Lawsonia intracellularis exploits β-catenin/Wnt and Notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation

Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE.

胞内劳森菌（Lawsonia intracellularis）是一种专性胞内细菌病原体，可引发猪增生性肠病（PE）。该菌感染会导致肠道隐窝细胞广泛增殖，并抑制分泌细胞与吸收细胞的分化。然而，介导增生性肠病发生的宿主上游细胞通路仍未明晰。β-连环蛋白/Wnt信号通路（β-catenin/Wnt signalling）对维持肠干细胞（ISC）的增殖与自我更新能力至关重要，而Notch信号通路（Notch signalling）则调控分泌细胞系与吸收细胞系的分化命运。因此，本研究采用免疫荧光（IF）与定量逆转录聚合酶链反应（RTqPCR）技术，检测了攻毒后3、7、14、21及28天的未感染与胞内劳森菌感染猪回肠组织中β-连环蛋白/Wnt通路与Notch-1信号通路的活化水平。研究结果显示，尽管胞内劳森菌感染峰值期隐窝内Ki67阳性细胞核数量显著增加，提示细胞增殖增强，但细胞生长启动因子c-MYC与肠干细胞增殖促进因子ASCL2的表达均出现下调。感染峰值期同时伴随β-连环蛋白的胞质与膜定位染色增强，以及AXIN2与SOX9转录本的诱导表达——这两个基因均编码β-连环蛋白/Wnt信号通路的负调控因子，提示β-连环蛋白/Wnt通路的活化水平可能发生改变，且其靶基因的表达呈现差异化调控。研究发现，HES1与OLFM4的表达上调、ATOH1转录本水平下调，与感染峰值期隐窝内Notch-1信号通路的活化增强相一致。值得注意的是，ATOH1转录本水平的显著下调与14天攻毒后MUC2表达的耗竭相吻合，这符合ATOH1促进杯状细胞成熟的功能。感染峰值期LGR5转录本水平无显著变化，提示隐窝增生并非源于肠干细胞群体的扩增。总体而言，在胞内劳森菌感染峰值期，Notch-1信号通路的同时活化与β-连环蛋白/Wnt通路的衰减，似乎与杯状细胞成熟受抑制及隐窝细胞增殖增强相关。此外，隐窝与肠腔细胞间凋亡调控的明显差异，结合14天攻毒后Notch-1信号通路的强活化以及沿隐窝分布的SOX9表达上调，提示活跃增殖的转运扩增细胞和/或吸收祖细胞群体发生了扩增，这为理解增生性肠病的发生与维持机制提供了潜在的理论基础。