Identification of a Chemokine Network That Recruits FoxP3⁺ Regulatory T Cells Into Chronically Inflamed Intestine

Kinetics and localization of chronic intestinal inflammation in SAMP1/YP mice. (A) The histopathology of SAMP1/YP mice at various ages (original magnification 100×). (B) Infiltration of mononuclear cells and polymorphonuclear cells, hyperplasia of goblet cells, and thickening of muscularis externa in the terminal ileum were measured with age. Combined data were obtained from 10 high-power fields per affected tissue site and mouse, and data from 4–6 mice were combined for each time point. (C) The inflammation is limited to the ileum of ∼30-week-old SAMP1/YP mice. Intestinal tissue sections were stained with H&E (original magnification 100×), and representative areas are shown from 4–6 mice for each group.

The gut chemokine network of SAMP1/YP mice. AKR/J control mice and SAMP1/YP mice were examined by (A) real-time reverse-transcription PCR, (B and C) conventional reverse-transcription PCR, and (D) ribonuclease protection assay. Thirty-week-old mice were used for A, B, and D. A representative set of data from at least 3 independent experiments using different mice is shown.

CCL5 protein is overexpressed in the inflamed intestinal lesion of SAMP1/YP mice. (A) CCL5 protein is up-regulated in the terminal ileum of SAMP1/YP mice compared with AKR/J mice (n = 4). Terminal ileum tissues were examined for CCL5 protein by ELISA. CCL5 expression levels were normalized per milligram of total protein. (B) Frozen sections of terminal ileum tissues from SAMP1/YP mice (30 weeks old) were examined for CCL5 expression by immunohistochemistry. Representative data from 8 separate experiments are shown.

CCL5 protein is overproduced by intestinal CD8⁺ cells in SAMP1/YP mice upon T-cell activation. MLN T cells were cultured for 48 hours, and the culture media were assayed for CCL5 by ELISA. T cells were cultured in the presence of (A) the polyclonal activator concanavalin A for indicated time or (B and C) anti-CD3 and/or anti-CD28 for 24 hours followed by ELISA to quantitate CCL5 produced in culture media. (A) One representative kinetic datum is shown, or (B and C) data from 3 independent experiments were combined and averages and SE are shown.

Activated T cells of SAMP1/YP mice are more responsive to CCL5 in chemotaxis than their counterparts from control mice. (A) In vitro chemotaxis assays were performed using the Transwell system (5-μm pores) with T cells isolated from MLNs of SAMP1/YP mice and AKR/J mice and activated for 4 days in the presence of IL-2. (B) T cells were cultured in the presence of IL-2 and IL-12 for 4 days and examined for their chemotactic responses to CCL5 (100 ng/mL). Data from 3 independent experiments were combined, and averages and SE are shown. *Significant differences between T cells from AKR/J and SAMP1/YP mice.

CCR5 is the main receptor for CCL5, and the CCR5-CCL5 interaction is specifically blocked by TAK-779. (A) Reverse-transcription PCR analysis of CCR1 and CCR5. Representatives from 3 independent experiments are shown. (B) The TAK-779 effect on CCL5 binding to chemokine receptors. Chemokine receptors were overexpressed in MLN CD4⁺ T cells prepared from AKR/J mice after retroviral gene transfer. CCL5-Fc protein was examined for its binding capacity to the chemokine receptors. TAK-779 was tested for its blocking effect on interactions between the CCL5-Fc protein and chemokine receptors. (C) TAK-779 was also tested for its blocking effect on the migration of CCR5⁺ CD4⁺ T cells generated by retroviral gene transfer. Representative data from 3 independent experiments (error bars are differences of 2 measurements).

CCR5⁺ T cells preferentially migrate to the inflamed lesion. (A) CCR5⁺ CD4⁺ T cells, generated by retroviral gene transfer from the MLN cells of SAMP1/YP mice, were labeled with CFSE, while CCR5⁻ control CD4⁺ T cells were labeled with tetramethylrhodamine-5-(and-6)-isothiocyanate. The 2 T-cell subsets were coinjected into 25- to 30-week-old SAMP1/YP or AKR/J mice via a tail vein. TAK-779 (100 μg/mouse) or control medium (5% d-mannitol) was injected intraperitoneally 12 hours before and right after the cell injection. T cells were isolated from various tissues of the mice 20–24 hours postinjection and analyzed by flow cytometry analysis. The tissues examined were spleen, MLNs, peripheral lymph nodes, Peyer’s patches (PP), lamina propria of a normal intestinal segment (the distal part of jejunum right before ileum [LP(N)]), lamina propria of the inflamed terminal ileum lesion (the last 7–8 cm of ileum before the ileocecal junction [LP(I)]), and peritoneal cavity. (B) Homing indexes were calculated according the formula described in Material and Methods. Migration of CCR5⁺ CD4⁺ T cells in SAMP1/YP and AKR/J mice is compared. Representative data from 3 independent experiments. Significant differences (P < .05) from TAK-779 (*) and AKR/J controls (**). (C) CD4⁺ T cells isolated from various tissue sites were stained for CCR5 surface expression.

