Jagged1 and Notch1 help edit M cell patterning in Peyer's patch follicle epithelium

Abstract

Mucosal epithelium M cells are dispersed across Peyer's patch follicle associated epithelium (PPFAE) with minimal clustering. Since Notch signaling can influence patterning in epithelia, we examined its influence on PPFAE M cell distribution. Conditional deletion of Notch1 in intestinal epithelium increased PPFAE M cells and also increased M cell clustering, implying a role for Notch in both M cell numbers and lateral inhibition. By contrast, conditional deletion of the ligand Jagged1 also increased M cell clustering, but with a paradoxical decrease in M cell density. In vitro, inhibition of Notch signaling reduced expression of an M cell associated gene CD137, consistent with cis-promoting effects on M cell development. Thus, Jagged1 may have a cis-promoting role in committed M cells, but a trans-inhibitory effect on neighboring cells. In sum, Jagged1-Notch signaling may edit the pattern of M cells across the PPFAE, which may help optimize mucosal immune surveillance.

Figure 1. Goblet cells on small intestine villi are greatly increased in the absence of Notch1

A. Control villi (single floxed Notch1) stained for goblet cells using Alcian Blue, showing normal dispersed distribution and density of goblet cells on intestinal villi. (Scale bar: 50 um) B. FloxNotch/VilCre villi stained with Alcian Blue. Here, there is greatly increased goblet cell density, with many goblet cells directly adjacent to each other. (Scale bar: 50 um) C. Quantitation of goblet cell density in wild type and Notch1 conditional knockout villi. Goblet cell density was measured by goblet cell counts per villous basement membrane length. The Notch1 conditional knockout villi showed approximately double the density of goblet cells versus control villi. (single flox Notch N=22; FloxNotch N=15; ***, P<0.0005)

Figure 2. Peyer’s patch M cells show increased numbers and clustering in floxNotch mice

A. Quantitation of M cell density across Peyer’s patch epithelium, measured by direct counting of M cells per Peyer’s patch follicle epithelium surface area. Notch1 conditional knockout Peyer’s patches contained a higher density of M cells. (single flox Notch N=11; FloxNotch N=10; **, P<0.005) B. Quantitation of goblet cell density across Peyer’s patch epithelium. In Notch1 conditional knockout mice, goblet cell density was increased in Peyer’s patch in similar fashion to that seen in intestinal villi, and in parallel to the increase seen for M cells. (single flox Notch N=11; FloxNotch N=10; *, P<0.05) C. Calculation of M cell clustering across Peyer’s patch epithelium. Adjacent cells with more than 3 microns in direct contiguous contact are considered clustered, and counts show the proportion of M cells in clustered arrangements. (single flox Notch N=11; FloxNotch N=10; ***, P<0.0005) D. Confocal image of Peyer’s patch whole mount showing dispersal of M cells in control Peyer’s patch and occasional M cell cluster (arrow). (Scale bar: 30 um) E. Image showing increased clustering in FloxNotch Peyer’s patch. Clustered M cells with increased contacts along sides (outlined by dotted lines) are identified with arrows. (Scale bar: 30 um)

Figure 3. FloxJagged1 Peyer’s patches have fewer M cells but increased clustering

A. Direct quantitation of M cell density across Peyer’s patch epithelium showed that, in contrast to Notch1 conditional knockout mice, there were reduced numbers in Jagged1 conditional knockout mice compared to wild type. (single flox Jag1 N=32; FloxJag1 N=32;**, P<0.005) B. Quantitation of M cell clustering (measured as described for Notch1 conditional knockout). Despite the decrease in M cell density, Jagged1 conditional knockout Peyer’s patch epithelium showed increased M cell clustering. (single flox Jag1 N=32; FloxJag1 N=32; **, P<0.005) C. Confocal image of FloxJagged1 Peyer’s patch whole mount showing M cell clustering (outlined by dotted lines, arrows). Two goblet cells are identified with black asterisks. (Scale bar: 25 μm) D. Goblet cell density counts showed a slight reduction in Jagged1 conditional knockout Peyer’s patch compare to wild type, though not as strong as the reduction in M cell density. (single flox Jag1 N=32; FloxJag1 N=32; *, P<0.05)

Figure 4. Studies on Caco-2BBe cell gene expression show coordinate regulation of M cell genes Jagged1 and CD137

A. TNFα plus LTβR agonist (“Cytokine”: 100ng/ml TNFα and 5μg/ml of LTβR agonist antibody) induced upregulation of Jagged1 with no additional influence of soluble CD137L agonist. (t-test analysis; **, P<0.005) B. Confocal images of untreated Caco-2BBe cells stained for CD137 (green) and Jagged1 (red). Nuclei are blue. (Scale bar: 30 μm) C. Confocal images of cytokine treated Caco-2BBe cells shows upregulation of CD137 (arrows), but there is no co-localization of CD137 (green) with Jagged1 (red). (Scale bar: 30 μm) D. Cytokine treatment of Caco-2BBe cells induces expression of CD137, but expression shows a dose-dependent inhibition by the Notch signaling inhibitor DAPT. One tailed paired t- test comparing DMSO control to 100μm DAPT was significant (P<0.05).

Figure 5. Model of Jagged-Notch interactions and M cell patterns in Peyer’s patch epithelium

A. A low magnification view of the overall distribution of M cells (green) across Peyer’s patch follicle epithelium. Note that at this low magnification, dispersion of M cells derived from crypt stem cell progenitors is evident, though the rare M cell clusters are not as visible. B. Simplified cartoon model of M cell editing by Jagged1 and Notch interactions, indicating a possible role for Jagged1 in both lateral inhibition of M cell development and cis-reinforcement of M cell lineage decisions.