Volume 19, Issue 1, Pages 38-51 (July 2016) Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer  Matthias Schewe, Patrick F. Franken, Andrea Sacchetti, Mark Schmitt, Rosalie Joosten, René Böttcher, Martin E. van Royen, Louise Jeammet, Christine Payré, Patricia M. Scott, Nancy R. Webb, Michael Gelb, Robert T. Cormier, Gérard Lambeau, Riccardo Fodde  Cell Stem Cell  Volume 19, Issue 1, Pages 38-51 (July 2016) DOI: 10.1016/j.stem.2016.05.023 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2016 19, 38-51DOI: (10.1016/j.stem.2016.05.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Pla2g2a Expression Modulates ISC Function and Paneth Cell Differentiation (A) Organoid assays performed with crypts from indicated Mom-1-sensitive or Mom-1-resistant strains. Differences are statistically significant (p < 0.001) except for TgPla2g2a versus CBA/J and FVB/N versus BALB/c. (B) Organoid assays performed on C57BL/6J (Pla2g2a−/−) and Tg-Pla2g2a male and female mice (n = 5, ∗p < 0.001). (C) Lysozyme IHC analysis of duodenal sections of C57BL/6J (Pla2g2a−/−) and Tg-Pla2g2a mice. (D) Ki67 IHC analysis of small intestinal crypts (duodenum and ileum) from Pla2g2a−/− (C57BL/6J) and Tg-Pla2g2a mice. (E) Heatmap representation qRT-PCR expression analysis of Paneth- and stem-cell-specific genes from FACS-purified CD24/SSC lineages. Cell Stem Cell 2016 19, 38-51DOI: (10.1016/j.stem.2016.05.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Pla2g2a Expression Modulates Wnt Signaling through Yap1 Phosphorylation (A) TOP-Flash luciferase reporter analysis of Wnt signaling activity in the colon cancer cell lines SW480, HCT116, and HT29 upon transient transfection with a PLA2G2A expression vector in the presence/absence of Wnt3a conditioned medium (n = 3, ∗p < 0.05). (B) Yap1 qRT-PCR analysis in Paneth cells (CD24hiSSChi), secretory precursors (CD24medSSClo), and stem cells (Lgr5+CD24medSSClo) sorted by FACS from C57BL/6J (Pla2g2a−/−) and Tg-Pla2g2a mice in the Lgr5EGFP-IRES-creERT2 (Lgr5+) background (left). CD24hiSSChi Paneth cells were also sorted from Pla2g2a+/+ (FVB/N) mice and compared with the corresponding subpopulations sorted from Pla2g2a−/− (C57BL/6J) and Tg-Pla2g2a mice (right) (n = 3, ∗∗p < 0.001). (C) Phospho-Yap1 IHC analysis of small intestinal crypts from C57BL/6J (Pla2g2a−/−; left panel) and Tg-Pla2g2a mice (right panel). The black and white arrows indicate an Lgr5+ stem cell (positive for Yap1) and a Paneth cell (negative for Yap1), respectively. Cell Stem Cell 2016 19, 38-51DOI: (10.1016/j.stem.2016.05.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Reconstitution Organoid Assays Highlight the Role of Pla2g2a in Inhibiting Wnt Signaling through Yap1 (A) Flowchart of the reconstitution organoid assay. Single-cell suspensions were obtained from small intestinal crypts isolated from C57BL/6J (Pla2g2a−/−) and Tg-Pla2g2a mice previously bred with Lgr5-EGFP reporter animals (Lgr5EGFP-IRES-creERT2). Purified Lgr5+ stem cells (Lgr5+CD24medSSClo) and Paneth cells (CD24hiSSChi) were mixed and plated in matrigel for organoid growth. (B) Results of the reconstitution organoid assay with Paneth cells (PCs) and Lgr5+ stem cells (SCs) from C57BL/6J (B6) and Tg-Pla2g2a (Tg) mice previously bred with Lgr5-EGFP reporter animals (Lgr5EGFP-IRES-creERT2) (n = 3, ∗p < 0.05; ∗∗p < 0.001). (C) Organoid formation following reconstitution of Lgr5+stem cells (SCs) and Paneth cells (PCs) sorted from Pla2g2a−/− (B6), Tg-Pla2g2a (Tg), and FVB/N (FVB) mice. When indicated, PCs were pretreated with siRNA oligonucleotides directed against Pla2g2a, Yap1, or a “scrambled” (SCR) control sequence (n = 3, ∗p < 0.05; ∗∗p < 