CD4+CD25+ regulatory cells contribute to the regulation of colonic Th2 granulomatous pathology caused by schistosome infection

Abstract

Eggs of the helminth Schistosoma mansoni accumulate in the colon following infection and generate Th2-biassed inflammatory granulomas which become down- modulated in size as the infection proceeds to chronicity. However, although CD4+CD25+FoxP3+ regulatory T cells (T(regs)) are known to suppress Th1-mediated colitis, it is not clear whether they control Th2-associated pathologies of the large intestine which characterise several helminth infections. Here we used a novel 3D-multiphoton confocal microscopy approach to visualise and quantify changes in the size and composition of colonic granulomas at the acute and chronic phases of S. mansoni infection. We observed decreased granuloma size, as well as reductions in the abundance of DsRed+ T cells and collagen deposition at 14 weeks (chronic) compared to 8 weeks (acute) post-infection. Th2 cytokine production (i.e. IL-4, IL-5) in the colonic tissue and draining mesenteric lymph node (mLN) decreased during the chronic phase of infection, whilst levels of TGF-β1 increased, co-incident with reduced mLN proliferative responses, granuloma size and fibrosis. The proportion of CD4+CD25+FoxP3+T(regs): CD4+ cells in the mLN increased during chronic disease, while within colonic granulomas there was an approximate 4-fold increase. The proportion of CD4+CD25+FoxP3+T(regs) in the mLN that were CD103+ and CCR5+ also increased indicating an enhanced potential to home to intestinal sites. CD4+CD25+ cells suppressed antigen-specific Th2 mLN cell proliferation in vitro, while their removal during chronic disease resulted in significantly larger granulomas, partial reversal of Th2 hypo-responsiveness and an increase in the number of eosinophils in colonic granulomas. Finally, transfer of schistosome infection-expanded CD4+CD25+T(regs) down-modulated the development of colonic granulomas, including collagen deposition. Therefore, CD4+CD25+FoxP3+T(regs) appear to control Th2 colonic granulomas during chronic infection, and are likely to play a role in containing pathology during intestinal schistosomiasis.

Figure 1. Colonic granuloma size and fibrosis is reduced in the chronic phase of schistosome infection.

A). Accumulation of eggs /gram of colon tissue (n = 4 mice/time point); mean eggs (± SEM). B) Representative photomicrographs of the colon at the acute (8 wks) or chronic (14 wks) stage of infection stained with H&E. Scale bars are 200 µm: egg denoted ‘Sm’. C) Granuloma area at the acute or chronic stage; bars are mean / animal (n = 4) from three separate histological sections. D) Granuloma volumes calculated from isolated granulomas at the acute or chronic stage calculated from three separate granulomas per individual animal. E) Cross sections of colon at the acute (8 wks) or chronic (14 wks) stage of infection stained with haematoxylin / Van Geison (collagen fibres  =  pink). All scale bars are 200 µm. Parasite egg is denoted ‘Sm’. F) 3D images of multiphoton confocal stacks of colonic tissue from infected hCD2-VaDsRed-B.6 mice sampled in situ during acute or chronic infection. DsRed fluorescent cells are >90% CD3⁺; blue fluorescence is second harmonic generation of type 1 collagen; green/yellow auto-fluorescence are schistosome eggs. Grid squares are 63.9 µm². Quantification of multiphoton confocal stacks from hCD2-VaDsRed-B.6 mice: G) granuloma half-volumes, H) DsRed⁺ cell counts and I) collagen half-volumes. Data calculated from four separate granulomas from individual animals (n = 4). J) Salt-soluble collagen from colonic tissues of naïve, acute, and chronic mice (n = 4). Data is mean (±SEM) collagen concⁿ–/mg of tissue (left), or adjusted for numbers of eggs/mg (right).

Figure 2. Egg antigen-specific Th2, but not TGF-β1 responses become down-modulated within the mLN and colon during chronic infection.

A) Proliferative responses and B) cytokine release (pg/ml) by mLN cells from naïve, acute, or chronic mice (n = 4/group) to anti-CD3 mAb, or SEA. C) Cytokine levels within colonic tissues (pg/mg tissue) and D) adjusted for numbers of eggs / mg of tissue. Data are mean proliferative response / cytokine concentration ±SEM.

Figure 3. Frequencies of CD4⁺FoxP3⁺ T_regs increase at the chronic phase in both the mLN and colonic granulomas.

