Fig 1.
Intestinal epithelial Lsd1 deletion induces proliferation and a repair-like state in epithelium of mice.
(A) Immunofluorescent images of Ki67+ (green) in colonic epithelium of WT and cKO adult mice. DAPI was used as a nuclear counterstain. (B) Quantification of crypt length in naïve WT and cKO adult mice. Quantification of Ki67+ cells in colonic crypts of WT and cKO adult mice. n = 4 mice in each group, line is mean. (C) Organoids from WT and cKO mice were cultured for 3 days. The expression of Ki67+ (green) was determined by immunofluorescence. (D) Quantification of Ki67 intensity per organoid area in budding organoids from WT and cKO mice. Each dot represents 1 organoid. (E) Crypts cells were isolated from colon of WT and cKO adult mice and flow cytometric analysis of SCA1 was carried out. (F) Quantification of SCA1+ positive cells in crypts from WT and cKO adult mice, lines indicate littermate controls. n = 5 mice (G) Organoids were treated with GSK-LSD1 for 4 days followed by IL-22 stimulation for 30 minutes. Whole cell lysates were prepared and subjected to immunoblot analysis of p-STAT3, STAT3, and GAPDH. (H) Band intensity of pSTAT3 and STAT3 was measured using Image Studio Lite by LICOR. Each dot represents one biological replicate. (I) Organoids were co-cultured with GSK-LSD1 (5μM) and IL-22 (10 ng/ml) for 4 days in ENR (EGF, NOGGIN, R-SPONDIN) medium. The mRNA expression of Reg3b and Reg3g was determined by RT-qPCR relative to housekeeping gene Hprt. n = 3 biological replicates of a representative experiment. Unpaired two-tailed Student’s t test (B, D,H & I) was performed to observe significant differences. ns = not significant, * P ≤ 0.05, ** P < 0.01, *** P ≤ 0.001.
Fig 2.
LSD1-deficient epithelium renders mice susceptible to C. rodentium.
(A) Survival plot of WT and cKO mice after C. rodentium infection. Kaplan-Meier survival curves was used and tested with a Gehan-Breslow-Wilcoxon test to identify significant differences between wild-type and cKO mice (p value = 0.0405). (B) Body weight loss comparing Naive, WT and cKO mice during C. rodentium infection. n = 9 (naive), n = 16 (WT) and n = 14 (cKO) mice, three pooled experiments. Data points are mean and SD. (C) Stool CFU (colony forming units) counts in WT (n = 22) and cKO (n = 21) infected mice using plating on agar after 6 days of C. rodentium infection. (D) CFU counts in liver and spleen tissue of WT (n = 12) and cKO (n = 11) mice at day 6 post C. rodentium infection. (E) RT-qPCR for Il22, Il17a and Ifng was carried out in colon tissue from naive (n = 10), WT (n = 12) and cKO (n = 11) mice infected with C. rodentium for 6 days. Expression is relative to Hprt. (F) ELISA for IL-22, IL-17A and IFN-γ in re-stimulated mesenteric lymph node cells after 6 days of C. rodentium infection. (G) Measurement of FITC dextran fluoroscence in serum derived from (naive = 4), WT (n = 4) and cKO (n = 4) infected with C. rodentium for 6 days. * P ≤ 0.05, ** P ≤ 0.01, Each dot represents one mouse, and means are depicted.
Fig 3.
Intestinal-epithelial LSD1 is required for appropriate crypt hyperplasia and goblet cell responses to infection with C. rodentium.
