Goblet cell hyperplasia is not epithelial-autonomous in the Cftr knockout intestine

Abstract

Goblet cell hyperplasia is an important manifestation of cystic fibrosis (CF) disease in epithelial-lined organs. Explants of CF airway epithelium show normalization of goblet cell numbers; therefore, we hypothesized that small intestinal enteroids from Cftr knockout (KO) mice would not exhibit goblet cell hyperplasia. Toll-like receptors 2 and 4 (Tlr2 and Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum compared with wild-type (WT) mice. IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2, and Tlr4 protein expression were increased in Cftr KO intestine relative to WT. In contrast, WT and Cftr KO enteroids did not exhibit differences in basal or IL-13-stimulated goblet cell numbers, or differences in expression of Tlr2, Tlr4, and Spdef. Ileal goblet cell numbers in Cftr KO/Tlr4 KO and Cftr KO/Tlr2 KO mice were not different from Cftr KO mice, but enumeration was confounded by altered mucosal morphology. Treatment with Tlr4 agonist LPS did not affect goblet cell numbers in WT or Cftr KO enteroids, whereas the Tlr2 agonist Pam3Csk4 stimulated goblet cell hyperplasia in both genotypes. Pam3Csk4 stimulation of goblet cell numbers was associated with suppression of Notch1 and Neurog3 expression and upregulated determinants of goblet cell differentiation. We conclude that goblet cell hyperplasia and inflammation of the Cftr KO small intestine are not exhibited by enteroids, indicating that this manifestation of CF intestinal disease is not epithelial-automatous but secondary to the altered CF intestinal environment.NEW & NOTEWORTHY Studies of small intestinal organoids from cystic fibrosis (CF) mice show that goblet cell hyperplasia and increased Toll-like receptor 2/4 expression are not primary manifestations of the CF intestine. Intestinal goblet cell hyperplasia in the CF mice was not strongly altered by genetic ablation of Tlr2 and Tlr 4, but could be induced in both wild-type and CF intestinal organoids by a Tlr2-dependent suppression of Notch signaling.

Keywords: Toll-like receptor; cystic fibrosis; enteroid; goblet cell; small intestine.

Graphical abstract

Figure 1.

Goblet cell hyperplasia and markers of inflammation in Cftr KO mouse jejunum. A: mean goblet cell numbers per 100 cell nuclei in villous epithelium of proximal jejunum from WT and Cftr KO sex-matched littermate mouse pairs. Individual data points are shown. PEG laxative: mice consumed a mouse chow diet and were provided 3,350 m. wt. polyethylene glycol in their drinking water to prevent intestinal impaction in the Cftr KO mice. Liquid diet: mice consumed a nutritionally complete liquid diet (Peptamen) for 2 wk to prevent intestinal impaction in the Cftr KO mice. *Significantly different from WT, P = 0.0123 (PEG laxative), P = 0.0026 (liquid diet) by unpaired t test. +Significantly different from PEG laxative, P = 0.00026 by unpaired t test; n = 7 WT-Cftr KO mouse pairs (3 males, 4 females) and three WT-Cftr KO mouse pairs (1 male, 2 females), respectively (see Supplemental Fig. S1; https://doi.org/10.6084/m9.figshare.16613464.v1, for images of PAS-stained images of WT and Cftr KO intestine under the two conditions). B: mRNA expression of IL-13 in freshly isolated jejunal crypts from WT and Cftr KO sex-matched littermate mouse pairs consuming PEG osmotic laxative since weaning (left) or after switching to Peptamen liquid diet for 2 wk (right). Individual data points are shown. *Significantly different from WT, P = 0.029 by Mann–Whitney rank sum test; n = 4 WT-Cftr KO mouse pairs. *Significantly different from WT, P < 0.005 by unpaired t test; n = 3 WT-Cftr KO mouse pairs. C: densitometry of immunoblots of Toll-like receptor Tlr2, Tlr4, and SAM pointed domain-containing ETS transcription factor (Spdef). WT densitometry is set at 1.0. Individual data points are shown. *Significantly different from WT by Mann–Whitney rank sum test, P = 0.001 for Tlr2, n = 8 WT-Cftr KO mouse pairs (4 males and 4 females); P = 0.0252 for Tlr4, n = 5 WT-Cftr KO mouse pairs (3 males and 2 females); P = 0.001 for Spdef, n = 8 WT-Cftr KO mouse pairs (4 males and 4 females). KO, knockout; PAS, periodic acid-Schiff; PEG, polyethylene glycol; Tlr, Toll-like receptor; WT, wild-type.

Figure 2.

Goblet cell hyperplasia and intestinal inflammation are normalized in Cftr KO enteroids. A: photomicrographs of PAS-stained enteroids under basal (left) and IL-13-treated (right) conditions. Yellow arrows, mucus anchored to goblet cells in Cftr KO enteroids. Scale bars = 150 µm. B: goblet cell count/100 nuclei in the villus-like region (between crypts) in jejunal enteroids from WT and Cftr KO sex-matched littermate mice under basal and IL-13-treated conditions. Individual data points are shown. ns, not significant WT vs. Cftr KO; *Significantly different from basal by unpaired t test. WT basal vs. WT IL-13, P = 0.024, Cftr KO basal vs. Cftr KO IL-13, P = 0.009; n = 4 WT-Cftr KO mouse pairs (3 males and 1 female). Average from 5 to 15 enteroids counted per mouse. C: densitometry of immunoblots of Toll-like receptor Tlr2, Tlr4, and SAM pointed domain-containing ETS transcription factor (Spdef) in passage 1, days 5–7 enteroids from WT, and Cftr KO sex-matched littermate mice. WT densitometry is set to 1. Individual data points are shown. ns, not significant from WT by Mann–Whitney rank sum test for Tlr2 (8 mouse pairs, 4 males and 4 females), Tlr4 (5 mouse pairs, 2 males, 3 female), and Spdef (6 mouse pairs, 3 maless and 3 female). KO, knockout; PAS, periodic acid-Schiff; WT, wild-type.

