Notch-1 signaling regulates intestinal epithelial barrier function, through interaction with CD4+ T cells, in mice and humans

Abstract

Background & aims: Interactions between lymphocytes and intestinal epithelial cells occur in the subepithelial space of the gastrointestinal tract. Normal human lamina propria lymphocytes (LPLs) induce differentiation of intestinal epithelial cells. The absence of LPLs in mice, such as in RAG1(-/-) mice, results in defects in epithelial cell differentiation. We investigated the role of lymphoepithelial interactions in epithelial differentiation and barrier function.

Methods: We used adoptive transfer to determine if CD4(+) T cells (CD4(+)CD62L(+)CD45Rb(Hi) and/or CD4(+)CD62L(+)CD45Rb(Lo)) could overcome permeability defect (quantified in Ussing chambers). Immunofluorescence staining was performed to determine expression of cleaved Notch-1, villin, and claudin 5 in colon samples from mice and humans. Caco-2 cells were infected with a lentivirus containing a specific Notch-1 or scrambled short hairpin RNA sequence. Tight junction assembly was analyzed by immunoblot and immunofluorescence analyses, and transepithelial resistance was monitored.

Results: Expression of cleaved Notch-1, villin, or claudin 5 was not detected in RAG1(-/-) colonocytes; their loss correlated with increased intestinal permeability. Transfer of CD45Rb(Hi) and/or CD45Rb(Lo) cells into RAG1(-/-) mice induced expression of cleaved Notch, villin, and claudin 5 in colonocytes and significantly reduced the permeability of the distal colon. Loss of Notch-1 expression in Caco-2 cells correlated with decreased transepithelial resistance and dysregulated expression and localization of tight junction proteins. Levels of cleaved Notch-1 were increased in colonic epithelium of patients with Crohn's disease.

Conclusions: LPLs promote mucosal barrier function, which is associated with activation of the Notch-1 signaling pathway. LPLs maintain intestinal homeostasis by inducing intestinal epithelial cell differentiation, polarization, and barrier function.

Figure 1

Colonic tissues sections from C57/BL6 WT, RAG1 deficient mice, as well as RAG1 deficient mice who received either CD4+CD62L+CD45RbHi T cells or CD4+CD62L+CD45RbHi and CD4+CD62L+CD45RbLo T cells were immunostained using an anti-cleaved Notch-1 (red) antibody. The nuclei were stained with Hoechst 33342 (blue). All samples were analyzed with a Leica SP5-DM Confocal microscope at 40× and 63× magnification. These data are representative of 5 experiments.

Figure 2

Colonic tissues sections from C57/BL6 WT, RAG1 deficient mice, as well as RAG1 deficient mice who received either CD4+CD62L+CD45RbHi T cells or CD4+CD62L+CD45RbHi and CD4+CD62L+CD45RbLo T cells were immunostained using an anti-villin (left panel) (red) or anti-claudin 5 (right panel) antibody. The nuclei were stained with Hoechst 33342 (blue). All the samples were analyzed with a Leica SP5-DM Confocal microscope at 40× magnification. These data are representative of 5 experiments.

Figure 3

Assessment of changes in epithelial resistance (top panel) as a measure of passive transcellular and paracellular ion transport, and changes in paracellular permeability (lower panel) for small molecules. WT, non-reconstituted RAG−/− mice, RAG−/− mice reconstituted with CD4+CD62L+CD45RbHi T cells, RAG−/− mice reconstituted with CD4+CD62L+CD45RbHi and CD4+CD62L+CD45RbLo T cells or RAG−/− mice reconstituted with whole CD4+ T cells were sacrificed after 6 weeks. The distal colon was harvested and the resistance was measured along with paracellular permeability (assessed by flux measurements of Dextran-FITC from the mucosal to serosal compartment, measured by spectrofluorometry). Data represent the mean ± SEM of at least 5 mice/group. The symbols *, **, *** refer to statistical significance, respectively <0.05, <0.01, <0.001.

Figure 4

a. GFP (control shRNA) and Notch-1 KD Caco-2 lines were immunostained using an anti-Notch-1 (red) antibody. The presence of GFP (green) was observed at a wavelength of 488nm. The nuclei were staining with Hoechst 33342 (blue). All the samples were analyzed with a Leica SP5-DM Confocal microscope at 63× magnification. These data are representative of 5 experiments. b. Transepithelial resistance (TER) in GFP and Notch-1 KD Caco-2 cell monolayers seeded on Transwells for one week. TER was measured using an Ohm-meter. The symbol *** reflects a statistical significance of p<0.001.

Figure 5

a. Tight junction protein expression in GFP and Notch-1 KD Caco-2 cell membranes. Immunoblotting for claudin-2, claudin-5, claudin-8, occludin, and ZO-1 in membrane protein extracts obtained from GFP and Notch-1 KD Caco-2 cells. b. GFP and Notch-1 KD Caco-2 lines were immunostained using anti-claudin-2, claudin-5, occludin, and ZO-1 antibodies (red). The nuclei were stained with Hoechst 33342 (blue). All the samples were analyzed with a Leica SP5-DM Confocal microscope at 63× magnification. These data are representative of 5 experiments.

Figure 6

Nor, CD, and UC colonic tissue sections were immunostained using an anti-cleaved Notch-1 antibody. The slides were counter-stained with Mayer’s Hematoxylin solution, and examined with a Zeiss Axioskop light microscope at 20X magnification. These data are representative of 5 experiments.