Notch-1 signaling regulates intestinal epithelial barrier function, through interaction with CD4+ T cells, in mice and humans.
OBJECTIVE
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.
Immunology Institute, Mount Sinai School of Medicine, New York, New York 10029, USA. stephanie.dahan@mssm.edu
Stephanie
S
Dahan
Keren M
KM
Rabinowitz
Andrea P
AP
Martin
M Cecilia
MC
Berin
Jay C
JC
Unkeless
Lloyd
L
Mayer
D019264:Adoptive Transfer; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D018938:Caco-2 Cells; D062507:Claudin-5; D003424:Crohn Disease / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000276:immunology / Q000473:pathology / Q000502:physiology*; D019041:L-Selectin / Q000032:analysis / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000276:immunology; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000378:metabolism; D051881:Receptor, Notch1 / Q000032:analysis / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D019108:Tight Junctions / Q000276:immunology / Q000502:physiology
C107466:CLDN5 protein, human; D062507:Claudin-5; C491511:Cldn5 protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; C497761:NOTCH1 protein, human; C497762:Notch1 protein, mouse; D051881:Receptor, Notch1; C026615:villin; D019041:L-Selectin; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; C516770:Ptprc protein, mouse