Parasite Proximity Drives the Expansion of Regulatory T Cells in Peyer's Patches following Intestinal Helminth Infection

Abstract

Helminth infections are typically chronic in nature; however, the exact molecular mechanisms by which these parasites promote or thwart host immunity remain unclear. Worm expulsion requires the differentiation of CD4(+) T cells into Th2 cells, while regulatory T cells (Tregs) act to dampen the extent of the Th2 response. Priming of T cells requires drainage or capture of antigens within lymphoid tissues, and in the case of intestinal helminths, such sites include the mucosa-associated Peyer's patches (PPs) and the draining mesenteric lymph nodes (MLN). To gain insight into when and where the activation of the adaptive T cell response takes place following intestinal helminth infection, we analyzed Th2 and Treg responses in the PPs and MLN following infection with the murine intestinal helminth Heligmosomoides polygyrus bakeri. Protective Th2 responses were observed to be largely restricted to the MLN, while a greater expansion of Tregs occurred within the PPs. Interestingly, those PPs that formed a contact with the parasite showed the greatest degree of Treg expansion and no evidence of type 2 cytokine production, indicating that the parasite may secrete products that act in a local manner to selectively promote Treg expansion. This view was supported by the finding that H. polygyrus bakeri larvae could promote Treg proliferation in vitro. Taken together, these data indicate that different degrees of Treg expansion and type 2 cytokine production occur within the PPs and MLN following infection with the intestinal helminth H. polygyrus bakeri and indicate that these organs exhibit differential responses following infection with intestinal helminths.

FIG 1

PPs exhibit an early Treg response and a decreased effector T cell response compared to those in MLN following H. polygyrus bakeri infection. (A and B) RT-qPCR analysis of IL-4, IL-5, and IL-13 mRNA expression levels in freshly isolated tissues from PPs (A) or MLN (B) of naive or H. polygyrus bakeri-infected mice at 3, 6, 12, and 21 dpi. Data are normalized to values for β-actin, GAPDH, and naive controls and are from one of two independent experiments (n = 3 per group). Data are expressed as means ± SEM (*, P < 0.05; **, P < 0.01 [as determined by one-way ANOVA with Dunnett's multiple-comparison test]). (C) RT-qPCR analysis of IL-4, IL-5, and IL-13 mRNA expression levels in the CD4⁺ fraction isolated from PPs or MLN of naive or H. polygyrus bakeri (Hpb)-infected mice (10 dpi). Results are normalized to β-actin and GAPDH values and were pooled from two experiments showing similar responses (cells from 8 to 9 mice per group were pooled for each data point). Data are expressed as means ± SEM (***, P < 0.001; ****, P < 0.0001; ns, not significant [as determined by two-way ANOVA with Tukey correction]). (E and F) Cells were isolated from PPs or MLN of naive or H. polygyrus bakeri-infected mice at 7, 14, 21, and 28 dpi and analyzed by flow cytometry. Data for CD25^high FoxP3⁺ (E) and CD103⁺ CD25^high FoxP3⁺ (F) cells are shown as a percentage of CD4⁺ cells. Data from one of two independent experiments are shown (n = 5 per group) and expressed as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [as determined by one-way ANOVA with Dunnett's multiple-comparison test]).

FIG 2

PC61 administration partially restores cytokine production in PPs at 7 dpi. Data from RT-qPCR analysis of IL-4, IL-5, and IL-13 mRNA expression levels in freshly isolated tissues from PPs (A) or MLN (B) of naive or H. polygyrus bakeri-infected mice at 7 and 14 dpi are shown. On the day of infection, mice were treated with PC61 or the isotype control antibody, as indicated. Data were normalized to values for β-actin and are from one of two independent experiments (n = 3 to 4 per group). Data are expressed as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [as determined by one-way ANOVA with Dunnett's multiple-comparison test]).

FIG 3

Intestinal bacteria do not contribute to the accumulation of CD25^high FoxP3⁺ cells in PPs of H. polygyrus bakeri-infected mice. Mice belonging to the antibiotic-treated (ABX) group were treated with enrofloxacin and co-amoxicillin for 4 weeks, as described in Materials and Methods. Cells were then isolated from PPs and MLN of naive or H. polygyrus bakeri-infected mice at 7 dpi and analyzed by flow cytometry. Data for CD25^high FoxP3⁺ cells (A) and CD103⁺ CD25^high FoxP3⁺ cells (B) are shown as a percentage of CD4⁺ cells. Results are pooled from data from two independent representative experiments of three (n = 4 to 5 per group, per experiment) and expressed as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [as determined by one-way ANOVA with Tukey's multiple-comparison test]).

FIG 4

CD25^high FoxP3⁺ cell accumulation and diminished Th2 responses in PPs correlate with contact with H. polygyrus bakeri larvae. (A to D) Cells were isolated from PPs of naive or H. polygyrus bakeri-infected mice at 7 dpi. In infected mice, PPs were divided according to the presence (+) or absence (−) of the parasitic larvae within the organ. Data from flow cytometric analysis of total CD25^high FoxP3⁺ cells (A) and CD103⁺ CD25^high FoxP3⁺ cells (C) are shown as a percentage of CD4⁺ cells. Representative fluorescence-activated cell sorter plots (B) and histograms of CD103 expression (D) are also shown; the same number of events is shown for each histogram. For panels A and C, results are pooled from data from two independent representative experiments of three (n = 4 to 5 per group, per experiment); data are expressed as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [as determined by one-way ANOVA with Tukey's multiple-comparison test]). (E to G) Data from RT-qPCR analysis of IL-4 (E), IL-5 (F), and IL-13 (G) mRNA expression levels from one experiment are shown and are representative of data from two independent experiments (n = 4 per experiment for naive mice, and n = 10 per group experiment for H. polygyrus bakeri-infected mice; data for PPs from 2 mice were pooled for each data point); data are expressed as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [as determined by one-way ANOVA with Tukey's multiple-comparison test]).

FIG 5

H. polygyrus bakeri larvae promote the proliferation of CD25^high FoxP3⁺ cells in vitro. (A to D) Ki-67 expression was assessed in cells isolated from PPs and MLN of naive or H. polygyrus bakeri-infected mice at 7 dpi. The percentages of Ki-67⁺ cells within the CD4⁺ CD25^high FoxP3⁺ (A) and CD4⁺ FoxP3⁻ (C) populations as well as representative histograms of Ki-67 expression (B and D) are shown. Results are pooled from data from two independent experiments (n = 3 to 6 per group) and expressed as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [as determined by two-way ANOVA with Tukey's multiple-comparison test]). (E to L) CD4⁺ splenocytes were isolated from naive C57BL/6 mice and incubated for 48 h in presence or absence of H. polygyrus bakeriL4. Percentages (E and I) and total numbers (F and J) of CD25^high FoxP3⁺ (E and F) and FoxP3⁻ (I and J) cells within the CD4⁺ population are shown. Percentages of Ki-67⁺ cells within the CD25^high FoxP3⁺ (G) and FoxP3⁻ (K) populations are also shown. Total numbers of Ki-67⁺ CD25^high FoxP3⁺ (H) and Ki-67⁺ FoxP3⁻ (L) cells are represented. Results are pooled from data from two independent experiments and expressed as means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [as determined by Student's t test]). Each condition was analyzed in triplicate.