RegIII proteins as gatekeepers of the intestinal epithelium

Mammalian RegIII proteins are expressed in the intestine and in the pancreas in response to inflammation or infection. In the mouse intestine, expression of RegIIIβ and RegIIIγ is increased by microbial colonization, inflammation and infection. At the outset of this thesis human PAP and mouse RegIIIγ were reported to be bactericidal for Gram-positive bacteria. Additionally, human PAP had been shown to attenuate NF-κbsignallingin human monocytes and epithelial cells and administration of anti-PAP antibodies increased inflammation in an experimental rat model of acute pancreatitis. The overarching goals of this thesis were to find out more about the protective role of mouse RegIIIβ and RegIIIγ in the intestine and explore their protective role in colitis and bacterial infection. In Chapter 2 we investigated expression of RegIIIβ and RegIIIγ in intestine of Muc2 knockout (-/-) mice, which develop colitis after about 4 weeks, due to the absence of a secreted mucus layer in the small intestine or colon. RegIII proteins were expressed in Paneth cells, enterocytes and goblet cells pointing to a new function for goblet cells in innate immunity. Ang4 expression was confined to Paneth cells and goblet cells. Absence of Muc2 increased expression levels of RegIIIβ, RegIIIγ, and Ang4 and colitis appeared first in the distal colon where the RegIII expression is lowest. In Chapter 3 we investigated the distinct phases of colitis development in Muc2-/- mice from before weaning to 4 and 8 weeks of age, also taking into account the effect that mucin deficiency has in the ileum. Gene set enrichment approaches showed increased expression of innate and adaptive immune pathways associated with colitis over time, whereas in the ileum many immune signalling pathways were down-regulated. Nevertheless, RegIIIβ and RegIIIγ were significantly upregulated, suggesting their proposed antimicrobial and/or anti-inflammatory activities might be related to the suppression of immune pathways and avoidance of immune-mediated damage. Furthermore, we showed that RegIIIβ could specifically bind to mucin and fucosylated glycans in vitro, which may serve to inhibit bacterial binding to membrane bound mucins on the epithelium, and also enable RegIIIβ to be retained in the secreted mucin. An in vitro approach was used in Chapter 4, where we investigated the activities of RegIIIγ and RegIIIβby expressing and purifying recombinant proteins. Both proteins were insoluble when expressed in E. coli but RegIIIβ could be expressed and secreted in baculovirus as a soluble protein. As previous work reported that RegIII proteins were bactericidal even when produced as inclusion bodies in E. coli and refolded, we followed similar procedures to obtain soluble RegIII proteins. In our hands both the E. coli and baculovirus produced proteins bound strongly to both Gram-positive and Gram-negative bacteria after processing of an N-terminal pro-peptide by trypsin, but lacked any appreciable bactericidal activity. Furthermore these proteins did not influence the growth of Salmonella enteritidis andListeria monocytogenes. Attempts to crystallize the proteins were unsuccessful but structural models of the protein were generated based on the crystal structure of human PAP. These models were used to dock known ligands of RegIIIγ or RegIIIβ. Only one ligand is known for RegIIIγ, which is peptidoglycan, but for RegIIIβ the ligands include peptidoglycan, lipid A and the fucose-containing glycans identified in chapter 3. RegIIIβ was predicted to have two different binding sites which would allow it to bind to mucins and bacteria simultaneously, thereby preventing penetrating of the mucus. In Chapter 5 a RegIIIβ-/- mouse was used to study the role of the protein during infection with Gram-negative Salmonella enteritidis or Gram-positive Listeria monocytogenes. Whereas recovery of S. enteritidis orL. monocytogenes from faeces was similar in RegIIIβ-/- and wild type (WT) mice, significantly higher numbers of viable S. enteritidis, but not L. monocytogenes, were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the RegIIIβ-/- than the WT mice. The results suggest that mouse RegIIIβ plays a protective role against intestinal translocation of the Gram-negative bacterium S. enteritidis but not against the Gram-positive bacterium L. monocytogenes. In Chapter 6, the generation of a RegIIIγ-/- mouse is described. One of the main phenotypic differences between the RegIIIγ-/- and WT was an altered distribution of the ileal mucus and increased bacterial contact with the epithelium. Additionally, measurement of innate immune markers in the mucosa suggested heightened inflammation in the RegIIIγ-/- mice. Compared to WT mice, RegIIIγ-/- mice infected with S. enteritidis and L. monocytogenes showed an increase of mucosal inflammatory markers indicating protective, anti-microbial roles of RegIIIγ in defense against both Gram-positive and Gram-negative bacteria. Chapter 7summarizes and discusses the key results of the thesis in the context of the wider literature and possible directions for future research</p

