Interleukin-7 links T lymphocyte and intestinal epithelial cell homeostasis

Abstract

Interleukin-7 (IL-7) is a major survival factor for mature T cells. Therefore, the degree of IL-7 availability determines the size of the peripheral T cell pool and regulates T cell homeostasis. Here we provide evidence that IL-7 also regulates the homeostasis of intestinal epithelial cells (IEC), colon function and the composition of the commensal microflora. In the colon of T cell-deficient, lymphopenic mice, IL-7-producing IEC accumulate. IEC hyperplasia can be blocked by IL-7-consuming T cells or the inactivation of the IL-7/IL-7R signaling pathway. However, the blockade of the IL-7/IL-7R signaling pathway renders T cell-deficient mice more sensitive to chemically-induced IEC damage and subsequent colitis. In summary, our data demonstrate that IL-7 promotes IEC hyperplasia under lymphopenic conditions. Under non-lymphopenic conditions, however, T cells consume IL-7 thereby limiting IEC expansion and survival. Hence, the degree of IL-7 availability regulates both, T cell and IEC homeostasis.

Figure 1. Elevated levels of IL-7 expression and IEC hyperplasia in the colon of Rag⁻ mice.

(A) Representative bioluminescence (BL) images for Rag-competent (Rag⁺; n = 31) and Rag-deficient IL-7GCDL mice (Rag⁻; n = 21) are shown. BL was determined (B) in the intestine and (C) thymus, heart, lung, liver, skin and kidney of Rag⁺ (n = 12) and Rag⁻ IL-7GCDL mice (n = 12). (A–C) BL is shown in photons per s per cm² per steradian. (D, E) Colon sections from Rag⁺ (n = 5–8) and Rag⁻ IL-7GCDL mice (n = 6–8) were stained with (D) periodic acid-Schiff (PAS)/Alcian blue (AB) or (E) DAPI and antibodies for IL-7 and EpCam. (D) Differentiated goblet cells stain positive for PAS (red) and appear purple/magenta. Acidic mucopolysaccharides/glycosaminoglycans are visualized by AB. Arrows indicate the distance between the basis of the crypts and the colon lumen. (D, E) Data are representative for 3 independent experiments and 2–3 staining reactions per mouse.

Figure 2. IL-7R signaling promotes lymphopenia-associated IEC hyperplasia and alters colon function.

(A) Colon sections from WT (n = 13–16), Rag⁻ (n = 16–37) and Rag⁻IL-7R⁻ mice (n = 13) were stained with DAPI and antibodies for Ki67, EpCam, cleaved caspase 3 (Casp3) or Gob5. (B) Colon wall thickness (µm) and (C) the percentage of Ki67⁺ cells in crypts were determined for WT (n = 6), Rag⁻ (n = 12) and Rag⁻IL-7R⁻ (n = 6) mice. (B, C) 30–60 individual measurements were performed. (A–C) Data are representative for 4 independent experiments and 2–3 staining reactions per mouse. (D) Transepithelial resistance (Ω·cm²), and apparent permeabilities (P) for (E) Na⁺ and (F) Cl⁻ were determined for colon samples from WT (n = 6), Rag⁻ (n = 5) and Rag⁻IL-7R⁻ (n = 5) mice. Five to twelve independent measurements per experimental group were performed. (B–F) Shown are mean values+SEM. Statistically significant values are indicated: ** p<0.01 and *** p<0.001 (Student's t test).

Figure 3. IEC express functional IL-7R.

(A, B) IEC were isolated from the colon of (A) Rag⁻IL-7R⁻ and (A, B) Rag⁻ mice. (B) Rag⁻ IEC were stimulated with 20 ng/ml rec. IL-7 for 15 minutes or were left untreated. The levels of IL-7R expression and Stat5 phosphorylation (p-Stat5) were determined by flow cytometry. (A, B) Shown are relative cell numbers and relative fluorescence intensities for (A) IL-7R and (B) p-Stat5 after gating on viable (A) (7AAD⁻), (A, B) CD45⁻, EpCam⁺ IEC. Results are representative for 2–3 independent experiments.

Figure 4. IL-7 promotes IEC proliferation and survival.

