mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake

Abstract

How adult tissue stem and niche cells respond to the nutritional state of an organism is not well understood. Here we find that Paneth cells, a key constituent of the mammalian intestinal stem-cell (ISC) niche, augment stem-cell function in response to calorie restriction. Calorie restriction acts by reducing mechanistic target of rapamycin complex 1 (mTORC1) signalling in Paneth cells, and the ISC-enhancing effects of calorie restriction can be mimicked by rapamycin. Calorie intake regulates mTORC1 in Paneth cells, but not ISCs, and forced activation of mTORC1 in Paneth cells during calorie restriction abolishes the ISC-augmenting effects of the niche. Finally, increased expression of bone stromal antigen 1 (Bst1) in Paneth cells—an ectoenzyme that produces the paracrine factor cyclic ADP ribose—mediates the effects of calorie restriction and rapamycin on ISC function. Our findings establish that mTORC1 non-cell-autonomously regulates stem-cell self-renewal, and highlight a significant role of the mammalian intestinal niche in coupling stem-cell function to organismal physiology.

Figure 1. Calorie restriction augments the capacity of Paneth cells to boost ISC function

a. Olfm4+ ISCs and Cryptdin4+ Paneth cells were increased in CR mice (in situ hybridization, proximal jejunum, n=3). b. Crypt base columnar cells (CB-cells) showed a 2-fold increase in BrdU incorporation and TA-cells revealed a reduction after a 4-hour pulse in CR mice (n=3). c. CR-crypts were 2-fold more capable of forming organoids (n=8). Representative AL and CR-organoids are shown at 5 days (red arrowhead marks organoids and yellow asterisk indicates aborted crypts; scale bar = 50 μm). d–e. CR increased the number of surviving (d) and proliferating (e, Ki67+) crypts after irradiation induced damage (n=3 for d and e). f. Schematic demonstrating dark green Lgr5^hi ISCs, red Paneth cells, and light green EGFP^low progenitors. CR increased ISCs (dark green) and Paneth cells (red) by 1.5-fold, and reduced EGFP^low (light green) progenitors by 20% (CR, n=27; AL, n=26). g. A schematic illustrating the mixing of ISCs with Paneth cells in matrigel. h. Organoid formation per Lgr5^hi ISCs cocultured with Paneth cells from CR mice was significantly increased (n=5). Representative image of primary organoids at day 7. i. Dissociated organoids derived from CR-Paneth cells gave rise to larger secondary organoids at day 18 (n=5). j. Sorted ISC-Paneth cell doublets plated at clonal density (50–100 doublets per 30 ul droplet of matrigel) demonstrated that CR-doublets had nearly 3-fold more organoid potential (n=3). k. EGFP^low progenitors harbored little organoid potential (n=4). l. Subcloning of individual CR-Paneth derived organoids gave rise to 3-fold more secondary organoids (27 organoids from 3 independent mice per condition were analyzed, shades of grey or blue denote separate mice). m. Paneth cells isolated from mice that had been on CR, but were returned to an AL diet for 3 days, also retained an augmented capacity to promote organoid formation (n=3). (Unless other wise indicated, in all panels: values = mean; error bars = s.d.; scale bars= 50 μm; * indicates P<0.05; ** P<0.01; and *** P<0.001).

Figure 2. Nutritional regulation of mTORC1 in Paneth cells

a. Overnight fasted mice treated without or with rapamcyin were refed for 5 hours and intestinal sections immunostained for phospho-S6 (P-S6). Refeeding or administration of insulin activates mTORC1 in Paneth cells (arrow heads) but not ISCs (arrows, n=3–5, scale bar = 20 μm). b. Similar results were observed in cytospun preparations of sorted Paneth cells that were confirmed to be greater than 95% positive for the Paneth cell marker Lysozyme (n=3). P-S6 was reduced in sorted Paneth cells from fasted mice and was induced 20 minutes after injection with insulin. c. Immunoblots of isolated crypts from CR mice show reduced phosphorylation of known mTORC1 substrates phospho-S6K1 (P-S6K1) and P-S6.

Figure 3. mTORC1 signaling in Paneth cells mediates the effects of calorie restriction on ISC function

a. Schematic of the Tet-ON human Rheb2 transgene (Rheb-tg). b. Doxycycline (dox) induced Rheb2 protein and S6 phosphorylation in the liver (background band: B.G.). c–d. Fasted Rheb-tg mice demonstrated increased immunostaining of P-S6 in intestinal epithelium (c) and in isolated Paneth cells (d) upon dox injection. e–f. Induction of Rheb2 from the start of CR abrogated the enhancement in organoid formation per crypt (n=3) (e) and per Lgr5^hi ISC cocultured with Paneth cells (n=5) (f). g–h. Rapamycin (R) (n=13) increased the frequency of Lgr5^hi ISCs (g) and Paneth cells (h) compared to vehicle (V) (n=14) as measured by flow cytometry. i–j. Organoid potential of intestinal crypts (n=3) (i) and AL Lgr5^hi ISCs with Paneth cells (n=5) (j) from R, CR, or CR+R treated mice. k. Rapamycin increased crypt organoid formation in Rictor deleted Rictor^fl/flUBC-CreERT2 mice (n=3) to a similar extent as in wild-type (WT) mice. l. Organoid formation of Lgr5^hi ISCs combined with Paneth cells from rapamycin treated mice was significantly increased in comparison to Paneth cells from vehicle treated mice (n=5). m. Dissociated primary organoids derived from R-Paneth cells gave rise to more secondary organoids (n=5). (Unless otherwise indicated, in all panels: values = mean; error bars = s.d.; scale bars= 20 μm; * indicates P<0.05; ** P<0.01; and *** P<0.001).

Figure 4. Calorie restriction enhances expression of bone stromal antigen 1 (Bst-1) in Paneth cells, whose product cyclic ADP ribose (cADPR) enhances ISC function

a. Transcriptional profiling of Paneth cells from CR and AL mice (n=4 and 3, respectively) identified significant expression changes in 401 genes, 57 of which encode plasma membrane associated or secreted proteins. b. Validation of the increased transcription of Bst-1 by qRT-PCR (n=3). c–d. Increased Bst-1 protein in crypts from CR mice and rapamycin treated mice detected via immunoblotting (c) and in CR-Paneth cells by immunostaining (d). e. Exogenous cADPR increased the organoid potential of AL-crypts to an extent similar as those of CR-crypts (n=3). f–g Inhibition of Bst-1 by siRNA-mediated knock-down (n=3) (f) abrogated the enhanced potential of CR-crypts to form organoids (n=3) (g). i. A model of intestinal adaptation to calorie restriction by non-cell autonomous regulation of ISC self-renewal. CR attenuates mTORC1 activity in the Paneth cells, resulting in increased expression of Bst-1, whose paracrine product cADPR promotes ISC self-renewal. * indicates P<0.05; ** P<0.01, *** P<0.001. Values = means and error bars = s.d.