0.001). (1) B6-PCs + B6-SCs, (2) Tg-PCs + Tg-SCs, (3) Tg-PCs [siRNA-Pla2g2a] + B6-SCs, (4) Tg-PCs [siRNA-Yap1] + B6-SCs, (5) Tg-PCs [siRNA-SCR] + B6-SCs, (6) Tg-PCs, (7) B6-PCs + B6-SCs, (8) FVB-PCs + B6-SCs, (9) FVB-PCs [siRNA-Pla2g2a] + B6-SCs, (10) FVB-PCs [siRNA-Yap1] + B6-SCs, (11) FVB-PCs [siRNA-SCR] + B6-SCs, and (12) FVB-PCs. (D) TOP-Flash reporter analysis of Wnt signaling activity in the colon cancer cell line SW480 upon transient transfection with a PLA2G2A expression vector in the presence/absence of siRNA-driven YAP1 downregulation (n = 3, ∗p < 0.05). Cell Stem Cell 2016 19, 38-51DOI: (10.1016/j.stem.2016.05.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Secreted Pla2g2a Enhances ISC Function through PGE2 Synthesis and Wnt Signaling upon Inflammation (A) C57BL/6J organoid formation in the absence/presence of recombinant Pla2g2a and soluble Pla2r1 receptor (n = 5, ∗p < 0.001). (B) Quantified organoid formation following DSS treatment of C57BL/6J (Pla2g2a−/−), Tg-Pla2g2a, and FVB/N (Pla2g2a+/+) mice, in the absence/presence of soluble Pla2r1 (n = 3, ∗p < 0.001; ∗∗p < 0.05). (C) Results of organoid assays performed on Pla2g2a−/− (C57BL/6J) mice in the presence of agonists (AA, PGE2) and antagonists (NSAIDs; ketoprofen and diclofenac), the latter in the presence of recombinant Pla2g2a (n = 5, ∗p < 0.001). (D) Results of organoid assays from C57BL/6J versus Pla2r1−/− mice in the absence/presence of recombinant Pla2g2a (n = 5, ∗p < 0.001). (E) qRT-PCR expression analysis of Pla2r1 in sorted Lgr5+, Paneth, and p16 (Paneth-like) cells of the small (SI) and large (LI) intestine. Control qRT-PCR analyses were performed to validate the quality and identity of the sorted stem (Lgr5), Paneth (Lyz), and p16 (Pla2g10) cells. (F) Left panel (2–9): organoid numbers following reconstitution of Paneth cells (PCs) (2–5) or Lgr5+ stem cells (SCs) (6–9) sorted from the small intestine of Lgr5-reporter C57BL/6J (B6) mice pretreated with recombinant Pla2g2a. When indicated, sorted cells were also treated with siRNA oligonucleotides specific for Pla2r1 and Cox2, or scrambled sequences (SCR) (n = 3, ∗p < 0.05; ∗∗p < 0.001). (1) B6-PCs + B6-SCs (negative control; no rec. Pla2g2a), (2) B6-PCs + B6-SCs, (3) B6-PCs [siRNA-SCR] + B6-SCs, (4) B6-PCs [siRNA-Pla2R1] + B6-SCs, (5) B6-PCs [siRNA-Cox2] + B6-SCs, (6) B6-PCs + B6-SCs, (7) B6-PCs + B6-SCs [siRNA-SCR], (8) B6-PCs + B6-SCs [siRNA-Pla2R1], and (9) B6-PCs + B6-SCs [siRNA-Cox2]. Right panel (10–12): organoid numbers upon reconstitution of B6-PCs with B6-SCs in the presence of 1 μM PGE2 for 30 min. (10) Both B6-SCs and B6-PCs pretreated with PGE2, (11) only PCs pretreated with PGE2, and (12) only PCs pretreated with PGE2. (G) Organoid assays from C57BL/6J (Pla2g2a−/−) mice in the presence/absence of pyrrophenone (PP) and recombinant Pla2g2a (left panel), and from C57BL/6J versus Pla2g4a−/− in the absence/presence of recombinant Pla2g2a (right panel) (n = 5, ∗p < 0.001). Cell Stem Cell 2016 19, 38-51DOI: (10.1016/j.stem.2016.05.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Pla2g10 Is Expressed by the Paneth-like Secretory Cell Lineage in the Mouse Colon (A) CD24/cKit/SSC FACS sorting strategy employed for colonic epithelial cells from Lgr5-EGFP reporter animals (Lgr5EGFP-IRES-creERT2). Upper panel, whole colon; lower panel, GFP+ (Lgr5+) fraction. Sorting gates were defined as follows: p11 (CD24med/cKitlo), p15 (CD24med/cKitmed), p16 (CD24hi/cKithi), and p18 (CD24med/cKithi). (B) Organoid formation assay upon reconstitution of 1,000 cells from the p15/16/18 gates (after depletion of GFP-positive doublets by side scatter) with 1,000 Lgr5+ stem cells (p11) (n = 3, ∗p < 0.001). (C) Confocal microscopy images of doublets constituted by smaller Lgr5+ (GFP+) stem cells and larger Lgr5-negative and c-Kit+ (red) cells found in p16. (D) Heatmap representation of the results of the qRT-PCR expression analysis of sorted p16, p15, and p11 cells for Paneth- and goblet-specific genes. Cell Stem Cell 2016 19, 38-51DOI: (10.1016/j.stem.2016.05.