Mitochondria complex III-generated superoxide is essential for IL-10 secretion in macrophages. Mitochondria complex III-generated superoxide is essential for IL-10 secretion in macrophages

Mitochondrial electron transport chain (ETC) function modulates macrophage biology, however, mechanisms underlying mitochondrial ETC control of macrophage immune responses are not fully understood. Here we report that mutant mice with mitochondrial ETC complex III (CIII)-deficient macrophages exhibit increased susceptibility to influenza A virus and LPS-induced endotoxic shock. Cultured bone marrow-derived macrophages (BMDMs) isolated from these mitochondrial CIII-deficient mice released less IL-10 than controls following TLR3 or TLR4 stimulation. Surprisingly, restoring mitochondrial respiration without generating superoxide using alternative oxidase (AOX) was not sufficient to reverse LPS-induced endotoxic shock susceptibility or restore IL-10 release. However, activation of protein kinase A (PKA) rescued IL-10 release in mitochondrial CIII-deficient BMDMs following LPS stimulation. Additionally, mitochondrial CIII deficiency did not affect BMDM responses to interleukin-4 (IL-4) stimulation. Thus, our results highlight the essential role of mitochondrial CIII generated superoxide in the release of anti-inflammatory IL-10 in response to TLR stimulation Overall design: Bone-marrow derived macrophages from mice with either intact QPC expression or QPC KO were treated with either LPS, IL-4, or vehicle. BMDMs were stimulated with 0.1µg/mL ultrapure O5:B55 LPS (Invivogen tlrl-pb5lps) or IL-4 10ng/mL for 18h prior to collection.

线粒体电子传递链（ETC）功能可调控巨噬细胞生物学特性，然而线粒体ETC调控巨噬细胞免疫应答的具体机制尚未完全阐明。本研究报道，巨噬细胞线粒体电子传递链复合物III（CIII）缺陷的突变小鼠，对甲型流感病毒感染及脂多糖（LPS）诱导的内毒素休克易感性显著升高。从该线粒体CIII缺陷小鼠体内分离培养的骨髓来源巨噬细胞（BMDMs），经Toll样受体3（TLR3）或Toll样受体4（TLR4）刺激后，其分泌的白细胞介素10（IL-10）水平较对照组更低。令人意外的是，通过交替氧化酶（AOX）在不产生超氧阴离子的前提下恢复线粒体呼吸功能，既不足以逆转LPS诱导的内毒素休克易感性，也无法恢复IL-10的分泌水平。然而，在LPS刺激后，蛋白激酶A（PKA）的激活可挽救线粒体CIII缺陷BMDMs的IL-10分泌能力。此外，线粒体CIII缺陷并不会影响BMDMs对白细胞介素4（IL-4）刺激的应答反应。综上，本研究结果凸显了线粒体CIII产生的超氧阴离子，在Toll样受体刺激下介导抗炎性IL-10分泌过程中的关键作用。

实验整体设计：分别对QPC基因正常表达与QPC基因敲除（KO）的小鼠骨髓来源巨噬细胞，施以脂多糖、白细胞介素4或溶剂对照处理。具体刺激方案为：以0.1μg/mL超纯O5:B55型脂多糖（Invivogen货号tlrl-pb5lps）或10ng/mL白细胞介素4刺激BMDMs，孵育18小时后收集样本。