SAMP1/YitFc mice develop ileitis via loss of CCL21 and defects in dendritic cell migration

Abstract

Background & aims: The lymphatic chemokine CCL21 is required for dendritic cell (DC) migration from tissues to lymph nodes, which helps establish tolerance to foreign yet harmless antigens. We demonstrate that CCL21 is almost completely absent from SAMP1/YitFc (SAMP) mice, which spontaneously develop chronic ileitis that resembles Crohn's disease, and that DC migration is severely impaired in these mice compared with AKR mice (controls). Toll-like receptor agonists like the Toll-like receptor 7 agonist R848 induce DC maturation and mobilization.

Methods: We collected intestinal and other tissues and mesenteric lymph nodes (MLN) from SAMP mice. Expression of CCL21 was measured by quantitative reverse transcription polymerase chain reaction and immunofluorescence analyses; spontaneous and induced migration of DCs were assessed by flow cytometry. We analyzed production of retinoic acid by DCs and their ability to induce development of regulatory T cells. Mice were fed R848 to determine its effects on migration of DCs and development of ileitis in SAMP mice.

Results: SAMP mice expressed almost no CCL21 in any tissue tested. Their CD11b(+)CD103(+) DCs were defective in migration from the ileal lamina propria to the MLN. DCs from SAMP mice also had a greatly reduced ability to produce retinoic acid and induce development of regulatory T cells compared with control mice. Young SAMP mice had reduced CCL21 expression and decreased DC migration before developing ileitis. Administration of R848 to adult SAMP mice increased migration of DC to the MLN and development of regulatory T cells there, and reduced the severity of ileitis.

Conclusions: Loss of CCL21 signaling and DC migration is required for development of ileitis in SAMP mice. Reagents such as R848, which activate DC migration to the MLN, may be developed as treatments for patients with Crohn's disease.

Keywords: CD; Immune Regulation; Oral Tolerance; Small Intestine.

Figure 1. Defective MLN DC in SAMP mice

(A) Single cell suspensions were prepared by incubating minced tissues in collagenase and DNAse I. Sequential gates were set to exclude debris, doublets, dead cells, cells of B and T cell lineage and granulocytes (SSC^hi). (B) Representative flow cytometry analysis of DC in AKR and SAMP MLN from 10–20 weeks old mice. Two populations of CD11c⁺MHCII^hi and CD11c^hiMHCII^int MLN DCs were detected. (C) MHCII^hi cells contained 3 major subpopulations: CD103⁺CD11b⁺, CD103⁺CD11b⁻ and CD103⁻CD11b⁻ cells. Mean of percentages of parent gate ± SEM are indicated on the plots, ^# denotes statistically significant differences between AKR and SAMP mice. (D) The fraction of MHCII^hi and MHCII^int cells and (E) subpopulations of MHCII^hiCD11c⁺ cells was calculated as % of all live cells. Results are the mean ± SEM of 3 experiments with n=5 in AKR and n=6 in SAMP groups. n.s – not significant, **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05 by two-sided Student’s t test

Figure 2. Accumulation of CD103⁺CD11b⁺ mDC in the terminal ileum LP of SAMP mice

LP cells were prepared from 5–7 weeks old AKR and SAMP mice by digestion and gated (A) on live single CD45⁺MHCII⁺CD103⁺CD11c⁺ cells. (B) Representative flow cytometry plots of CD103⁺CD11c⁺CD11b⁺ and CD11b⁻ population in AKR and SAMP LP. Percentages of parent gate (mean ± SEM) are indicated on the plots, ^# denotes significant differences between AKR and SAMP mice. (C) Frequency of gated populations among all live leukocytes. Results are the mean ± SEM of 2 experiments with n=4 for AKR and n=3 for SAMP groups. (D) Expression of β₇ integrin on CD103⁺ (red) and CD103⁻ (blue) MHCII^high MLN DCs. Isotype control (grey) was done on MHCII^high cells. (E) Sections of terminal ileum from 20 weeks old AKR and SAMP mice were stained with antibodies to β₇ integrin (green), CD11b (red), and Lyve1 (Lymphatic Vessel Endothelial Receptor 1, magenta). Shown is maximum intensity projection of a 6 μm confocal stack. β7⁺CD11b⁺ cells (arrows) are enriched in SAMP LP. Control staining (insert) was done on tissue from β₇-deficient mouse. Representative of 2 experiments with at least 2 mice per group. Bar = 20 μm. ** p<0.01, ^# p<0.05

