Listeria monocytogenes is a major foodborne pathogen that causes listeriosis and has a high mortality rate ranging from 20 to 30% (de Noordhout et al., 2014). Its severity is attributed to the fact that it can cross the intestinal, placental, and blood-brain barriers in immunocompromised individuals, inducing gastroenteritis, abortions, and meningitis (Doganay, 2003).

As the first defense barrier, the intestine plays a vital role in L. monocytogenes defense (Gahan and Hill, 2014; Becattini and Pamer, 2018). Under normal conditions, mucosal epithelial cells, mucus-secreting cells, and immune cells form a protective barrier against invading pathogens (Ruch and Engel, 2017). Related studies mainly focused on the protective effects of immune cells during L. monocytogenes infection (Cho et al., 2017; Wilharm et al., 2021). However, the intestinal epithelial cells compose numerous cell types that help maintain intestinal homeostasis and defend against enteric pathogen invasion. Specifically, goblet cells can produce mucin glycoproteins and form mucus, and Paneth cells, at the bottom of intestinal crypts, can secret antimicrobial peptides (AMPs). It had been found that L. monocytogenes could damage intestinal homeostasis and affect the differentiation of intestinal epithelial cells. A study on intestinal infection by L. monocytogenes demonstrated a significant effect on the differentiation of epithelial cells by increasing the number of goblet cells in mice (Pian et al., 2020). In addition, it was reported that the counts of Paneth cells in organoids decreased after L. monocytogenes infection for 1 h but increased after infection for 18 h through the regulation of Wnt signaling pathway (Huang et al., 2021). Therefore, these studies indicated that the differentiation of epithelial cells, especially the secretory cells, could be affected by bacteria invasion in vivo or in vitro.

In literature, the proliferation and differentiation of intestinal epithelial cells were confirmed to be regulated by a variety of signaling pathways, such as Wnt, EGF, BMP, and Notch signaling pathways (Date and Sato, 2015). Among them, the Notch signaling pathway played a crucial role in regulating the differentiation of intestinal secretory cells, such as goblet and Paneth cells. Recently, some studies have demonstrated that mice exposed to Cadmium or Salmonella infection led to a loss of goblet cells through the activation of Notch-signaling pathway (Wu et al., 2018; Xie et al., 2020). Conversely, blockade of the Notch pathway using γ-secretase inhibitors led to the conversion of all intestinal epithelial cells into goblet cells (Wong et al., 2004; van der Flier and Clevers, 2009). These studies indicated that the Notch signaling pathway regulated the differentiation of secretory cells under physiological and pathological conditions. However, researches on the influence of intestinal infection caused by L. monocytogenes on the Notch pathway are insufficient.

Animals and cells models are often used to investigate the infection mechanism of the enteric pathogens in the intestine. However, most traditional intestinal cell models have immortalized 2-D cell lines, such as Caco-2 cells containing a single cell type, which cannot reproduce some characteristics of natural infection (Wilson et al., 2015). Recently, intestinal organoids were served as a more effective model to study pathogen-host interactions (Hill and Spence, 2017). In contrast to traditional cell lines, intestinal organoids occupied 3-dimensional space. They formed complex microenvironments that facilitated differentiation and persistence of epithelial subtypes and the formation of villus-like structures, comprised of Paneth cells, Goblet cells, enterocytes, enteroendocrine cells, and stem cells (Watson et al., 2014; Finkbeiner et al., 2015). In addition, it was reported that intestinal organoids were used to visualize the invasiveness of Salmonella and the morphologic changes of the organoids (Zhang et al., 2014). And intestinal organoids also were used to model the infection of L. monocytogenes (Huang et al., 2021), and pathogenic Escherichia coli strains (Vandussen et al., 2014; Rajan et al., 2018), which provided important insights into the pathogenesis of the intestine.

Therefore, in this study, mice and intestinal organoids were used to establish an invasion model of L. monocytogenes to explore its influence on the differentiation of secretory cells and differentiation-related Notch pathway, which will provide more information on the mechanism of L. monocytogenes infection in the intestine.

L. monocytogenes infection is a severe foodborne disease worldwide, reported in more than 30 countries, including the USA, Canada, Germany, Portugal, and Austria (Desai et al., 2019; Jamshidi and Zeinali, 2019). During L. monocytogenes infection, the intestine is a vital defense line. This study showed that L. monocytogenes infection could lead to villi damage with H&E staining, which was closely related to the increase of pro-inflammatory cytokines TNF-α secretion. This corroborated the study of Alkhuriji et al. that L. monocytogenes infection in mice could cause epithelial cells exfoliation and degeneration of the lamina propria and induce the production of TNF-α in the intestine (Alkhuriji et al., 2020). In addition, L. monocytogenes increased the level of LPS in the serum, which indicated that the intestinal permeability was increased (Mokkala, K. et al., 2017).