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Pla2g10, Analogous to Pla2g2a in the Small Intestine, Has Opposing Roles in Regulating Wnt and ISC Function in the Colon during Homeostasis and Inflammation (A) Colon organoid assay of C57BL/6J (Pla2g2a−/−/Pla2g10+/+) and Pla2g10-knockout (Pla2g2a−/−/Pla2g10−/−) mice (n = 3, ∗p < 0.001). (B) TOP-Flash reporter assay of Wnt activity in colon cancer cell lines SW480 and HCT116 upon transient transfection with a PLA2G10 expression plasmid (n = 3, ∗p < 0.001). (C) Organoid formation following reconstitution of colonic Lgr5+ stem cells (SCs) with Pla2g10-proficient (B6) and Pla2g10- deficient (Pla2g10KO) Paneth-like cells (p16). When indicated, p16 cells were pretreated with siRNAs against Pla2g10, Yap1, or a “scrambled” (SCR) control sequence (n = 3, ∗p < 0.05; ∗∗p < 0.001). (1) B6-p16, (2) B6-p16 + B6-SCs, (3) B6-p16 [siRNA-SCR] + B6-SCs, (4) B6-p16 [siRNA-Pla2g10] + B6-SCs, (5) B6-p16 [siRNA-Yap1] + B6-SCs, (6) Pla2g10KO-p16, (7) Pla2g10KO-p16 + B6-SCs, (8) Pla2g10KO-p16 [siRNA-SCR] + B6-SCs, (9) Pla2g10KO-p16 [siRNA-Pla2g10] + B6-SCs, and (10) Pla2g10KO-p16 [siRNA-Yap1] + B6-SCs. (D) TOP-Flash Wnt reporter assay in the colon cancer cell line SW480 upon transient transfection with a PLA2G10-expression plasmid and siRNA-driven YAP1 downregulation (n = 3, ∗p < 0.05). Cell Stem Cell 2016 19, 38-51DOI: (10.1016/j.stem.2016.05.023) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Pla2g2a and Pla2g10 Are Genetic Modifiers of IBD and Colon Cancer (A) Results of colon organoid assay of C57BL/6J (Pla2g2a−/−/Pla2g10+/+) and Pla2g10-knockout (Pla2g2a−/−/Pla2g10−/−) mice in the presence/absence of recombinant mGX sPLA2 (both wild-type and the H48Q catalytic mutant proteins were employed), and of the recombinant (soluble) Pla2r1 receptor (n = 3, ∗p < 0.001; ∗∗p < 0.05). (B) Results of colon organoid assays performed on C57BL/6J mice in the presence of recombinant mGX sPLA2 alone or in combination with an antibody directed against the mouse Pla2r1 receptor (Rouault et al., 2007), pyrrophenone, a specific cPLA2α inhibitor (Lambeau and Gelb, 2008), and NSAIDs (ketoprofen and diclofenac) (n = 3, ∗p < 0.001). (C) Quantification of the response to DSS-induced inflammation in mice of the four Pla2g2a/Pl2g10 genotypes as measured by percentage of weight loss (compared with starting weight). See also Table S1 for more details. (D) Results of colon organoid assay of C57BL/6J (Pla2g2a−/−/Pla2g10+/+) and Pla2g10-knockout (Pla2g2a−/−/Pla2g10−/−) mice upon DSS-driven inflammation in the presence/absence of the soluble Pla2r1 receptor (n = 3, ∗p < 0.001). (E) Quantification of the response to DSS-induced inflammation and varespladib administration (from day 8 onward) in mice of the four Pla2g2a/Pl2g10 genotypes as measured by percentage of weight loss (compared with starting weight). Varespladib treatment of C57BL/6J animals prevented their recovery, and the mice had to be euthanized at day 10. See also Table S2 for more details. (F) Incidence of colonic tumors in Pla2g2a−/−/Pla2g10+/+ (C57BL/6J), Pla2g2a−/−/Pla2g10−/+, and Pla2g2a−/−/Pla2g10−/− mice upon AOM-DSS treatment (n = 10, ∗p < 0.001). Cell Stem Cell 2016 19, 38-51DOI: (10.1016/j.stem.2016.05.023) Copyright © 2016 Elsevier Inc. Terms and Conditions