A) Representative flow cytograms showing the frequencies of labelled CD4⁺ and FoxP3⁺ cells in suspensions of the mLN and spleen. Values in italics are quadrant percentages. Values in bold, upper right-hand quadrant, are mean (±SEM) CD4⁺FoxP3⁺ cells as a % of total CD4⁺ cells (relevant quadrants outlined in bold). Histograms show mean (±SEM) total CD4⁺ (open) and CD4⁺FoxP3⁺ (closed), mLN or spleen cell or numbers (n = 3 mice). Significant differences compared with naïve cell numbers are indicated. Bar indicates significant difference between CD4⁺FoxP3⁺ mLN numbers at the chronic compared with acute stage. B) Representative images of CD4^{+ (}green) and FoxP3⁺ (red) cells in labelled cryosections of mLN and, C) proportion of CD4⁺FoxP3⁺ / CD4⁺ cells/ field of view (2 fields of view / animal, n = 3) Scale  = 14 µm. D) Representative images of CD4⁺ and FoxP3⁺ cells in colonic granulomas, with high power insert also shown. E) Frequencies of labelled CD4⁺FoxP3⁺ / CD4⁺ colonic granuloma cells / field of view (2 fields of view / animal, n = 3). F) qRT-PCR analysis of FoxP3 mRNA in colonic tissue plotted as arbitrary units of FoxP3 normalised to CD3 transcript in colonic tissue. Bars are mean FoxP3 A.U. transcript per group. G). Flow plots of FoxP3 and CD103, and FoxP3 and CCR5 expression, gated on CD4⁺ mLN cells of naïve or chronic mice (n = 4). Values in italics are quadrant percentages. Values in bold, upper right-hand quadrant, are mean (± SEM) CD4⁺FoxP3⁺CD103⁺ cells, or CD4⁺FoxP3⁺CCR5⁺ cells as a % of total CD4⁺FoxP3⁺ cells (relevant quadrants outlined in bold).

Figure 4. CD4⁺CD25⁺ mLN cells suppress antigen-specific CD4⁺ Th2 responses in vitro.

A) Flow plots of mLN cell suspensions (n = 3 mice) labelled with anti-CD25 and anti-FoxP3, gated on CD4 expression. Values in italics are quadrant percentages. Values in bold, upper right-hand quadrant, are mean (± SEM) CD4⁺CD25⁺FoxP3⁺ cells as a % of total CD4⁺CD25⁺ cells (relevant quadrants outlined in bold). B) Antigen-specific proliferation of CD4⁺CD25^- effector cells from acute mice and CD4⁺CD25⁺T_regs from chronic mice, or co-cultured together in a 2∶1 ratio. C) Co-culture of CD4⁺CD25^- effector cells derived from acute mice cultured with CD4⁺CD25⁺T_regs derived from naïve, acute or chronic mice. D) CD4⁺CD25^- effector cells from mice with an acute or chronic infection, either depleted of CD4⁺CD25⁺ cells, or with CD4⁺CD25⁺ cell ‘add-back’ co-cultures from the same stage of infection at a 2∶1 ratio. All histograms are mean (± SEM) cpm ³H-thymidine incorporation.

Figure 5. In vivo ablation of CD25⁺ cells impairs regulation of colonic granulomas and antigen-specific Th2 responses.

A) Percentage of CD25⁺ or CTLA-4⁺ mLN lymphocytes from mice with chronic infection after treatment with anti-CD25 mAb, or isotype control. Antibodies given at 2 week intervals from wk 9 to wk 13, tissues sampled at wk 14. Data are mean % positive (± SEM). B) Photomicrographs of colonic tissue and quantification of granuloma areas after antibody treatment. Scale bar  = 200 µm; egg denoted ‘Sm’. Data is granuloma area (µm²) at wk 14 calculated from 3-4 separate histological sections per individual animal (n = 4). C) Colonic granulomas (x100) stained with H&E showing eosinophils (closed arrow) and large mononuclear cells (open arrow) in isotype mAb treated (left) and anti-CD25 treated (right|) infected mice. Scale bar  = 200 µm; egg denoted ‘Sm’. D) Numbers of eosinophils (left) and large mononuclear cells enumerated from the sections above; Bars are mean cell counts / group (n = 4) with 3–4 fields of view / mouse. E) Antigen-specific mLN responses in anti-CD25 mAb treated mice; data are means of cpm ³H-thymidine incorporation and IL-4 secretion pg/ml (n = 3).

Figure 6. Transfer of schistosome-expanded CD4⁺CD25⁺ T_regs modulates the development of acute-stage granulomas.

A) Isolated mLN CD4⁺CD25⁺T_regs from mice with chronic infection used for transfer. B) 3D images of multiphoton confocal stacks of colonic tissue viewed in situ at the acute stage of infection in hCD2-VaDsRed-B.6 control mice, or recipients of infection-expanded CD4⁺CD25⁺T_regs (2.5✕10⁶). Grid squares are 63.9 µm2. C) Granuloma area (left), DsRed⁺ cell counts (middle), and collagen half-volume (right) taken from confocal stacks (as above). Data are from 5–6 separate granulomas per mouse. Bars are mean / group (n = 3). D) Soluble collagen and cytokine in colonic extracts from infected control mice, or recipients of CD4⁺CD25⁺ cells. Bars are means / group (n = 3). Data is mean pg/ml (± SEM) per group.