As specialized intestinal epithelial cells, goblet and Paneth cells play an essential role in mucosal homeostasis and bacterial defense. During infection, goblet cell secretion, which contains mucopolysaccharides, critical in forming the first line of defense by protecting the intestinal barrier against pathogenic invasion, was described extensively (Pelaseyed et al., 2014). This study demonstrated that the number of goblet cells, in vivo and in vitro, and the relative expression of Muc2 gene were significantly increased, which was consistent with the previous study (Pian et al., 2020). Notably, Paneth cells, located at the bottom of the intestinal crypt, could produce various antibacterial peptides to kill pathogens, including regenerating 3γ (reg3γ), lysozyme, and defensin (Bevins and Salzman, 2011). This study showed that the count of Lyz⁺ cells was increased in mice, which was consistent with the protein and mRNA expression levels in mice and organoids. Moreover, it was reported that L. monocytogenes increased the number of Paneth cells during co-culture with organoids for 18 h in vitro (Huang et al., 2021). Although the increasing number of goblet and Paneth cells shielded against epithelial damage, excessive secretory cells may disrupt the intestinal homeostasis by consuming the stem cells (Putman et al., 2011), which could rejuvenate tissue homeostasis and repair injured tissues (Umar, 2002). Therefore, how to preserve the number and function of goblet and Paneth cells during the treatment of bacterial infection is imperative.

The Notch signaling pathway is a development switch for intestinal secretory and absorptive cells. However, its suppression leads to the inhibition of enterocytes differentiation and a dramatic expansion in goblet and Paneth cell numbers (van Es et al., 2005). The Notch signaling was activated when one of the Delta or Jagged Notch transmembrane receptors interacted with one of the five Notch ligands triggering proteolytic cleavage of the receptor (Scoville et al., 2008). The cleavage released the free Notch 1 intracellular domain (NICD) that translocated into the nucleus to upregulate target genes, mainly of Hes class, such as Hes1 in the intestine, which suppressed the Math1 gene (Yang et al., 2001). It was reported that constitutive overexpression of the Notch1 receptor reduced the differentiation of enteroendocrine and Paneth cells, thus decreasing their numbers (Robine et al., 2005). Also, deficiency in Hes1 mice led to an abundance of goblet, enteroendocrine, and Paneth cells, but a reduced number of enterocytes (Suzuki et al., 2005). Conversely, the intestine from Math1 deficient mice exhibited an intestinal epithelium formed only by enterocytes (Yang et al., 2001).

Furthermore, under pathological conditions, Wu et al. found that Salmonella infection induced the loss of goblet cells and reduced the mRNA expression of Muc2 by increasing the expression of Dll1, Dll4, and Hes1 genes, indicating the activation of Notch signaling pathway (Wu et al., 2018). However, to the best of our knowledge, the influence of L. monocytogenes infection on the Notch signaling pathway has not been documented in mice and organoids. Based on the increase in goblet and Paneth cells, our findings speculated whether L. monocytogenes promoted the differentiation of intestinal secretory cells by inhibiting the Notch signaling pathway. Consistent with this hypothesis, the relative expression of related genes of Notch pathway (Jag1, Dll4, Nocth1, and Hes1) were detected and decreased, while the mRNA relative expression of Math1 was upregulated in mice. Also, the intestinal organoids, an effective intestinal cell model, were used to establish an infection model in vitro. The mRNA relative expression of Jag1, Dll4, Nocth1, and Hes1 were down-regulated significantly, while the relative expression of Math1 gene was significantly increased, which was consistent with a previous study (Huang et al., 2021). As such, the decreased expression of the Notch signaling pathway genes demonstrated L. monocytogenes inhibition on the Notch pathway. Combined with these results, the Notch pathway inhibition may be the reason for the increased number of goblet and Paneth cells after L. monocytogenes infection in mice and organoids.

In summary, this study indicated that L. monocytogenes infection damaged the intestinal barrier and upregulated LPS level in serum, and increased the expression of TNF-α in the jejunum. Furthermore, L. monocytogenes infection inhibited the Notch pathway, which may lead to the increasing number of goblet and Paneth cells in the intestine of mice. Therefore, this study provides insight to study the mechanism of L. monocytogenes damage to the intestinal mucosal barrier, which is helpful to control L. monocytogenes pathogenic infection.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Nanjing Agriculture University Committee on Animal Resources Committee and the National Institutes of Health guidelines for the performance of animal experiments.

CZ: Data curation, Formal analysis, Writing - original draft. YYZ: Writing - original draft. AB: Writing - original draft. JH: Writing - original draft. YFZ: Writing - original draft. KY: Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing - original draft, Writing - review and editing. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (32172267) and Program for Student Innovation through Research and Training (202110307047).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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