(A, B) Rag⁻IL-7⁻ (IL-7⁻; n = 3) and Rag⁻IL-7R⁻ (IL-7R⁻; n = 3) mice were treated with PBS (white bars) or IL-7/anti-IL-7 (black bars) twice a week for 2 weeks. (A, C) Colon wall thickness (µm) and (B, D) the percentage of Ki67⁺ cells in crypts were determined in colon sections from (A, B) IL-7-treated Rag⁻IL-7⁻ and Rag⁻IL-7R⁻ mice and (C, D) untreated WT (n = 6) and IL-7tg (n = 5) mice. (A) 30–54, (B) 15–23, (C) 47–68 and (D) 29–32 individual measurements were performed per experimental group. Shown are mean values+SEM. Statistically significant values are indicated: * p<0.05 and ** p<0.01 (Student's t test). (E) Colon sections from PBS-treated (upper row) and IL-7/anti-IL-7-treated Rag⁻IL-7⁻ (lower row; n = 3) were stained with DAPI and antibodies for Ki67, EpCam, cleaved caspase 3 (Casp3) or β-catenin (βcat). (F) Colon sections from WT (n = 5) and IL-7tg mice (n = 6) were stained with DAPI and antibodies for β-catenin. (E, F) White arrows indicate nuclei. Bar diagrams show the percentage of luminal IEC with nuclear β-catenin. 130–280 nuclei per experimental group were counted. Shown are mean values+SEM. Statistically significant values are indicated: * p<0.05 and ** p<0.01 (Student's t test). (A–F) Data represent one experiment with a total of 23 individual mice and 2–3 independent staining reactions per mouse.

Figure 5. T lymphocytes regulate IEC homeostasis in an antigen-independent, IL-7R-dependent fashion.

(A, B) Rag⁻ (n = 6) and Rag⁻IL-7R⁻ (n = 7) were reconstituted with 5×10⁶ polyclonal CD4⁺ and CD8⁺ T cells from Thy1.1-congenic mice. Untreated Rag⁻ (n = 8) and Rag⁻IL-7R⁻ (n = 7) served as controls. Eighty-five days after transfer, colon sections were prepared and analyzed for (A) colon wall thickness (µm) and (B) the percentage of Ki67⁺ cells in crypts. Data are representative for 2 independent experiments. (A, B) Colon sections from untreated Rag⁻OT-I⁺ mice (n = 5) were analyzed in parallel. (A) 45–62 and (B) 26–43 individual measurements were performed per experimental group. (C) Relative IL-7 mRNA levels were determined in relation to β-actin mRNA levels in the colon of the indicated groups (Rag⁻ n = 12; Rag⁻+T cells n = 8; Rag⁻ IL-7R⁻ n = 8; Rag⁻ IL-7R⁻+T cells n = 8; Rag⁻ OTI⁺ n = 5). (A–C) Shown are mean values+SEM. Statistically significant values are indicated: * p<0.05, ** p<0.01, *** p<0.001 (Student's t test). (D) Colon sections from untreated Rag⁻ (n = 37), T cell-reconstituted Rag⁻ (n = 6) and Rag⁻OT-I⁺ mice (n = 5) were stained with DAPI and antibodies for Ki67, EpCam, cleaved caspase 3 (Casp3), Gob5 or β-catenin (βcat). White arrows indicate nuclei. Data are representative for at least two independent experiments and 2–3 staining reactions per mouse. (E) Rag⁻OT-I⁺ spleen cells were analyzed by flow cytometry. Shown are relative cell numbers and fluorescence intensities for IL-7R on CD8⁺ OT-I T cells (Rag⁻OT-I⁺). In control samples, primary antibodies were omitted. Results are representative for 2 independent experiments.

Figure 6. IL-7R signaling protects Rag⁻ mice from DSS-induced colitis.

(A, B) WT (n = 4), Rag⁻ (n = 8), Rag⁻IL-7R⁻ (n = 7) and Rag⁻OT-I⁺ mice (n = 6) received dextran sulfate sodium (DSS) via the drinking water. From day 5 on, DSS-free drinking water was provided. (A) Body weight was determined every day and calculated in relation to the initial body weight. Shown are the mean relative body weight ± SEM and the time after onset of DSS treatment. (B, C) Colon samples were taken at day 8 and analyzed histologically. Shown are histological scores for groups of untreated (open symbols; n = 4) and DSS-treated mice (closed symbols; n = 6–8). (C) Shown are representative colon sections from the indicated mice. (A–C) Data represent one experiment.