文献学习101–IL-33诱导的代谢重编程控制选择性激活巨噬细胞的分化与炎症的消退

1. IL-33 induces the delayed differentiation of a pro- resolving AAM phenotype

在anterior tibial muscle胫骨前肌注射cardiotoxin (CTX)可以引起局部肌肉坏死和组织损伤炎症反应。表现为大量的单核细胞及单核衍生的巨噬细胞的浸润。而坏死组织的清除、炎症的消退和肌纤维的重生都非常依赖于subsequent differentiation of pro-resolving AAMs。
Fig 1a-c： 为了探究IL33的作用，作者首先使用了Il1rl1^-/-的小鼠，发现敲除鼠在CTX注射后损伤加重，恢复减慢。提示IL-33-IL1RL1 signaling axis在抑制免疫损伤和促进组织修复中发挥作用。
Fig 1d-f： 作者对WT和Il1rl1^-/-小鼠的BMDM给予了IL-33干预，并进行了转录组测序。This analysis confirmed that the IL-33- IL1RL1 axis specifically induced the concomitant expression of pro- and anti-inflammatory genes. （Arg1和Retnla主要参与向修复巨噬的转变）
Fig 1g-i： qpcr, western和elisa也得到了一致的结果。
Fig 1j： In accordance with a switch toward a pro-resolving macrophage phenotype, IL-33-activated macrophages also displayed an increased capacity to ingest and clear necrotic cells.
Fig 1k： Other pro-inflammatory alarmins and IL-1 family members such as IL-1b and IL-18 that equally use MyD88-dependent signaling, in turn, showed no impact on AAM markers (K), while IL-4 similarly induced markers of AAM differentiation (G and H) but failed to induce pro-inflammatory cytokines.

而且IL33诱导的促炎基因和抗炎基因的表达具有不同的kenetics （早期释放促炎细胞因子，稍晚则转变为促修复细胞因子）

2. IL-33 and IL-4 induce distinct AAM phenotypes

为了探究损伤时IL33在不依赖于IL4的情况下诱导促修复AAM表型的特征，作者对IL33和IL4诱导的AAMs进行了转录组测序。
Fig 2a-d： IL33和IL4诱导的AAM具有不同的转录特征。
Fig 2e-f： 敲除IL4受体基因阻断了IL4诱导的促修复基因表达，但不影响IL33介导的促修复基因表达。
Fig 2g-i： 然而，deletion of the gene encoding for the signaling adaptor MyD88，阻断了IL33诱导的促修复基因表达，但不影响IL4介导的促修复基因表达。
These results confirm that IL-33 and IL-4 use different receptors and proximal signaling adaptors to imprint an AAM phenotype.

3. IL-33-induced AAM differentiation depends on mitochondrial uncoupling

Fig 3a： 对IL33诱导基因的功能富集提示IL33影响了巨噬细胞的代谢。
由于代谢与巨噬细胞极化关系密切，作者随后dissect these IL-33-induced metabolic changes in macrophages in more detail。
Fig 3b-d： 与IL4和LPS不同，IL33刺激6h后不直接影响巨噬细胞的extracellular acidification rate 和 basal nor the maximal respiratory capacity。
Fig 3c-f： IL-33-treated macrophages rapidly developed a proton leak after IL-33 stimulation, which indicated an IL-33- induced and IL1RL1-dependent uncoupling of the mitochondrial respiratory chain in macrophages.
Fig 3g： Uncoupling was not associated with transcriptional changes in the expression of genes encoding for uncoupling proteins (UCPs) such as UCP1 or UCP2. 然而，作者在IL33处理的巨噬中观察到UCP2蛋白在线粒体的积聚。在Il1rl1^-/-细胞中则没有出现这种现象，而且IL33处理的巨噬中UCP2蛋白在线粒体的积聚在使用p38 MAPK信号通路的抑制剂 SB 203580抑制后消失。提示IL-33- induced and p38 MAPK-mediated translocation of preformed UCP2 into the mitochondrial compartment.

