RegIII proteins as gatekeepers of the intestinal epithelium (2013)

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