤细胞系和致癌物诱导的肝细胞癌的体内模型，以检验这些假设，共同更好地阐明正常组织动态平衡和肿瘤发生中依赖CSF1的巨噬细胞和基质细胞之间的信号机制。

DESCRIPTION (provided by applicant): All tissues contain stromal and parenchymal cells and tissue-resident macrophages, which are critical for tissue homeostasis, repair and immunity. Macrophage colony stimulating factor is a critical cytokine for macrophage growth, differentiation, and development, and its deficiency leads to substantial macrophage deficits in the CSF1op/op mouse. While stromal cell-secreted CSF1 is known to be upregulated in response to various cytokines and hormones, very little is understood about how it may be regulated locally by interactions with macrophages, the primary MCSF consumer. Less is understood about how tumor cells can manipulate MCSF synthesis, except for a putative EGF-CSF1 paracrine positive feedback loop between macrophages and invasive breast carcinoma cells. Such a feedback loop cannot explain the evident stability of tissue-specific ratios of macrophages to stromal cells in the tissue microenvironment, nor how adaptive physiologic demands for macrophage functions or macrophage-produced growth factors affect the local MCSF production set point. We hypothesize that macrophages produce a signal reporting their MCSF requirements to stromal cells, and that stromal cells respond to this signal by inducing CSF1 transcription, inhibiting macrophage signal production once through feedback inhibition, and postulate that a reciprocal negative feedback loop exists reporting stromal cell demand for macrophage functions or growth factors. Consistent with our hypothesis, our preliminary data in a mouse embryonic fibroblast - macrophage co-culture system suggests that stromal cell CSF1 expression may be induced significantly by as yet unknown intercellular signals, and conditioned media transfer experiments suggest that this CSF1-inducing signal may be mediated by a soluble factor. Furthermore, little is known of how a functional mismatch between MCSF production by tumors and tumor- associated macrophage (TAM) MCSF requirements leads to tumor pathology. Although some tumors are known to overexpress CSF1 successfully to recruit TAMs, we hypothesize that tumors fail to adequately regulate local MCSF production to TAM demands, leading to increased necrosis, hypoxia, pro-inflammatory TAM and tumor cell death. In this project, we combine in vitro macrophage and stromal cell co-culture systems, transcriptional analysis, with biophysical and biochemical methods for molecular signal isolation. Additionally, we develop in vivo models of CSF1-attenuated tumor cell lines and carcinogen-induced hepatocellular carcinoma on a liver-specific CSF1 conditional knockout model to test these hypotheses, to together better elucidate the CSF1-dependent signaling mechanisms between macrophages and stromal cells in normal tissue homeostasis and tumorigenesis.