线粒体质子漏（Proton Leak）是指在线粒体内质膜上存在的一种通透性蛋白通道，允许质子从线粒体膜间隙返回线粒体内膜内，从而绕过线粒体ATP合酶（ATP synthase）。正常情况下，质子在线粒体内膜上形成质子梯度，通过ATP合酶驱动ADP和磷酸转化为ATP。然而，质子漏会导致质子梯度的损失，减少ATP的产生效率。
质子漏可以通过调节线粒体内膜上的蛋白通道来发生。一个重要的质子漏通道是线粒体内膜上的UCP（uncoupling protein）家族蛋白。UCP通道可以被激活，使质子通过线粒体内膜，而不经过ATP合酶。这样一来，质子梯度的能量将被释放为热量，而不是用于ATP合成。质子漏的发生可以增加线粒体呼吸过程中产生的热量，并影响细胞能量代谢和体温调节。
质子漏可作为线粒体损伤的标志，也可被认为是调节线粒体ATP合成的一种机制。

此前文献报道，Mitochondrial uncoupling可以限制 reverse electron transport 来源的过度ROS产生。而ROS产生则是炎性巨噬细胞的特征。例如在LPS诱导的巨噬细胞中，过量的ROS可以引起线粒体损伤，损害线粒体功能 and/or 阻断TCA) cycle。

Fig 3h： 随后作者使用药物compound genipin (GNP, 抑制细胞中的 UCP2)阻断了mitochondrial uncoupling，we observed that GNP treatment of IL- 33-stimulated macrophages resulted in increasingly impaired mitochondrial function and a decrease in the oxygen consumption rate after prolonged stimulation for 24 h。

In summary, these data thus indicated that IL-33-activated macrophages displayed a metabolic rewiring that was distinct from IL-4- or LPS-treated macrophages and characterized by UCP2- mediated uncoupling of the respiratory chain, which blocked the generation of ROS and allowed sustained mitochondrial respiration as well as an intact TCA cycle in otherwise pro-inflammatory macrophages.

Fig 3i-l： 为了进一步验证前面的结果，作者使用IL33和IL4对GNP处理的巨噬和Ucp2^-/-巨噬进行了诱导。发现解偶联后IL33对修复性巨噬细胞的诱导能力消失，而IL4对修复性巨噬细胞的诱导能力则没有受到影响。
Fig 3j： Forced uncoupling induced by 2,4-dinitrophenol (DNP) further increased IL-33-mediated ARG1 expression but did not promote AAM differentiation in the absence of IL-33.
Fig 3m： IL-33诱导的促炎性细胞因子如TNF等的表达则不依赖于UCP2。

4. IL-33-mediated mitochondrial uncoupling results in a shift of metabolites and a subsequent expression of the transcription factor GATA3

前面的数据显示UCP2介导的线粒体呼吸链解耦连对线粒体功能、巨噬细胞plasticity、和IL33诱导下巨噬细胞的AAM分化十分重要，作者想要去探究其背后的机制。
Fig 4a-b： 作者对IL-33刺激的WT, GNP-treated, 和 Ucp2^-/-�巨噬进行了MS质谱检测。In accordance with a central metabolic role of mitochondrial uncoupling during mitochondrial fitness and function, this approach confirmed significant IL-33- induced and UCP2-dependent shifts in intracellular metabolites. 这些UCP2-依赖的改变包括IL33诱导的衣康酸和其它TCA代谢物如琥珀酸和延胡索酸的增加，都在阻断或UCP2-/-后下降。（ITAC就是衣康酸）
Fig 4c-d： Irg1^-/-影响IL-33诱导的AAM分化，不影响IL-4诱导的AMM分化。
Fig 4e： 为了探究IL33驱动AAM分化的转录因子，作者使用TRANSFAC进行了转录因子预测。结果显示除了与IL4诱导AAM分化一致，存在着NF-kB, AP-1, IRF, and NFAT家族转录因子的活化，GATA家族的转录因子同样是IL33诱导的巨噬细胞转录变化的mediators。
Fig 4f-g： IL33而不是IL4诱导下Gata3的表达上调。
Fig 4h： 而且IL-33诱导的 GATA3表达是依赖于UCP2-mediated mitochondrial uncoupling 和 ACOD1介导的衣康酸产生。因为GNP 和 Ucp2 或 Irg1 的敲除抑制了 IL-33 介导的巨噬细胞GATA3的表达。
These data accordingly suggest a central role of GATA3 as metabolic signaling hub in macrophages that responds to an IL-33-induced mitochondrial reprogramming and translates it into global changes in gene expression.

