Data_Sheet_1_Locally generated C3 regulates the clearance of Toxoplasma gondii by IFN-γ-primed macrophage through regulation of xenophagy.ZIP

Exogenous pathogen infection can induce autophagy in cells. Autophagy is essential for cell survival, development, and homeostasis. It not only regulates cell defense and stress, but also has a close relationship with innate and adaptive immunity. Complement is an important part of innate immunity, which could be activated by three approaches, including classic, alternative, and lectin pathways. All the three pathways result in the activation of C3, and generate anaphylatoxin fragments C3a and C5a, and formation of the membrane attack complex. Either C3a or C5a induces the inflammatory cytokines through binding to C3aR or C5aR, respectively. However, it is still unknown whether the complement could regulate the autophagy of intracellular microorganisms or not. In this study, we constructed a Toxoplasma gondii (T. gondii) and macrophages co-culture experimental model using T. gondii expressing enhanced green fluorescence protein (EGFP) fluorescence and C3−/-C57BL/6 J mice for that T. gondii invaded peritoneal macrophages in mice. Western blot, laser confocal microscopy (LCM), and transmission electron microscopy (TEM) were used to observe the changes of autophagy between the macrophages from wild-type (WT) and C3−/− mice. Flow cytometry and LCM were used to investigate the effect of autophagy on the killing ability of macrophages against T. gondii. Here, we found that local C3 could suppress not only the canonical autophagy of macrophage, but also the xenophagy to T. gondii. Interestingly, the inhibition of C3 on host cell autophagy could significantly suppress the clearance of T. gondii by the IFN-γ-primed macrophage. Finally, we investigated the mechanism of the autophagy regulation of C3 that the effect of C3 on the macrophage-specific autophagy against T. gondii depends on mTOR. And, there is C3a but not C5a/C5aR involved in regulating macrophage xenophagy against T. gondii. Collectively, our findings suggest locally generated C3 regulates the clearance of T. gondii by Macrophage through the regulation of the non-canonical IFN-γ-dependent autophagy pathway, and paint a clearer picture in the regulation of autophagy by innate immune components.

外源性病原体感染可诱导细胞发生自噬。自噬对于细胞的存活、发育及稳态维持至关重要。它不仅调控细胞的防御和应激反应，而且与先天性和适应性免疫紧密相关。补体是先天免疫的重要组成部分，可通过经典途径、替代途径和凝集素途径三种方式被激活。三种途径均导致C3的活化，并生成过敏性毒素片段C3a和C5a，以及形成膜攻击复合物。C3a或C5a分别通过与C3aR或C5aR结合，诱导炎症细胞因子。然而，补体是否能够调节细胞内微生物的自噬尚不明确。在本研究中，我们构建了采用表达增强型绿色荧光蛋白（EGFP）荧光的弓形虫（Toxoplasma gondii）和巨噬细胞共培养实验模型，并使用C3−/-C57BL/6 J小鼠，以观察弓形虫在感染小鼠腹腔巨噬细胞的过程。通过蛋白质印迹、激光共聚焦显微镜（LCM）和透射电子显微镜（TEM）观察野生型（WT）和C3−/−小鼠巨噬细胞中自噬的变化。利用流式细胞术和LCM探究自噬对巨噬细胞杀灭弓形虫能力的影响。我们发现，局部C3不仅能抑制巨噬细胞的经典自噬，还能抑制针对弓形虫的外源性自噬。有趣的是，C3对宿主细胞自噬的抑制能够显著抑制由IFN-γ预处理的巨噬细胞清除弓形虫的能力。最终，我们研究了C3调节自噬的机制，发现C3对巨噬细胞特异性自噬作用的影响依赖于mTOR。此外，在调节巨噬细胞针对弓形虫的外源性自噬过程中，仅涉及C3a，而不涉及C5a/C5aR。总体而言，我们的研究结果表明，局部生成的C3通过调节非经典IFN-γ依赖的自噬途径，调节巨噬细胞清除弓形虫，并在先天免疫成分调节自噬的过程中描绘了一幅更为清晰的图景。