SUSTAINED TL1A (TNFSF15) EXPRESSION ON BOTH LYMPHOID AND MYELOID CELLS LEADS TO MILD SPONTANEOUS INTESTINAL INFLAMMATION AND FIBROSIS

Abstract

TL1A is a member of the TNF superfamily, and its expression is increased in the mucosa of inflammatory bowel disease patients. Moreover, patients with certain TNFSF15 variants over-express TL1A and have a higher risk of developing strictures in the small intestine. Consistently, mice with sustained Tl1a expression in either lymphoid or myeloid cells develop spontaneous ileitis and increased intestinal collagen deposition. Transgenic (Tg) mice with constitutive Tl1a expression in both lymphoid and myeloid cells were generated to assess their in vivo consequence. Constitutive expression of Tl1a in both lymphoid and myeloid cells showed increased spontaneous ileitis and collagen deposition than WT mice. T cells with constitutive expression of Tl1a in both lymphoid and myeloid cells were found to have a more activated phenotype, increased gut homing marker CCR9 expression, and enhanced Th1 and Th17 cytokine activity than WT mice. Although no differences in T cell activation marker, Th1 or Th17 cytokine activity, ileitis, or collagen deposition were found between constitutive Tl1a expression in lymphoid only, myeloid only, or combined lymphoid and myeloid cells. Double hemizygous Tl1a-Tg mice appeared to have worsened ileitis and intestinal fibrosis. Our findings confirm that TL1A-DR3 interaction is involved in T cell-dependent ileitis and fibrosis.

Keywords: fibrosis; ileitis; mucosal inflammation; transgenic.

Fig. 1.

Phenotypic characterization of Tl1a Tg mice. (a) Body weights for WT (filled circle), L-Tg (open square), M-Tg (filled triangle) or L+M-Tg mice (filled square) are shown. Data are expressed as mean weight in grams (g) ± s.d. There are six mice in each group. Stool consistency (b) and fecal blood (c) were determined using standard measurements [21]. Data are expressed as mean ± s.d., n = 6 for each mouse group. Total number of cells were isolated from MLN (d) and lamina propria mononuclear cells (e) from the distal 10 cm of small intestine and represented as absolute cell number ×10⁶. Each data point represents an independent mouse. *P < 0.05, **P < 0.01

Fig. 2.

Tl1a Tg mice developed ileitis. (a) Myeloperoxidase (MPO) activity is measured on the distal 3 cm of ilea and mid-colon. Data are expressed as arbitrary unit (U) per gram (g) of protein. (b) Representative H&E-stained ilea sections obtained from 12-month-old mice at ×200 magnification are shown. (c) Quantative histology scores for ileum and colon at 12 months of age are shown and data are expressed as mean ± s.d., n = 6 per mouse group. *P < 0.05, **P < 0.01

Fig. 3.

Sustained Tl1a expression led to an increased expression of activation and gut homing marker on Tl1a-Tg T cells. Representative flow cytometry plot of cells isolated from MLN from 12 months old mice is shown for CCR9 in (a) and CD44 in (b). The percentages of CCR9 and CD44 from MLN CD4⁺ T cells are quantitated and represented as mean ± s.d., n = 6 per mouse group. *P < 0.05, **P < 0.01

Fig. 4.

Constitutive Tl1a expression enhanced Th-1 and Th-17 immune responses. (a) Representative flow cytometry plots of gated CD4⁺ cells from MLN isolated from 12-month-old mice were stained for intracellular Ifn-γ and Il-17 expressions are shown on top left panel. The percentages of CD4⁺IFNγ⁺, and CD4⁺Il17⁺ T cells are represented as mean ± s.d. and shown on top right panel. n = 6 per mouse group. (b) Isolated mononuclear cells from MLN were stimulated with anti-CD3 and anti-CD28 and the levels of secreted Ifnγ, Il-17, and Il-13 were assessed by ELISA. Each data point represents an independent mouse. *P < 0.05, **P < 0.01

Fig. 5.

Increased intestinal fibrosis and fibrogenesis in Tl1a-Tg mice. (a) Representative Masson Trichrome staining of collagen deposition in mid-colon tissue sections is shown in left panels. Thickness of collagen deposition is quantitated and shown in right panel. Fields at ×200 magnification were scored. (b) The expression of collagen (col1a2) and pro-fibrogenic factors (Tgfβ1, Igf1) are measured by RT-PCR and represented as mean ± s.d., n = 6 per mouse group. *P < 0.05