5. GATA3 controls the differentiation of AAMs in response to IL-33

Fig 5a： 随后作者使用Cx3cr1 cre构建了巨噬特异性Gata3敲除鼠(Gata3^Δmac mice)。免疫荧光结果显示在WT小鼠BMDM中，IL33诱导了GATA3在细胞核的聚集。而这个现象在L-33刺激下Gata3^Δmac mice的BMDMs中消失。
Fig 5b-d： RNAseq的结果显示GATA3缺陷确实abrogated IL-33-mediated induction of a subset of IL-33-responsive genes in macrophages.
Fig 5e-f： WB和QPCR做了验证
Fig 5g： Notably, IL-33 failed to increase the capacity of GATA3-deficient macrophages to ingest and clear necrotic cells demonstrating that this transcription factor coordinated various pro-resolving properties of macrophages in response to IL-33.
Fig 5h： The defective IL-33-induced AAM differentiation in GATA3-deficient BMDMs, however, did not alter expression of IL-33-induced Irg1 mRNA and of pro-inflammatory cytokines such as TNF, IL- 6, or IL-1b. Production of IL-1b was even increased.

6. Tissue damage results in the differentiation of pro- inflammatory and pro-resolving mononuclear phagocytes

Fig 6a-c： 随后作者对Gata3^Δmac 鼠和同窝对照进行了肌炎造膜，分选了肌肉中的CD45⁺CD11B⁺LY6G^-单核巨噬细胞，进行了单细胞测序。结果得到6个细胞群，包括2群Ccr2⁺单核和2群Cd68⁺巨噬。5群是DC，6群是增殖巨噬。
Fig 6d-e： 轨迹分析结果提示新招募的 Ccr2^hi单核最先分化为炎性 Il1b⁺单核，随后分化为Trem2^hi和Mylpf⁺巨噬。 从单核分化为促修复的巨噬过程与Ccr2的下调和Cd68和Trem2的上调平行。促炎因子如Il1b, Ccl4, 和 Cxcl2 的表达在intermediary stage，也就是pro-inflammatory Il1b⁺ monocytes达到峰值，而在分化为Trem2^hi 和 Mylpf⁺巨噬时下降。mRNAs such as Mylpf and Des encoding for muscle cell markers accordingly appeared primarily in differentiated pro-resolving macrophages.

7. GATA3 controls the differentiation of pro-resolving macrophages

Fig 7a-b： GATA3的缺陷并没有改变总的单核巨噬细胞数目，但是影响了亚群的比例。CTX诱导的肌肉损伤下，Gata3^Δmac 鼠的l1b⁺ monocytes增加，促修复的Mylpf⁺ macrophages则出现缺失。与前面轨迹分析的结果一致，GATA3的缺陷影响l1b⁺ monocytes向Mylpf⁺ macrophages的分化，因此损伤了对坏死肌肉的吞噬作用。
Fig 7c-d： 促炎巨噬不能向促修复巨噬分化的现象与Gata3^Δmac 鼠巨噬的促炎细胞因子Il1b等增加相一致。
Fig 7e： comparative pseudotime analysis也证实Gata3^Δmac鼠肌肉中的单核吞噬细胞在分化为巨噬的过程中存在exacerbated and prolonged inflammatory response。Accumulation of skeletal muscle mRNAs (e.g., Mylpf) in differentiated macrophages as readout for their pro-resolving and phagocytic activity, in turn, was reduced in Gata3Dmac mice, confirming the decreased efferocytosis observed in the absence of GATA3 in vitro。
Fig 7f-h： GATA3缺陷的小鼠表型更重，修复更慢。
These data thus demonstrated the functional relevance of GATA3 as transcriptional hub in macrophages that controlled their pro-resolving and reparative properties in response to tissue damage.