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

Abstract

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.

A novel inactivated vaccine against Lawsonia intracellularis induces rapid induction of humoral immunity, reduction of bacterial shedding and provides robust gut barrier function

“…Similarly, the level of cleaved-CASP3 in lumen of the ileum of vaccinated pigs was, at least in part, reduced to the level observed in sentinel pigs. This may indicate a possible reduction in apoptosis mechanisms as previously described [16] . In contrast, we found that the induction of CP, a marker of neutrophil and macrophage infiltration, was still noticeably expressed in the ileum of vaccinated pigs, perhaps indicating that innate immune cells were actively patrolling the lamina propria at 21 dpc [24] .…”

“…Bacterial loads were also significantly lower in the feces of vaccinated animals, both in terms of reduced severity (bacteria shed per day) and in reduction of the duration of fecal shedding. Reduced proliferation of enterocytes in vaccinated pigs compared to controls, as demonstrated by H&E staining of ileum tissues, coincided with significantly greater numbers of MUC2-expressing goblet cells and IAP-expressing brush border cells than controls indicating a restoration of these essential intestinal cell lineages [6] , [16] , [18] , [19] , [20] . Similarly, the level of cleaved-CASP3 in lumen of the ileum of vaccinated pigs was, at least in part, reduced to the level observed in sentinel pigs.…”

“…In particular, molecular markers that identify secretion (PAS signal and Mucin2, MUC2), absorption and homeostasis (Intestinal alkaline phosphatase, IAP), apoptosis (Cleaved-caspase3, CASP3) and immune cell distribution (Calprotectin, CP) were monitored ( Figs. 5 and 6 ) [6] , [16] , [18] , [19] , [20] . In the vaccinated group (n = 23) the level of PAS, MUC2 as well as IAP was significantly higher than controls (n = 25; p < .01, p < .01, p < .001, respectively) .…”

“…For quantitative analysis of PAS staining for Fig. 6 , images were acquired at 20× magnification from at least three random fields per condition (Sentinels, Controls, Vaccinates) and processed using Image J 1.49S as described previously [6] , [16] . Briefly, the green channel was selected and the intensity threshold was adjusted to highlight goblet cells.…”

Lawsonia intracellularis: Revisiting the Disease Ecology and Control of This Fastidious Pathogen in Pigs

“…It is now understood that apoptosis is not involved in the pathogenesis of PE, even though contradicting views were reported earlier ( 16 , 21 ). A recent study suggests that a combination of Notch-1 signaling and disruption of the β-catenin/Wnt pathway may be associated with immature crypt cell proliferation ( 24 ). Failure of crypt cells to differentiate into goblet and absorptive cells results in a severe decrease of MUC2 glycoprotein in the affected epithelium at the peak of infection ( 24 ).…”