(A) RT-qPCR for antimicrobials Reg3b and Reg3g in colon tissue of Naive (WT n = 10), and WT (n = 12) and cKO (n = 11) mice infected with C. rodentium for 6 days. (B&C) H&E staining and quantification of crypt length at distal colon in WT (n = 6) and cKO (n = 5) mice infected with C. rodentium for 6 days (scale bar = 50 μm). (D) Ki67 staining to measure proliferation in distal colon tissue of WT and cKO mice infected with C. rodentium for 6 days. (E) RT-qPCR for goblet-cell specific antimicrobials Retnlb in colon tissue of Naive (WT n = 10), WT (n = 12) and cKO (n = 11) mice infected with C. rodentium for 6 days. (F&G) Representative confocal images and quantification of RELMβ+ cells (green) in distal colon tissue of WT (n = 6) and cKO (n = 5) infected mice. DAPI (blue) was used as a nuclear counter stain. Scale bar = 50 μm. Unpaired two-tailed Student’s t test (A, C, E & G) was performed to observe significant differences among experimental groups. ns = not significant, * P ≤ 0.05, **, P < 0.01, *** P ≤ 0.001. Each dot represents one mouse and means are depicted.
Fig 4.
Intestinal-epithelial LSD1 is required for effective immunity against the whipworm Trichuris muris.
(A) WT (n = 9), cKO (n = 9) and Rag1KO (n = 3) mice were infected with 200 embryonated eggs of T. muris. Worm burden was quantified in caecum after 21 days of infection. Pooled from 2 independent experiments. (B) PAS staining for goblet cells was done in ceacal epithelium of WT and cKO mice after 21 days of T. muris infection. (C) Quantification of PAS+ cells in caecal epithelium of WT (n = 8) and cKO (n = 9) mice after 21 days of T. muris infection. (D) RT-qPCR for Retnlb and Clca1 in proximal colon of WT (n = 8) and cKO (n = 8) after 21 days of T. muris infection. (E&F) Representative confocal images and quantification of RELMβ+ cells (green) in caecum tissue of WT (n = 8) and cKO (n = 9) mice infected with T. muris for 21 days. DAPI (blue) was used as a nuclear counter stain. Scale bar = 50 μm. (G) Measurement of cecal crypts length in WT (n = 8) and cKO (n = 9) mice infected with T. muris for 21 days. (H) Sandwich ELISA for quantification of IL-13 and IFN-γ expression in restimulated mesenteric lymph node cells after 21 days of T. muris infection. Unpaired two-tailed Student’s t test (A, C, D, F&G) and one-way ANOVA (H) was performed to observe significant differences. ns = not significant, * P ≤ 0.05, **, P < 0.01, *** P ≤ 0.001.
Fig 5.
LSD1 controls (immune-driven) goblet cell responses.
(A) Colon sections of WT and cKO mice were stained for MUC2 (green), Ulex europaeus agglutinin I (UEA1) (red). UEA1 is a lectin which binds to glycoproteins and glycolipids and used as an additional marker for goblet cells. DAPI (blue) used as a counterstaining. (B) Quantification of MUC2+ cells and UEA1+ cells in the colon of WT (n = 7) and cKO (n = 8) mice. (C) WT and cKO colon organoids were seeded in ENR for 4 days and immunofluorescent staining of MUC2 (green) and β- catenin (purple) was performed. (D) Quantification of MUC2 intensity per colon organoid is represented. Colon organoids were derived from WT (n = 2) and cKO (n = 2) mice. (E&G) Immunofluorescent confocal images for MUC2 (E, green) and RELMβ (G, green) in WT and cKO organoids left untreated in ENR or supplemented with IL-13 (10 ng/ml), IL-22 (5ng/ml) or DAPT 10 μM for 4 days. DAPI was used to stain nuclei (blue). (F&H), Quantification of MUC2 fluorescence intensity (F) and number of RELMβ positive cells (H) per organoid. (I) Representative bright field images of WT and cKO organoids treated with IL-13 (10 ng/ml), IL-22 (5ng/ml) and DAPT 10 μM for 4 days. Scale bar is 100μm. (J) Quantification of organoid area from minimal projections 4 days after seeding. Dots represents the mean of one well. Images are representative of 3 independent experiments. (K) cKO organoids were treated with IL-13 (10 ng/ml), IL-22 (5ng/ml) and combination of IL-13 + IL-22 for 4 days. Immunofluorescent images of RELMβ (green) and β-catenin (purple). (L) Quantification of RELMβ+ cells per organoid are shown. Unpaired two-tailed Student’s t test (B & D) and One-way analysis of variance with Tukey’s Multiple Comparison Test (F, H, J& L) was performed to observe significant differences. ns = not significant, * P ≤ 0.05, **, P < 0.01, *** P ≤ 0.001.
