Heightened turnover and failed maturation of monocyte-derived macrophages in Chronic Granulomatous Disease

Loss of NADPH oxidase activity in phagocytes leads to altered cellular responses and exaggerated inflammation in Chronic Granulomatous Disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild type (WT) peritonea were lavaged and characterized over time. Though the numbers harvested from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured with loss of Ly6C and gain of MHCII, CD206 and CD36, while MoMacs in CGD remained small and were mostly Ly6C+MHCII-. RNAseq analyses showed few intrinsic differences between genotypes in newly recruited MoMacs, but significant differences over time. WT MoMacs demonstrated changes in metabolism, adhesion and reparative functions, while CGD MoMacs remained inflammatory. Labeling with PKH dye demonstrated that while WT MoMacs were mostly recruited within the first 24 hours and remained in the peritoneum while maturing and enlarging, CGD monocytes continued to stream into the peritoneum for days with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in what appeared to be nascent granulomata. Importantly, these observations appeared to be entirely driven by the milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and behavior, and conversely, altered maturation/behavior of WT MoMacs was seen after adoptive transfer into inflamed peritonea of CGD mice. These data demonstrate heightened recruitment and fundamental failure of MoMac maturation in CGD. Overall design: Monocytes and monocyte-derived macrophages from C57BL/6 (WT) and gp91 phox -/y (CGD) mice were collected from peritoneal lavage at 3 timepoints after intraperitoneal zymosan injection. Peritoneal lavage cells were FACS sorted to purify monocyte/macropahges with differential expression of the phenotypic markers Ly6C and MHCII, yielding populations of Ly6C+MHCII-, Ly6C+MHCII+, Ly6C-MHCII- or Ly6C-MHCII+ monocyte/macrophages over time. Three replicate cohorts of mice were collected for each treatment condition, resulting in a total of 42 samples analyzed.

吞噬细胞中NADPH氧化酶活性缺失会引发慢性肉芽肿病（Chronic Granulomatous Disease, CGD）患者出现细胞应答异常与过度炎症反应。本研究旨在评估gp91phox-/y小鼠中Nox2缺失对酵母聚糖诱导的腹膜炎期间单核细胞衍生巨噬细胞（monocyte-derived macrophages, MoMacs）的影响。我们通过腹膜灌洗获取CGD与野生型（wild type, WT）小鼠腹膜内的MoMacs，并随时间对其进行表型特征分析。尽管两种基因型小鼠的细胞收获量几乎一致，但二者的成熟过程存在显著差异：新招募的WT型MoMacs会快速增大并成熟，表现为Ly6C表达丢失以及MHCII、CD206与CD36表达上调；而CGD小鼠的MoMacs始终维持较小体积，且多数呈现Ly6C+MHCII-表型。转录组测序（RNA sequencing, RNAseq）分析显示，新招募的MoMacs在两种基因型间仅存在少量固有差异，但随时间推移二者的差异显著升高。WT型MoMacs展现出代谢、黏附及修复功能相关的基因表达变化，而CGD型MoMacs则始终维持炎症表型。PKH染料标记实验表明，WT型MoMacs大多在诱导后的前24小时内完成招募，并在腹膜中留存以完成成熟与体积增大；而CGD型单核细胞会持续数天向腹膜内涌入，其中大量迁移至膈肌部位，聚集于中性粒细胞簇周围的纤维蛋白（原）（fibrin(ogen)）凝块中，该结构看似为早期肉芽肿。重要的是，上述表型差异完全由局部微环境所驱动：将CGD型MoMacs过继转移至WT小鼠的炎症腹膜中后，其免疫表型成熟过程与行为模式恢复正常；反之，将WT型MoMacs过继转移至CGD小鼠的炎症腹膜中后，其成熟过程与行为模式也出现异常改变。本研究数据表明，CGD小鼠体内MoMacs存在招募增强与成熟过程的根本性缺陷。整体实验设计：从C57BL/6（WT）与gp91phox-/y（CGD）小鼠中分离单核细胞及单核细胞衍生巨噬细胞，于腹腔注射酵母聚糖后的3个时间点通过腹膜灌洗获取样本。通过流式细胞术分选（FACS），根据表型标志物Ly6C与MHCII的差异表达纯化单核细胞/巨噬细胞，最终随时间获得Ly6C+MHCII-、Ly6C+MHCII+、Ly6C-MHCII-及Ly6C-MHCII+四类单核细胞/巨噬细胞群。每个处理条件设置3个重复小鼠队列，最终共获取42份样本进行分析。