Figure 3.

Goblet cell number and morphological changes in ileum from WT, Cftr KO, and Tlr4 KO crossbred mice. A: goblet cell counts/100 nuclei in villous epithelium of PAS-stained sections of ileum from WT/Tlr4^+/+, WT/Tlr4^+/−, WT/Tlr4^−/−, Cftr KO/Tlr4^+/+, Cftr KO/Tlr4^+/−, and Cftr KO/Tlr4^−/− mice. Individual data points are shown. ^a,b,cMeans with the same letter are not significantly different by one-way ANOVA and Holm–Sidak pairwise comparisons. Cftr KO/Tlr4^+/+ significantly different from all WT genotypes, P < 0.001 vs. WT/Tlr4^+/+, P = 0.004 vs. WT/Tlr4^+/−, and P = 0.003 vs. WT/Tlr4^−/−. Cftr KO/Tlr4^+/− significantly different from WT/Tlr4^+/+, P = 0.021; n = 4 WT/Tlr4^+/+ (2 males, 2 females), 3 WT/Tlr4^+/− (3 males), 3 WT/Tlr4^−/− (3 males), 4 Cftr KO/Tlr4^+/+ (2 females, 2 males), 3 Cftr KO/Tlr4^+/− (2 males, 1 female), and 2 Cftr KO/Tlr4^−/− mice (2 males). B: photomicrographs of PAS-stained ileum from WT (top left), Cftr KO (top right), Tlr4 KO (bottom left), and Cftr KO/Tlr4 KO double knockout (bottom right). White arrows, elongated crypts. Black arrows, shortened villi. Blue arrow, lamina propria upfolding. Scale bars = 50 µm. KO, knockout; PAS, periodic acid-Schiff; Tlr, Toll-like receptor; WT, wild-type.

Figure 4.

Goblet cell number and morphological changes in ileum from WT, Cftr KO, and Tlr2 KO crossbred mice. A: goblet cell counts/100 nuclei in villous epithelium of PAS-stained sections of ileum from WT/Tlr2^+/+, WT/Tlr2^−/−, Cftr KO/Tlr2^+/+, and Cftr KO/Tlr2^−/−, and WT/Tlr2^−/− vs. individual data points shown. ^a,bMeans with the same letter are not significantly different by one-way ANOVA and Holm–Sidak pairwise comparisons. P = 0.004; WT/Tlr2^+/+ vs. Cftr KO/Tlr2^+/+, P = 0.014; WT/Tlr2^+/+ vs. Cftr KO/Tlr2^−/−, P = 0.014; WT/Tlr^−/− vs. Cftr KO/Tlr2^+/+, P = 0.044; WT/Tlr2^−/− vs. Cftr KO/Tlr2^−/−, P = 0.038; n = 4 WT/Tlr2^+/+ (1 male, 3 females), 5 WT/Tlr2^−/− (3 males, 2 females), 4 Cftr KO/Tlr2^+/+ (1 male, 3 females), and 3 Cftr KO/Tlr4^−/− mice (2 males, 1 female). B: photomicrographs of PAS-stained ileum from WT (left), Tlr2 KO (middle), and Cftr KO/Tlr2 KO (right) mice. Black arrows, widened villi. Yellow arrows, villus fusion. Blue arrow, lamina propria upfolding. Scale bars = 50 µm. KO, knockout; PAS, periodic acid-Schiff; Tlr, Toll-like receptor; WT, wild-type.

Figure 5.

Goblet cell numbers in LPS and Pam3Csk4-treated enteroids from WT and Cftr KO mice. A: Passage 1, 7-day-old WT and Cftr KO small intestinal enteroids that were untreated (control) or treated with LPS (100 µg/mL) for 72 h. Individual data points shown. ns, not significantly different by unpaired t test; n = 3 WT and Cftr KO sex-matched littermate mouse pairs (2 males, 1 female). Average from 4 to 18 enteroids counted per mouse. B: Passage 1, 7-day-old WT, Cftr KO, and Tlr2 KO enteroids that were untreated (control) or treated with Pam3Csk4 (100 µM) for 72 h. Individual data points shown. *Signficantly different from Control by unpaired t test, P < 0.0002 for WT and P < 0.0007 for Cftr KO. n = 4 WT and Cftr KO sex-matched littermate mouse pairs (3 males, 1 female). Average from 5 to 23 enteroids counted per mouse. C: Passage 1, 7-day-old WT that were treated with DMSO vehicle (control) or treated with vehicle + Pam3Csk4 (100 µM) or Pam3Csk4 (100 µM) + CAPE (50 µg/mL) for 72 h. Individual data points shown. ^a,bMeans with the same letter are not significantly different by one-way ANOVA and Holm–Sidak vs. control comparisons. P = 0.035; n = 4 WT mice (3 males, 1 female). Average from 4 to 22 enteroids counted per mouse. D: Passage 1, 7-day-old WT that were treated with DMSO vehicle (control) or treated with vehicle + Pam3Csk4 (100 µM) or Pam3Csk4 (100 µM) + SB202190 (50 µM) for 72 h. Individual data points shown. ^a,bMeans with the same letter are not significantly different by one-way ANOVA and Holm–Sidak vs. control comparisons. P = 0.022; n = 4 WT mice (3 males, 1 female). Average from 3 to 11 enteroids counted per mouse. KO, knockout; LPS, lipopolysaccharide; PAS, periodic acid-Schiff; PEG, polyethylene glycol; Tlr, Toll-like receptor; WT, wild-type.