Intestinally secreted C-type lectin Reg3b attenuates salmonellosis but not listeriosis in mice

The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal infection with pathogens increase the expression of Reg3g and Reg3b in the murine ileum. Reg3g is directly bactericidal for Gram-positive bacteria, but the exact role of Reg3b in bacterial infections is unknown. To investigate the possible protective role of Reg3b in intestinal infection, Reg3b knockout (Reg3b-/-) mice and wild-type (WT) mice were orally infected with Gram-negative Salmonella enteritidis or Gram-positive Listeria monocytogenes. At day 2 after oral Listeria infection and at day 4 after oral Salmonella infection, mice were sacrificed to collect intestinal and other tissues for pathogen quantification. Protein expression of Reg3b and Reg3g was determined in intestinal mucosal scrapings of infected and noninfected mice. In addition, ex vivo binding of ileal mucosal Reg3b to Listeria and Salmonella was investigated. Whereas recovery of Salmonella or Listeria from feces of Reg3b-/- mice did not differ from that from feces of WT mice, significantly higher numbers of viable Salmonella, but not Listeria, bacteria were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the Reg3b-/- mice than from those of WT mice. Mucosal Reg3b binds to both bacterial pathogens and may interfere with their mode of action. Reg3b plays a protective role against intestinal translocation of the Gram-negative bacterium S. enteritidis in mice but not against the Gram-positive bacterium L. monocytogenes

The role of innate signaling in the homeostasis of tolerance and immunity in the intestine.

In the intestine innate recognition of microbes is achieved through pattern recognition receptor (PRR) families expressed in immune cells and different cell lineages of the intestinal epithelium. Toll-like receptor (TLR) and nucleotide-binding and oligomerization domain-like receptor (NLR) families are emerging as key mediators of immunity through their role as maturation factors of immune cells and triggers for the production of cytokines and chemokines and antimicrobial factors. At the mucosal surface chronic activation of the immune system is avoided through the epithelial production of a glycocalyx, steady-state production of antimicrobial factors as well as the selective expression and localization of PRRs. Additionally, the polarization of epithelial TLR signaling and suppression of NF-¿B activation by luminal commensals appears to contribute to the homeostasis of tolerance and immunity. Several studies have demonstrated that TLR signaling in epithelial cells contributes to a range of homeostatic mechanisms including proliferation, wound healing, epithelial integrity, and regulation of mucosal immune functions. The intestinal epithelium appears to have uniquely evolved to maintain mucosal tolerance and immunity, and future efforts to further understand the molecular mechanisms of intestinal homeostasis may have a major impact on human healt

Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids

An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology

Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids

An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology

REG3¿-deficient mice have altered mucus distribution and increased mucosal inflammatory responses to the microbiota and enteric pathogens in the ileum

REG3¿ is considered to have a protective role against infection with Gram-positive bacteria due to its bactericidal activity, but evidence from in vivo studies is lacking. We generated a REG3¿-/- mouse, and investigated the effect of lack of REG3¿ on intestinal mucus distribution, spatial compartmentalization of bacteria, and expression of innate immunity genes. Infection studies were also performed with Gram-positive and Gram-negative pathogens to investigate the antimicrobial role of REG3¿. REG3¿-/- mice display altered mucus distribution, increased bacterial contact with the epithelium, and elevated inflammatory markers in the ileum without histological evidence of pathology. Infection response pathway genes were differentially expressed in both Listeria monocytogenes and Salmonella enteritidis infected REG3¿-/- and wild-type (wt) mice. Higher amounts of myeloperoxidase and interleukin-22 transcripts were present in the ileal mucosa of REG3¿-/- than wt mice, but translocation to the organs was unaffected. We concluded that REG3¿ has a protective role against mucosal infection with pathogenic Listeria and Salmonella in vivo. REG3¿ is equally distributed throughout the mucus and its absence results in increased epithelial contact with the microbiota resulting in low-grade inflammation. REG3¿ can bind to Gram-negative and Gram-positive bacteria and influence mucus distribution in the ileum, properties which may contribute to mucosal protection