Macrophage-intrinsic regulators of tissue type 2 inflammation

巨噬细胞在2型免疫反应中发挥多种调节和效应功能，但控制这种功能可塑性的因素还不是很清楚。巨噬细胞的转录图谱由转录因子和表观遗传机制/修饰物塑造，这些转录因子和表观遗传机制/修饰物允许快速反应，在某些情况下，允许稳定的重新编程和“先天记忆”。转谷氨酰胺酶-2(TG2)和转录因子芳香烃受体(AhR)参与了2型免疫反应的调节，但它们在2型巨噬细胞激活和功能中的作用尚不清楚。我们观察到来自屋尘螨(HDM)过敏性哮喘患者的单核细胞或HDM致敏小鼠的骨髓细胞的巨噬细胞在体外培养7天后表现出炎症基因的表达和反应性增加。引人注目的是，HDM致敏小鼠的骨髓祖细胞与幼稚小鼠的骨髓祖细胞相比，TG2的表达增加。这表明，在2型炎症期间，外周血巨噬细胞前体细胞可以稳定地重新编程，TG2可能在这一过程中发挥作用。鉴于最近报道的TG2在组蛋白(H3K4me3Q5)5羟色化和转录激活(在神经元中)中的作用，我们旨在研究在蠕虫感染或HDM引发的过敏性呼吸道炎症(AAI)期间，髓系/造血TG2缺陷是否会导致体内2型激活的巨噬细胞基因表达改变。我们还旨在确定哮喘患者和HDM致敏或蠕虫感染小鼠巨噬细胞基因表达的差异是否与H3K4me3Q5和其他组蛋白标记和/或染色质可及性相关。此外，我们还发现，在两种变态反应性哮喘的临床前模型中，缺乏AhR或其直接下游靶酶细胞色素P450CyP1B1会导致更严重的AAI。在小鼠和人肺中，细胞色素P1B1主要通过在非造血细胞(上皮细胞)中的表达来调节AAI，而AhR则在刺激时调节上皮细胞和髓系细胞中的基因表达。值得注意的是，缺乏AhR的肺泡样巨噬细胞不能在屋尘螨或脂多糖刺激下上调抗炎前列腺素(PGE2)和白三烯(LTB4、LTC4、LTD4、LTE4)。因此，我们现在的目标是在全球范围内描述AhR在调节巨噬细胞内脂质介质合成中的作用，并在体内/体外AAI和蠕虫感染的背景下验证这一功能。这两个目标都将通过下一代测序方法(RNAseq、ChIPseq、ATACseq)实现，这些方法已经在几个FOR2599项目的框架内针对巨噬细胞进行了优化。综上所述，我们将确定AhR和TG2在2型免疫中作为巨噬细胞激活和重编程的关键调节因子的作用。

Macrophages fulfil a multitude of regulatory and effector functions during type 2 immune responses, but the factors that govern this functional plasticity are poorly defined. Transcriptional profiles of macrophages are shaped by transcription factors and epigenetic mechanisms/ modifiers that allow for rapid responses and – in some contexts - stable reprogramming and “innate memory”. The enzyme transglutaminase-2 (TG2) and the transcription factor aryl hydrocarbon receptor (AhR) have been implicated in the regulation of type 2 immune responses, but their role in “type 2” macrophage activation and function has remained obscure. We have observed that macrophages derived from blood monocytes of house dust mite (HDM) allergic asthma patients or from bone marrow cells of HDM-sensitized mice show increased inflammatory gene expression and responsiveness after 7 days of in vitro culture. Strikingly, bone marrow progenitors of HDM-sensitized mice showed an increased expression of TG2 compared to progenitors from the bone marrow of naïve mice. This suggested that peripheral macrophage progenitors are stably reprogrammed during type 2 inflammation and that TG2 may play a role in this process. Given the recently reported role of TG2 in histone (H3K4me3Q5) serotonylation and transcriptional activation (in neurons), we aim to investigate whether a myeloid/ hematopoietic TG2 deficiency results in altered gene expression in type 2 activated macrophages in vivo during helminth infection or allergic airway inflammation (AAI) triggered by HDM. We also aim to determine whether differences in gene expression in macrophages from asthma patients and from HDM-sensitized or helminth infected mice correlate with H3K4me3Q5 and other histone marks and/ or chromatin accessibility. Moreover, we have found that lack of the AhR or its immediate downstream target enzyme cytochrome P450 CYP1B1 leads to more severe AAI in two preclinical models of allergic asthma. Whereas CYP1B1 regulates AAI primarily through its expression in non-hematopoietic (epithelial) cells in both murine and human lung the AhR regulates gene expression in epithelial and myeloid cells upon stimulation. Strikingly, alveolar-like macrophages deficient for AhR fail to upregulate anti-inflammatory prostaglandins (PGE2) and leukotrienes (LTB4, LTC4, LTD4, LTE4) upon stimulation with house dust mite or LPS. Therefore, we now aim to globally profile the role of the AhR in the regulation of lipid mediator synthesis within macrophages and validate this function in settings of AAI and helminth infection in vivo/ex vivo. Both of these objectives will be addressed by next generation sequencing approaches (RNAseq, ChIPseq, ATACseq) that have been optimized for macrophages within the frame of several FOR2599 projects. In summary, we will determine the role of AhR and TG2 as key regulator of macrophage activation and reprogramming in type 2 immunity.