Macrophage programming is regulated by a cooperative interaction between fatty acid binding protein 5 and peroxisome proliferator-activated receptor gamma

The resolution of inflammation is an active process that is tightly regulated to achieve repair and tissue homeostasis. In the absence of resolution, chronic inflammation underlies the pathogenesis of chronic lung disease including Chronic Obstructive Pulmonary Disease (COPD) with recurrent exacerbations. Over the course of inflammation, macrophage programming transitions from inflammatory to pro-resolving, and is in part regulated by the nuclear receptor Peroxisome Proliferator-Activated Receptor ? (PPAR?). In our previous work, we demonstrated an association between Fatty Acid Binding Protein 5 (FABP5) expression and PPARg activity in PBMCs. However, the role of FABP5 in macrophage programming is not yet defined, thus, we further investigated its interaction with PPAR? and its role in macrophage programming. Here, we demonstrate that FABP5 is necessary to initiate PPAR? activation and macrophage pro-resolving programming. We used the RNAscope tool in combination with co-immunofluorescence, immunoprecipitation, PPAR? activity assay, and chromatin immunoprecipitation to show that FABP5 and PPAR? not only interact but also regulate one another. By integrating flow cytometry, ELISA, assay for transposase-accessible chromatin by sequencing (ATAC-seq) and the Seahorse technology, we extend our findings to illustrate that lack of FABP5 expression promotes a glycolytic pro-inflammatory macrophage programming. Taken together, these data provide evidence that FABP5 and PPAR? reciprocally regulate each other's expression and function, consistent with a positive feedback regulatory loop between the two to promote macrophage pro-resolving programming. Overall design: The Omni method for the Assay for transposase-accessible chromatin by sequencing (Omni-ATAC-seq) was performed in primary mouse bone marrow-derived macrophages for WT or FABP5-deficient mice. All samples were sequenced in duplicate.

炎症消退是一个受严格调控的主动过程，旨在实现组织修复与内稳态。若炎症消退过程缺失，慢性炎症将成为多种慢性肺部疾病的发病基础，其中包括伴反复急性加重的慢性阻塞性肺疾病（Chronic Obstructive Pulmonary Disease, COPD）。在炎症进程中，巨噬细胞编程会从促炎表型向促消退表型转变，这一过程部分受核受体过氧化物酶体增殖物激活受体γ（Peroxisome Proliferator-Activated Receptor γ, PPARγ）调控。既往研究中，我们团队已证实外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）中脂肪酸结合蛋白5（Fatty Acid Binding Protein 5, FABP5）的表达与PPARγ活性存在关联。但目前FABP5在巨噬细胞编程中的作用尚未明确，因此本研究进一步探究了其与PPARγ的相互作用，以及其在巨噬细胞编程中的功能。

本研究证实，FABP5是启动PPARγ激活与巨噬细胞促消退表型编程的必要条件。我们联合使用RNAscope技术、免疫荧光共染色、免疫沉淀、PPARγ活性检测以及染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验，证明FABP5与PPARγ不仅存在相互作用，还可彼此调控。通过整合流式细胞术、酶联免疫吸附实验（Enzyme-Linked Immunosorbent Assay, ELISA）、转座酶可及性染色测序（assay for transposase-accessible chromatin by sequencing, ATAC-seq）以及海马细胞能量代谢检测技术，我们进一步拓展了研究结论，证实FABP5表达缺失会促使巨噬细胞向糖酵解型促炎表型编程。

综上，本研究数据表明FABP5与PPARγ可相互调控对方的表达与功能，二者之间存在正反馈调控环路，从而促进巨噬细胞向促消退表型编程。

整体实验设计：本研究对野生型（Wild Type, WT）及FABP5基因敲除小鼠的原代骨髓源性巨噬细胞开展了Omni版转座酶可及性染色测序（Omni-ATAC-seq），所有样本均进行双份测序。