Abstract
Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10−/−, Cd14−/−, and Alpk1−/−) were then stimulated with either LPS or Escherichia coli Nissle 1917 (EcN). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14−/− organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after EcN stimulation. In contrast, compared to WT, Alpk1−/− organoids showed decreased expression of different TJ and cytokine genes in response to EcN but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14, but not Alpk1, alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge.
Highlights
Inflammatory bowel disease (IBD) onset involves the interplay between microbiota, environmental conditions, genetic factors, and a disrupted intestinal barrier [1]
We utilized colon-derived 3D organoids derived from WT, Il10-/, Cd14-/, and Alpk1-/- knockout mice to investigate the response to either purified LPS or whole Gram-negative bacteria
We show for the first time a role for ALPK1 in tight junction (TJ) component regulation after infection with the Gram-negative bacteria Escherichia coli Nissle 1917 (EcN)
Summary
Inflammatory bowel disease (IBD) onset involves the interplay between microbiota, environmental conditions, genetic factors, and a disrupted intestinal barrier [1]. Several mouse models of IBD have been established [2] including the wellstudied Il10 (interleukin-10) deficiency model This model is characterised by a dysregulated immune response to enteric microflora leading to the onset of colitis through a disrupted barrier due to increased levels of interferon gamma and tumor necrosis factor alpha (TNFα) [3]. Using the Il10-/mouse model and quantitative trait locus (QTL) analyses, several studies have identified genetic loci associated with susceptibility to IBD. These studies revealed ten cytokine deficiency-induced colitis susceptibility (Cdcs) loci and candidate genes with a potential influence on colitis onset [4,5,6,7,8,9]. We showed that in wild-type (WT) but not in Cd14-/- mice, monoassociation with Escherichia coli Nissle 1917 (EcN) leads to increased expression of
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