TNFSF13 insufficiency disrupts human colonic epithelial cell growth and associated B cell dynamics
Xianghui Ma, Shaneice K. Nettleford, Yuhua Tian, Noor Dawany, Ayano Kondo, Yalan Li, Kelly Maurer, Tatiana A. Karakasheva, Rawan Shraim, Patrick A. Williams, Louis R. Parham, Lauren A. Simon, Charles H. Danan, Maire A. Conrad, David A. Piccoli, Marcella Devoto, Neil Romberg, Kathleen E. Sullivan, Klaus H. Kaestner, Judith R. Kelsen, Kathryn E. Hamilton
Xianghui Ma, Shaneice K. Nettleford, Yuhua Tian, Noor Dawany, Ayano Kondo, Yalan Li, Kelly Maurer, Tatiana A. Karakasheva, Rawan Shraim, Patrick A. Williams, Louis R. Parham, Lauren A. Simon, Charles H. Danan, Maire A. Conrad, David A. Piccoli, Marcella Devoto, Neil Romberg, Kathleen E. Sullivan, Klaus H. Kaestner, Judith R. Kelsen, Kathryn E. Hamilton
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Research Article Gastroenterology Inflammation

TNFSF13 insufficiency disrupts human colonic epithelial cell growth and associated B cell dynamics

Abstract

Cytokines mediating epithelial and immune cell interactions modulate mucosal healing—a process that goes awry with chronic inflammation as in inflammatory bowel disease. TNFSF13 is a cytokine important for B cell maturation and function, but roles for epithelial TNFSF13 and putative contribution to inflammatory bowel disease are poorly understood. We evaluated functional consequences of a novel monoallelic TNFSF13 variant using biopsies, tissue-derived colonoids and induced pluripotent stem cell (iPSC)-derived colon organoids. TNFSF13 variant colonoids exhibited a >50% reduction in secreted TNFSF13, increased epithelial proliferation, and reduced apoptosis, which was confirmed in iPSC-derived colon organoids. Single cell RNA-sequencing and flow cytometry suggested FAS as the predominant colonic epithelial receptor for TNFSF13, which was confirmed by co-immunoprecipitation and binding assays. Imaging mass cytometry revealed an increase in epithelial-associated B cells in TNFSF13 variant colon tissue sections. Finally, TNFSF13 variant colonoids co-cultured with memory B cells demonstrated a reduction in immunoglobulin-producing plasma cells compared to control colonoid cocultures. Our findings support a role for epithelial TNFSF13 as a regulator of colonic epithelial growth and epithelial crosstalk with B cells.

Authors

Xianghui Ma, Shaneice K. Nettleford, Yuhua Tian, Noor Dawany, Ayano Kondo, Yalan Li, Kelly Maurer, Tatiana A. Karakasheva, Rawan Shraim, Patrick A. Williams, Louis R. Parham, Lauren A. Simon, Charles H. Danan, Maire A. Conrad, David A. Piccoli, Marcella Devoto, Neil Romberg, Kathleen E. Sullivan, Klaus H. Kaestner, Judith R. Kelsen, Kathryn E. Hamilton

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Figure 1

TNFSF13 variant colonoids/organoids exhibit enhanced colonoid formation efficiency and proliferation.

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TNFSF13 variant colonoids/organoids exhibit enhanced colonoid formation ...
(A) ELISA for secreted TNFSF13 in colonoid conditioned media (n = 3 patient lines for control and VEO-IBD; n = 3 passages for variant). (B) Representative TNFSF13 RNAscope in colonoids from control and variant participants (scale bar: 50 μm; n = 3 patient lines for control; n = 3 passages for variant). (C) Costaining of TNFSF13 and FAS RNAscope probes with Ki67 antibody in colon biopsies. Arrows indicate cells accumulated outside epithelial crypts (n = 3 patients for control; n = 3 tissue blocks for variant). (D) Representative images of colonoid formation assays at day 6 after seeding (scale bar: 300 μm). (E) Quantification of newly formed colonoids by size at day 6. Each passage included 2 technical replicates (n = 4 patient lines for control and VEO-IBD; n = 4 passages for variant). (F) TNFSF13 and FAS RNAscope with E-cadherin immunostaining in WT and variant iPSC-derived colon organoids at day 7 (scale bar: 50 μm; n = 3 passages). (G) Representative colonoid formation in WT and variant iPSC-derived organoids at day 9 (scale bar: 400 μm; n = 3 passages, each with ≥ 2 technical replicates). (H) Quantification of colonoid formation rate and area at day 9. Colonoid size calculated by maximum vertical projection area. (I and J) Percentage of EdU+ cells following IgG or TNFSF13 neutralizing antibody (nTNFSF13) treatment in (I) control tissue–derived colonoids (n = 3 patient lines) or (J) WT iPSC-organoids at day 7 (n = 3 passages). 2-way ANOVA with multiple comparisons was used for (A and E); 2-tailed Student’s t test for (H–J). P values shown on graphs unless P > 0.05.