Investigating the development of Peyer’s patches and the transcriptome of M cells in chickens

Abstract

Considering the approximately 8 billion world population in 2022 and its predicted growth rate of 1.1% per year, there is an increasing demand for food for this growing population. Poultry products such as meat and eggs are excellent sources of protein. Thus, the poultry industry has grown rapidly to meet the global demand for food. Poultry producing sectors heavily rely on vaccination to prevent poultry diseases, maintain welfare, and minimise the risk of disease outbreaks. Applying vaccines through mucosal surfaces such as drinking water and spray is a common approach on an industrial scale.

Understanding the mechanisms by which vaccines are taken up at mucosal surfaces and induce immune responses will help improve vaccine design and targeting.

Mucosal surfaces are protected by the immune responses that are generated by mucosa-associated lymphoid tissues (MALT). Peyer’s patches (PP) are the important parts of MALT which are located in the small intestine. PP are overlaid by follicle-associated epithelium (FAE) containing specialised epithelial cells called M cells that sample gut contents and deliver them to the underlying immune cells to induce antigen-specific immune responses. A better understanding of PP development could contribute to vaccination strategies in the poultry industry. In addition, the functional characteristics of M cells, i.e., translocating antigens across the epithelium to the gut-resident immune system, can be exploited for mucosal vaccine delivery. In chickens, developmental studies of PP has been hampered since chicken PP are not macroscopically as nodular as their mammalian counterparts. Furthermore, there is limited research on the immunobiology and the development of chicken intestinal M cells due to the lack of appropriate M cell-associated markers. Using CSF1R-eGFP reporter transgenic chickens, PP are detectable under the whole-mount microscope as PP contain the aggregations of CSF1R+ cells. Moreover, in these chickens, it was demonstrated that along with mononuclear phagocytes, M cells in the bursa of Fabricius, large intestine, and lung express CSF1R. Therefore, this thesis aimed to study the development of chicken PP and to identify the gene expression profile of chicken bursal M cells using CSF1R-eGFP reporter transgenic chickens, as well as to develop an in vitro model that facilitates chicken intestinal M cell studies. Using RNA sequencing (RNA-seq) analysis of isolated bursal M cells, M cell-associated genes were determined and used to detect M cells in the small intestine and in vitro models.

The spatiotemporal development of chicken PP was studied to examine the hypothesis that the organogenesis of all PP initiates during embryonic development and continues post-hatch. The results demonstrated that chicken PP anlagen are present at predetermined locations at embryonic day 18 (ED18) and their development and expansion continue post-hatch. The distribution and location of up to 7 PP were discovered in the chicken small intestine and PP structure was observed in 2- and 8-week-old chickens based on the germinal centre formation and the reduction of goblet cells in the overlying epithelium through histology.

In this thesis, it was hypothesised that M cell-associated gene signatures are conserved between mice and chickens and between M cells located in different parts of the chicken MALT. In contrast to the intestine, where finding a location with a high density of M cells is hard, FAE covering bursal follicles are enriched with M cells. Accordingly, using CSF1R-eGFP reporter transgenic chickens, the gene expression profile of bursal M cells was identified, from which shared M cell-associated genes between mice and chickens and chicken-specific M cell-associated genes were determined. The functional enrichment analysis of upregulated genes in bursal M cells indicated the association of these genes with translocating antigens and macromolecules, aligned with the function of M cells. This study also proposed putative cytokines involved in bursal M cell development and confirmed the protein expression of allograft inflammatory factor 1-like (AIF1L), SRY-box transcription factor 8 (SOX8) and Ets transcription factor PU.1 in bursal M cells. In addition, the co-localisation of SOX8 and CSF1ReGFP was demonstrated in the bursa of Fabricius, caecal tonsil, and lung, but not in the small intestinal PP.

In mammals, intestinal M cells are derived from crypt-based stem cells that are induced by the cytokine receptor activator of the nuclear factor-κB ligand (RANKL). Applying RANKL to 2D and 3D intestinal organoids or enteroids induces M cell differentiation and the expression of M cell-associated genes.

This study investigated whether the differentiation of intestinal M cells is induced by adding recombinant chicken RANKL to chicken 2D and 3D enteroids, similar to mammalian M cell models. Both the apical and basolateral application of RANKL did not consistently increase the expression of chicken M cell-associated gene signatures in 2D and 3D enteroids derived from ED18 villi. These observations may be linked to the embryonic origin of the chicken enteroids or the possible requirement of additional cytokines to induce chicken M cells in vitro.

In summary, this thesis has provided a better insight into PP distribution and development from ED18 to 8-week-old chickens that can be implemented in future research on intestinal mucosal immunity and in ovo or post-hatch vaccine targeting. Moreover, the RNA-seq and protein expression analysis in this study offer a valuable resource to better understand the molecular immunobiology, cellular development, and function of chicken M cells and to identify potential surface receptors for M cell-targeted vaccine strategy.