von Mücke-Heim Iven-Alex, Martin Jana, Uhr Manfred, Ries Clemens, Deussing Jan M
Molecular Neurogenetics, Max Planck Institute of Psychiatry, Munich, Germany.
Core Unit Analytics and Mass Spectrometry, Max Planck Institute of Psychiatry, Munich, Germany.
Front Pharmacol. 2023 Apr 10;14:1148190. doi: 10.3389/fphar.2023.1148190. eCollection 2023.
In recent years, purinergic signaling via the P2X7 receptor (P2X7R) on microglia has repeatedly been implicated in depression genesis. However, it remains unclear which role the human P2X7R (hP2X7R) plays in regulating both microglia morphology and cytokine secretion upon different environmental and immune stimuli, respectively. For this purpose, we used primary microglial cultures derived from a humanized microglia-specific conditional P2X7R knockout mouse line to emulate different gene-environment interactions between microglial hP2X7R and molecular proxies of psychosocial and pathogen-derived immune stimuli. Microglial cultures were subjected to treatments with the agonists 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP) and lipopolysaccharides (LPS) combined with specific P2X7R antagonists (JNJ-47965567, A-804598). Morphotyping revealed overall high baseline activation due to the in vitro conditions. Both BzATP and LPS + BzATP treatment increased round/ameboid microglia and decreased polarized and ramified morphotypes. This effect was stronger in hP2X7R-proficient (CTRL) compared to knockout (KO) microglia. Aptly, we found antagonism with JNJ-4796556 and A-804598 to reduce round/ameboid microglia and increase complex morphologies only in CTRL but not KO microglia. Single cell shape descriptor analysis confirmed the morphotyping results. Compared to KO microglia, hP2X7R-targeted stimulation in CTRLs led to a more pronounced increase in microglial roundness and circularity along with an overall higher decrease in aspect ratio and shape complexity. JNJ-4796556 and A-804598, on the other hand, led to opposite dynamics. In KO microglia, similar trends were observed, yet the magnitude of responses was much smaller. Parallel assessment of 10 cytokines demonstrated the proinflammatory properties of hP2X7R. Following LPS + BzATP stimulation, IL-1β, IL-6, and TNFα levels were found to be higher and IL-4 levels lower in CTRL than in KO cultures. Vice versa, hP2X7R antagonists reduced proinflammatory cytokine levels and increased IL-4 secretion. Taken together, our results help disentangle the complex function of microglial hP2X7R downstream of various immune stimuli. In addition, this is the first study in a humanized, microglia-specific model identifying a so far unknown potential link between microglial hP2X7R function and IL-27 levels.
近年来，小胶质细胞上通过P2X7受体（P2X7R）的嘌呤能信号传导反复被认为与抑郁症的发生有关。然而，目前尚不清楚人类P2X7R（hP2X7R）在分别调节不同环境和免疫刺激下小胶质细胞形态和细胞因子分泌中发挥何种作用。为此，我们使用了源自人源化小胶质细胞特异性条件性P2X7R基因敲除小鼠品系的原代小胶质细胞培养物，以模拟小胶质细胞hP2X7R与心理社会和病原体衍生免疫刺激的分子替代物之间不同的基因-环境相互作用。小胶质细胞培养物用激动剂2'（3'）-O-（4-苯甲酰苯甲酰）-ATP（BzATP）和脂多糖（LPS）联合特异性P2X7R拮抗剂（JNJ-47965567、A-804598）进行处理。形态学分析显示，由于体外条件，整体基线激活水平较高。BzATP和LPS + BzATP处理均增加了圆形/阿米巴样小胶质细胞，减少了极化和分支形态。与基因敲除（KO）小胶质细胞相比，这种效应在hP2X7R功能正常（CTRL）的小胶质细胞中更强。恰当地，我们发现用JNJ-4796556和A-804598拮抗仅在CTRL小胶质细胞中减少圆形/阿米巴样小胶质细胞并增加复杂形态，而在KO小胶质细胞中则不然。单细胞形状描述符分析证实了形态学分析结果。与KO小胶质细胞相比，在CTRL小胶质细胞中靶向hP2X7R的刺激导致小胶质细胞的圆度和圆形度更明显增加，同时长宽比和形状复杂度总体下降幅度更大。另一方面，JNJ-4796556和A-804598导致相反的动态变化。在KO小胶质细胞中观察到类似趋势，但反应幅度要小得多。对10种细胞因子的平行评估证明了hP2X7R的促炎特性。在LPS + BzATP刺激后，发现CTRL培养物中的IL-1β、IL-6和TNFα水平高于KO培养物，而IL-4水平低于KO培养物。反之，hP2X7R拮抗剂降低了促炎细胞因子水平并增加了IL-4分泌。综上所述，我们的结果有助于厘清各种免疫刺激下游小胶质细胞hP2X7R的复杂功能。此外，这是在人源化、小胶质细胞特异性模型中首次发现小胶质细胞hP2X7R功能与IL-27水平之间迄今未知的潜在联系的研究。
