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 2

FAS is a receptor for TNFSF13 in colonic epithelial cells.

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FAS is a receptor for TNFSF13 in colonic epithelial cells. Percentage of...
Percentage of FAS+ and HVEM+ cells by flow cytometry at day 7 after seeding in (A) colonoids (n = 3 patient lines for control and VEO-IBD; n = 3 passages for variant) or (B) iPSC-organoids (n = 3 passages per line). (C) Representative Western blot for FAS from coimmunoprecipitation supernatant in control and VEO-IBD colonoids at day 7. TNFSF13 antibody used for capture. Protein ladder lane (M) not shown in overlay (n ≥ 4 patient lines for control and VEO-IBD). (D) SPR binding analyses of FASL-FAS (upper) and TNFSF13-FAS (lower) interactions at various concentrations (n = 3 independent experiments). (E) Costaining of TNFSF13 and FAS RNAscope probes in control colonoids at day 7. Arrows indicate coexpression (scale bar: 100 μm; n = 3 experiments). (F) Co staining of TNFSF13 and FAS RNAscope probes with Ki67 antibody in control colonoids at day 7. Arrows indicate triple-positive cells (scale bar: 100 μm; n = 3 experiments). (G) Costaining of TNFSF13 and FAS RNAscope probes with FABP2 antibody in control colonoids at day 7. Arrows indicate triple-positive cells (scale bar: 100 μm; n = 3 experiments). (H and I) Percentage of EdU+ cells in control colonoids (left) or WT iPSC-organoids (right) at day 7 following treatment with (H) IgG or FAS neutralizing antibody (nFAS) or (I) IgG or recombinant human FAS ligand (rFASL). Two-way ANOVA with multiple comparisons for (A and B); 2-tailed Student’s t test for (H and I). P values shown unless P > 0.05.