Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis
Efren A. Reyes, … , Zev J. Gartner, Ophir D. Klein
Efren A. Reyes, … , Zev J. Gartner, Ophir D. Klein
Published August 29, 2023
Citation Information: J Clin Invest. 2023;133(20):e163591. https://doi.org/10.1172/JCI163591.
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Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis

Abstract

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell–derived TNF as an upstream regulator of mucin homeostasis.

Authors

Efren A. Reyes, David Castillo-Azofeifa, Jérémie Rispal, Tomas Wald, Rachel K. Zwick, Brisa Palikuqi, Angela Mujukian, Shervin Rabizadeh, Alexander R. Gupta, James M. Gardner, Dario Boffelli, Zev J. Gartner, Ophir D. Klein

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Figure 5

Epithelial TNF modulates CFTR-induced fluid pumping through TNFR1.

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Epithelial TNF modulates CFTR-induced fluid pumping through TNFR1. (A) E...
(A) Eighty-minute montage of WT and Tnf–/– organoids live imaged 8 hours after plating under normal growth conditions. Magenta masks outline lumens at 0 minutes and green masks outline lumens at 80 minutes. Scale bar: 50 μm. (B) Quantification of the inflation rate of organoids during 80 minutes without lumen collapse under normal growth conditions. Lumen size was normalized to t = 0 minutes and was measured for each individual organoid at 20-minute intervals. A simple linear regression was performed for compiled data. (C) Lumen size of organoids treated with 1 ng/mL rTNF and 50 μM CFTRinh-172 for 24 hours. Measurements were normalized to the size of individual lumens at t = 0 hours. NT, no treatment. (D) Lumen size of control or VillinCreERT2; Cftrfl/fl organoids treated with 1 ng/mL rTNF for 24 hours. (E) Lumen swelling of 2-day-old WT organoids costimulated with 0.4 μM forskolin and 1 ng/mL rTNF plus inhibitors of either CFTR (20 μM CFTRinh-172) or PKC (5 μM GF109203X). Plots represent mean of 30 organoids from 3 distinct organoid lines. FSK, forskolin. (F) Area under the curve of forskolin-induced swelling plots. Each point represents the mean AUC for an individual organoid line within the group. (G) Lumen size of control or Tnfr-KO organoids treated with 1 ng/mL rTNF for 24 hours. For all organoid experiments, n = 3 distinct established organoid lines per group with more than 20 organoids quantified per line. A 2-tailed P value was calculated testing the null hypothesis that the slopes between the 2 groups were equal in B. P values were calculated using an ordinary 1-way ANOVA with Dunnett’s multiple-comparison test comparing all groups to the control for C, D, and F and an ordinary 1-way ANOVA with Turkey’s multiple-comparison test for G.