Fig 6.
LSD1 epigenetically controls modulators of the cytoskeleton.
(A) Application of GSEA of a goblet cell gene set in WT and cKO small intestine RNA seq, and untreated vs GSK-LSD1 (24h) treated organoids RNA-seq data. (B) Top 500 genes upregulated in cKO crypts were selected and unbiased association with the top 20 GO terms is displayed. (C) Genes taken from in 6B bold-selected GO terms are displayed in a heatmap using Transcripts Per Million (TPM) values scaled to the max of the gene comparing WT and cKO crypts (left) and untreated and GSK-LSD1 (24h) treated organoids (right). (D) Mean ATAC signal of normalized peak density surrounding previously identified H3K4me1 peaks that are significantly up in cKO crypts compared to WT crypts. Representative graph from n = 2. (E) Representative Integrative Genomics Viewer (IGV) tracks of H3K4me1 and ATAC signal at the Flna locus (n = 2). (F) IGV tracks of H3K4me1 and LSD1 levels at the FLNA locus in a human cancer cell line. (G) Representative confocal images of FILAMIN A (FLNA) stained in WT and cKO organoids. Quantification of FLNA intensity in WT and cKO organoids. (H) WT organoids were untreated (ENR) or treated (GSK-LSD1- 5 μM) for 4 days and then stained for FLNA (green). DAPI was used as a counter stain (blue). Scale bar = 50 μm Organoids were quantified for FLNA fluorescent intensity. (I) FLNA staining in paraffin embedded colon sections of naive WT and cKO mice. White Arrow indicates the increased FLNA expression on the apical side of colonic crypts. Scale bar = 50 μm. Unpaired Student’s t test was performed to observe significant differences. *** P ≤ 0.001.
Fig 7.
Cytoskeleton manipulation partially rescues goblet cell maturation in LSD1-deficient organoids.
(A) cKO organoids were left untreated (ENR) or treated with Y-27632 (10 μM) for 4 days. Representative bright field images are shown. Scale bar is 100 μm. (B) Quantification of organoid area from minimal projections 4 days after seeding. Dots represents mean of one well, pooled from 3 independent experiments. (C&D) Representative immunofluorescent images of MUC2 (green) and β-catenin (purple). MUC2 fluorescence intensity relative to organoid area was measured. Scale bar is 50 μm. (E) cKO organoids were treated with IL-22 (5ng/ml) or IL22+ Y-27632) for 4 days. Representative bright field images are shown. Scale bar is 100 μm. (F) Mean organoid area of cKO organoids is depicted as well as the percentage of spheroids in each well 4 days after seeding. (G) cKO organoids were left untreated (ENR) or treated with IL-22 (5ng/ml), Y-27632 (10 μM) and combination of Y-27632 and IL-22 for 4 days and representative images for RELMβ (green) and β-catenin (purple)staining are shown. (H) Pooled quantification of RELMβ+ cells per organoid area from independent experiments. (I) WT organoids were pre-treated with GSK-LSD1 for 7 days. Next, these organoids were treated with combination of IL-22 (5ng/ml) + GSK-LSD1 and GSK-LSD1+ IL-22+ Y-27632 for 4 days. Immunofluorescence images of RELMβ (green) and β-catenin (purple) are shown. (J) Quantification of RELMβ+ cells per organoid area is shown. Unpaired two-tailed Student’s t test (B, D, F, H & J) was performed to define significance. * P ≤ 0.05, **, P < 0.01, *** P ≤ 0.001.