Macrophages target <i>Salmonella</i> by Lc3-associated phagocytosis in a systemic infection model

Innate immune defense against intracellular pathogens, like <i>Salmonella</i>, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of <i>Salmonella</i> during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. <i>enterica</i> serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages <i>in vivo</i>. We show that phagocytosis of live but not heat-killed <i>Salmonella</i> triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of <i>atg5</i> reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to <i>atg13</i> deficiency, indicating that Lc3-<i>Salmonella</i> association at this stage is independent of the autophagy preinitiation complex and that macrophages target <i>Salmonella</i> by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a <i>Salmonella</i> biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against <i>Salmonella</i> during systemic infection. <b>Abbreviations</b>: ATG: autophagy related gene; BECN1: Beclin 1; CFU: colony forming units; CYBA/P22PHOX: cytochrome b-245, alpha chain; CYBB/NOX2: cytochrome b-245 beta chain; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hfp: hours post fertilization; hpi: hours post infection; IRF8: interferon regulatory factor 8; Lcp1/L-plastin: lymphocyte cytosolic protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1A/1B-light chain 3; mCherry: red fluorescent protein; <i>mpeg1</i>: macrophage expressed gene 1; <i>mpx</i>: myeloid specific peroxidase; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX: neutrophil cytosolic factor 4; NTR-mCherry: nitroreductase-mCherry fusion; PTU: phenylthiourea; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB-1 inducible coiled coin 1; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; RUBCN/RUBICON: RUN and cysteine rich domain containing BECN1-interacting protein; SCV: <i>Salmonella</i>-containing vacuole; S. Typhimurium/S.T: <i>Salmonella enterica</i> serovar Typhimurium; TEM: transmission electron microscopy; <i>Tg</i>: transgenic; TSA: tyramide signal amplification; ULK1/2: unc-51-like autophagy activating kinase 1/2; UVRAG: UVRAG: UV radiation resistance associated; wt: wild type针对沙门氏菌（Salmonella）这类胞内病原体的先天免疫防御，高度依赖宿主的自噬（autophagy）机制。该应答在胃肠感染期间作为沙门氏菌侵染靶标的上皮细胞中得到了广泛研究。然而，目前对于自噬在巨噬细胞（macrophages）中的作用尚不清楚——巨噬细胞是系统性感染阶段该病原体的主要载体。本研究利用斑马鱼胚胎模型，在体内（in vivo）探究肠炎沙门氏菌鼠伤寒血清型（S. enterica serovar Typhimurium）与巨噬细胞巨自噬（macroautophagy/autophagy）机制的互作过程。 我们发现，活沙门氏菌（而非热灭活沙门氏菌）的吞噬作用会以多种模式触发自噬标记物绿色荧光蛋白-Lc3（GFP-Lc3）的招募，这些模式可标记包裹细菌的致密或疏松囊泡，该现象也可通过电子显微镜（electron microscopy）得到验证。中性粒细胞（neutrophils）也表现出类似的GFP-Lc3招募关联，但针对中性粒细胞/巨噬细胞平衡的基因调控实验以及细胞消融实验表明，巨噬细胞对于免疫防御应答至关重要。 与atg13基因缺陷不同，atg5基因缺陷会降低GFP-Lc3的招募水平并削弱宿主抵抗力，这表明该阶段Lc3与沙门氏菌的结合不依赖于自噬预起始复合物（autophagy preinitiation complex），且巨噬细胞通过LC3相关吞噬作用（Lc3-associated phagocytosis, LAP）靶向清除沙门氏菌。与此一致的是，RUN及富含半胱氨酸结构域的BECN1相互作用蛋白（Rubcn/Rubicon）基因缺陷会削弱GFP-Lc3的招募以及宿主抵抗力；该蛋白被报道为经典自噬（canonical autophagy）的负调控因子，同时也是LAP的诱导因子。 我们还发现该过程严格依赖于烟酰胺腺嘌呤二核苷酸磷酸氧化酶（nicotinamide adenine dinucleotide phosphate oxidase, NADPH oxidase）——这是另一种LAP发挥功能所必需的因子。Rubcn与NADPH氧化酶均为激活感染巨噬细胞内沙门氏菌活性氧（reactive oxygen species, ROS）生物传感器所必需。上述研究结果证实，LAP是系统性感染阶段巨噬细胞抵御沙门氏菌的主要宿主保护性自噬相关通路。 **缩写说明**： ATG：自噬相关基因（autophagy related gene） BECN1：Beclin 1 CFU：菌落形成单位（colony forming units） CYBA/P22PHOX：细胞色素b-245 α亚基（cytochrome b-245, alpha chain） CYBB/NOX2：细胞色素b-245 β亚基（cytochrome b-245 beta chain） dpf：受精后天数（days post fertilization） EGFP：增强型绿色荧光蛋白（enhanced green fluorescent protein） GFP：绿色荧光蛋白（green fluorescent protein） hfp：受精后小时数（hours post fertilization） hpi：感染后小时数（hours post infection） IRF8：干扰素调节因子8（interferon regulatory factor 8） Lcp1/L-plastin：淋巴细胞胞质蛋白1（lymphocyte cytosolic protein 1） LAP：LC3相关吞噬作用（LC3-associated phagocytosis） MAP1LC3/LC3：微管相关蛋白1A/1B轻链3（microtubule-associated protein 1A/1B-light chain 3） mCherry：红色荧光蛋白（red fluorescent protein） mpeg1：巨噬细胞表达基因1（macrophage expressed gene 1） mpx：髓系特异性过氧化物酶（myeloid specific peroxidase） NADPH oxidase：烟酰胺腺嘌呤二核苷酸磷酸氧化酶（nicotinamide adenine dinucleotide phosphate oxidase） NCF4/P40PHOX：中性粒细胞胞质因子4（neutrophil cytosolic factor 4） NTR-mCherry：硝基还原酶-mCherry融合蛋白（nitroreductase-mCherry fusion） PTU：苯硫脲（phenylthiourea） PtdIns3K：III型磷脂酰肌醇3-激酶（class III phosphatidylinositol 3-kinase） PtdIns3P：磷脂酰肌醇3-磷酸（phosphatidylinositol 3-phosphate） RB1CC1/FIP200：RB-1诱导卷曲螺旋蛋白1（RB-1 inducible coiled-coil 1） ROS：活性氧（reactive oxygen species） RT-PCR：逆转录聚合酶链反应（reverse transcriptase polymerase chain reaction） RUBCN/RUBICON：RUN及富含半胱氨酸结构域的BECN1相互作用蛋白（RUN and cysteine rich domain containing BECN1-interacting protein） SCV：沙门氏菌包含囊泡（Salmonella-containing vacuole） S. Typhimurium/S.T：肠炎沙门氏菌鼠伤寒血清型（Salmonella enterica serovar Typhimurium） TEM：透射电子显微镜（transmission electron microscopy） Tg：转基因（transgenic） TSA：酪酰胺信号扩增（tyramide signal amplification） ULK1/2：UNC-51样自噬激活激酶1/2（unc-51-like autophagy activating kinase 1/2） UVRAG：UV辐射抗性相关蛋白（UV radiation resistance associated） wt：野生型（wild type）