CCR5 blockade exacerbates the intestinal inflammation. (A and B) SAMP1/YP mice were injected intraperitoneally with TAK-779 (n = 8) or control medium (n = 7) for 11 weeks starting from 4 weeks of age. Similar numbers of age-matching AKR/J mice were also treated in the same manner. (C and D) SCID mice were injected with MLN cells of SAMP1/YP mice to induce inflammation and then treated with TAK-779 or control medium every 2 days for 6 weeks. Control mice did not receive MLN cells. Weight loss due to inflammation was examined (C). The average number of mice (n) for each group was 10. Tissue sections (distal colon) were stained with H&E and scored for inflammation (D and E). *Significant differences (P < .05) from controls.

CCR5⁺FoxP3⁺ T cells are greatly enriched in the inflamed intestine of SAMP1/YP mice. (A) The majority of FoxP3⁺ cells in the inflamed intestine of SAMP1/YP mice are CCR5⁺. FoxP3⁺ and Foxp3⁻ T cells, harvested from various organs of SAMP1/YP mice (total, n = 14) and AKR/J mice (total, n = 9) at 3, 7, and 30 weeks of age, were examined for expression of CCR5. (B) FoxP3⁺ cell frequencies among the CD4⁺ T cells of the intestine and MLNs of SAMP1/YP and AKR/J mice were determined. (C) CD4⁺CD25⁺ T cells of the MLNs of SAMP1/YP and AKR/J mice are equally competent in their suppressive activity in vitro on proliferation of CD4⁺CD25⁻ T cells. Indicated numbers of CD4⁺CD25⁺ T cells were added to the cultures of CD4⁺CD25⁻ T cells. A representative set of data obtained from 3 independent experiments are shown. *Significant differences (P < .05) from controls.

Preferential migration of FoxP3⁺ T cells into the inflamed intestine in a CCR5-dependent manner. (A) FoxP3 conditions T cells to overexpress CCR5 in response to IL-12. The mFoxP3 gene was retrovirally transferred into MLN CD4⁺ T cells. The T cells were cultured in the presence of IL-12 and retinoic acid, and expression of CCR5 was determined on the surface of transduced FoxP3⁺ and untransduced FoxP3⁻ CD4⁺ T cells. (B) FoxP3⁺ CD4⁺ T cells migrate better than FoxP3⁻ CD4⁺ T cells to inflamed intestine. Retrovirally generated FoxP3⁺ T cells were labeled with CFSE and injected intravenously into SAMP1/YP mice (6–7 weeks old). At 20 hours after cell injection, migration of FoxP3⁺ T cells into various organs was examined. FoxP3⁺ and FoxP3⁻ CD4⁺ T cells, migrated into various tissue sites, were identified by their staining patterns of CFSE and FoxP3. Homing index (HI) for this panel was calculated with the formula based on frequencies of the cells of interest determined by flow cytometry: HI = a ÷ b, where a represents (Migrated CFSE⁺FoxP3⁺ Cells Into Tissue Site A) ÷ (Migrated CFSE⁺ FoxP3⁻ Cells Into Tissue Site A) and b represents (Injected FoxP3⁺ Cells in Input) ÷ (Injected FoxP3⁻ Cells in Input). Data from 4 independent experiments (n = 4 in each group) were combined. (C) CCR5 blockade by TAK-779 suppresses FoxP3⁺ T cell migration to inflamed intestine. TAK-779 (or control medium) was injected intraperitoneally into SAMP1/YP mice 12 hours before and at the time of the injection of the cells to block the CCR5 pathway. Data from 3 separate experiments were combined (n = 3), and averages and SEM are shown. Percent HI was calculated using the formula: % HI = (a ÷ b) × 100, where a represents (Migrated CFSE⁺FoxP3⁺ Cells Into Tissue Site A of TAK-779–Injected Animals) ÷ (Resident CD4⁺ Cells in Tissue Site A of TAK-779–Injected Animals) and b represents (Migrated CFSE⁺FoxP3⁺ Cells Into Tissue Site A of Control-Injected Animals) ÷ (Resident CD4⁺ Cells in Tissue Site A of Control-Injected Animals).