Figure 3. Reduced CCL21 expression in SAMP mice

Quantitative RT-PCR for CCL21 (A) and CCL19 (B) in neonatal intestine, terminal ileum, mesenteric lymph nodes and skin of AKR and SAMP mice; mean±SEM for n=3–5 mice/group. Ribosomal protein L13A (Rpl13a) mRNA was used as a reference. (C) Whole mount staining of terminal ileum with antibodies to CCL21 (green) and Lyve1 (magenta). Confocal immunofluorescence demonstrates abundant presence of CCL21 protein (arrows) in Lyve1⁺ lymphatic vessel of AKR, while weak expression of CCL21 can be seen only sporadically in SAMP mice; Maximum intensity projection of 15 × 1 μm Z stacks. Bar = 100 μm Representative of 3 experiments with total n=4 mice/strain. Isotype control is shown in the insert. Bar = 30 μm. (D) Sections of MLN form AKR and SAMP mice were stained with antibodies to CCL21 (green), peripheral node addressin (red) and Lyve-1 (magenta). Diffuse and concentrated (arrows) pools of CCL21 in lymphatic vessels (L) and high endothelial venules (H) are absent in SAMP lymph nodes. Bar = 50 μm. Representative of 2 experiments.

Figure 4. SAMP DCs are defective in generating Foxp3⁺ cells in vitro

Capacity of SAMP and AKR MLN DCs to produce RA was measured by RALDH activity in Aldefluor assay. (A) Representative histograms of MLN MHCII^int (gray) and MHCII^hi (open) cells from AKR and SAMP mice (B) Aldefluor ΔMFI signal was calculated by subtracting median fluorescence intensity of MLN MHCII^hi cells treated with RALDH inhibitor DEAB from corresponding non-treated samples. Mean ± SEM from 3 experiments and n=4 mice for each group. (C–D) Naïve AKR CD4⁺CD45Rb^hiCD25⁻ T cells were incubated with magnetically enriched and FACS sorted CD11c+ DC from AKR or SAMP MLN for 5 days in the presence of 2 μg/ml anti-CD3ε and 5 ng/ml TGFβ1. (C) Representative flow cytometry plots of Foxp3 expression on cultured CD4⁺ cells. (D) Data from 2 experiments are presented and mean is indicated. **** p<0.0001, * p<0.05 by two-sided Student’s t test

Figure 5. Loss of MHCII^hi DC in MLN of SAMP mice is concomitant with the onset of clinical disease

(A–B) MLN MHCII^hi DCs and ileitis (C) were analyzed in 4–5, 6–7 and 10–11 weeks old mice (mean ± SEM, n≥3). (B) Diminished numbers of CD11c⁺MHCII^hiCD103⁺CD11b⁺ cells in the MLNs of >6 weeks old SAMP mice. (D) Sections of terminal ileum were stained with H&E. Bar = 100 μm. n.s – not significant, *** p<0.001, ** p<0.01, * p<0.05 by two-sided Student’s t test

Figure 6. TLR7 agonist R848 can overcome DC recruitment defect in SAMP mice

(A) Migration of CD11c⁺MHCII^hi DC into MLN was measured 18h after gavage of R848 (0.5 μg/g). (B) Treatment induced migration of CD103⁺ CD11b⁺ and CD11b⁻ cells in the AKR mice. Percentages of parent gate ± SEM are indicated on the plots, ^# denotes significant differences between vehicle and R848 treated mice of the same strain. (C) Frequency of migratory cells at baseline (vehicle treated mice) and after TLR induction (R848) was lower in MLN of SAMP mice. n=5–6 in each group. (E) Activity of RA-synthesizing enzyme in CD11c⁺MHCII^hi MLN cells following TLR7 ligand treatment was measured by flow cytometry. Filled histograms show vehicle-treated mice, black open histograms depict R848-treated mice. Control sample treated with RALDH inhibitor DEAB is shown as gray open histograms. Data are representative of 3 experiments. **** p<0.0001, ** p<0.01, * p<0.05, ^# p<0.05 by two-sided Student’s t test

Figure 7. Treatment with R848 reduces inflammation in the terminal ileum

(A–B) Effect of treatment with R848 on ileitis. Outcomes of a 7 day course of R848 treatment (0.5 μg/g body mass, by oral gavage) on >30 weeks old mice. Control animals received either vehicle (H₂O) by daily gavage or dexamethasone (Dex) by IP injection (100 μg every second day). (A) Ileal swiss-rolls were stained with H&E and (B) total inflammatory score was assessed. n=4–8 per group. (C–F) R848 induces mDCs and MLN Foxp3⁺ cells. (C) Representative flow cytometry plots and (D) frequency of DC subsets were analyzed among all live cells recovered from the lymph nodes. (E) Cells were gated to include CD4⁺ TCRβ⁺ T cells and expression of Foxp3 was determined by intracellular staining. Shown is percentage of CD4⁺Foxp3⁺ cells among all live MLN cells. Data from 2 experiments are presented as mean ± SEM, n=7–12 per group. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05 by one-way analysis of variance followed by Dunnett’s multiple comparisons test