Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory cytokines production in mouse macrophages

Giardia duodenalis is an important zoonotic protozoan that has a significant negative impact on public health worldwide. G. duodenalis activates Toll-like receptors (TLRs) and NOD-like receptors (NLRs)-dependent inflammation, but which parasite component activates these immune pathways remain to be further explored. G. duodenalis macrophage migration inhibitory factor (GdMIF) has close similarity with mammalian MIF. However, the immunomodulatory mechanisms of GdMIF remain poorly understood. Here, we found that G. duodenalis could secrete the GdMIF protein, and demonstrated that recombinant GdMIF (rGdMIF) acts as a potent immunostimulatory molecule in mouse peritoneal macrophages (PMϕs). rGdMIF stimulation significantly upregulated TLR2 and TLR4 expression and triggered robust secretion of cytokines (IL-6, IL-10, IL-12, TNF-α, and IL-1β). Mechanistically, rGdMIF activated the MAPK (p38, ERK), AKT, and NF-κB signaling pathways. TLR4 deficiency (TLR4-/-) markedly attenuated rGdMIF-induced IL-6, IL-10, IL-12, TNF-α and IL-1β secretion, and significantly reduced phosphorylation of p38, ERK, and AKT, whereas TLR2-/- had no effect. Subsequently, pretreatment of PMϕs with specific inhibitors of p38, ERK further suppressed, while AKT inhibition slightly enhanced rGdMIF-induced cytokines secretion. Besides, rGdMIF also potently activated the NLRP3 inflammasome, evidenced by increased NLRP3 expression, Caspase-1 cleavage (p20), mature IL-1β (p17) production, IL-1β secretion, LDH release, and cytoplasmic NLRP3 aggregation. The NLRP3 activation was abolished in NLRP3-/- and TLR4-/- PMϕs, but still observed in TLR2-/- PMϕs. These findings demonstrated that GdMIF activated TLR4-dependent MAPK and AKT signaling to regulate IL-6, IL-10, IL-12, TNF-α and IL-1β secretion, and drived TLR4-mediated NLRP3 inflammasome activation for IL-1β maturation and release. GdMIF is a novel G. duodenalis-derived pathogen-associated molecular pattern (PAMP) recognized by TLR4. This study provides new insights into the molecular mechanisms of G. duodenalis-host innate immune interactions.十二指肠贾第虫（Giardia duodenalis）是一种重要的人兽共患原生动物，对全球公共卫生具有显著负面影响。十二指肠贾第虫可激活依赖Toll样受体（Toll-like receptors，TLRs）和NOD样受体（NOD-like receptors，NLRs）的炎症通路，但目前尚不清楚哪种寄生虫组分可激活此类免疫通路，有待进一步探究。十二指肠贾第虫巨噬细胞移动抑制因子（Giardia duodenalis macrophage migration inhibitory factor，GdMIF）与哺乳动物MIF具有高度相似性，但其免疫调节机制仍有待深入阐明。本研究发现，十二指肠贾第虫可分泌GdMIF蛋白，并证实重组GdMIF（rGdMIF）在小鼠腹腔巨噬细胞（mouse peritoneal macrophages，PMϕs）中是一种强效免疫刺激分子。rGdMIF刺激可显著上调TLR2与TLR4的表达，并触发细胞因子（IL-6、IL-10、IL-12、TNF-α及IL-1β）的大量分泌。机制研究显示，rGdMIF可激活丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）（p38、ERK）、蛋白激酶B（AKT）及核因子κB（nuclear factor kappa-B，NF-κB）信号通路。TLR4缺陷（TLR4-/-）可显著减弱rGdMIF诱导的IL-6、IL-10、IL-12、TNF-α及IL-1β分泌，并显著降低p38、ERK及AKT的磷酸化水平，而TLR2缺陷（TLR2-/-）则无此类影响。后续实验中，采用p38、ERK的特异性抑制剂预处理巨噬细胞可进一步抑制rGdMIF诱导的细胞因子分泌，而AKT抑制剂则可小幅增强该效应。此外，rGdMIF还可强效激活NLRP3炎症小体，具体表现为NLRP3表达上调、半胱天冬酶-1（Caspase-1）剪切体（p20）生成增加、成熟IL-1β（p17）产生增多、IL-1β分泌及乳酸脱氢酶（lactate dehydrogenase，LDH）释放升高，以及胞质内NLRP3聚集。在NLRP3-/-及TLR4-/-巨噬细胞中，NLRP3的激活被完全消除，但在TLR2-/-巨噬细胞中仍可观察到该激活现象。上述研究结果证实，GdMIF可通过激活TLR4依赖的MAPK及AKT信号通路，调控IL-6、IL-10、IL-12、TNF-α及IL-1β的分泌，并介导TLR4依赖的NLRP3炎症小体激活以促进IL-1β的成熟与释放。GdMIF是一种新型的十二指肠贾第虫来源的病原相关分子模式（pathogen-associated molecular pattern，PAMP），可被TLR4识别。本研究为阐明十二指肠贾第虫与宿主先天免疫互作的分子机制提供了新的研究视角。