Regulatory T-cell Response to Enterotoxigenic Bacteroides fragilis Colonization Triggers IL17-Dependent Colon Carcinogenesis

Abstract

Many epithelial cancers are associated with chronic inflammation. However, the features of inflammation that are procarcinogenic are not fully understood. Regulatory T cells (Treg) typically restrain overt inflammatory responses and maintain intestinal immune homeostasis. Their immune-suppressive activity can inhibit inflammation-associated cancers. Paradoxically, we show that colonic Tregs initiate IL17-mediated carcinogenesis in multiple intestinal neoplasia mice colonized with the human symbiote enterotoxigenic Bacteroides fragilis (ETBF). Depletion of Tregs in ETBF-colonized C57BL/6 FOXP3(DTR) mice enhanced colitis but diminished tumorigenesis associated with shifting of mucosal cytokine profile from IL17 to IFNγ; inhibition of ETBF-induced colon tumorigenesis was dependent on reduced IL17 inflammation and was independent of IFNγ. Treg enhancement of IL17 production is cell-extrinsic. IL2 blockade restored Th17 responses and tumor formation in Treg-depleted animals. Our findings demonstrate that Tregs limit the availability of IL2 in the local microenvironment, allowing the Th17 development necessary to promote ETBF-triggered neoplasia, and thus unveil a new mechanism whereby Treg responses to intestinal bacterial infection can promote tumorigenesis.

Significance: Tregs promote an oncogenic immune response to a common human symbiote associated with inflammatory bowel disease and colorectal cancer. Our data define mechanisms by which mucosal Tregs, despite suppressing excessive inflammation, promote the earliest stages of immune procarcinogenesis via enhancement of IL17 production at the expense of IFNγ production.

Figure 1. ETBF induces distal colon tumorigenesis with parallel increases in IL-17A+ and Foxp3+ cells

A) Methylene blue-stained colon of 2 month ETBF-colonized Min mouse. The distal colon predominance of ETBF tumorigenesis is shown (white arrows). B) Lamina propria lymphocytes (LPLs) were isolated from distal colons of C57Bl/6 mice 7 days after sham (PBS) or ETBF inoculation and 4 hr culture with Cell Stimulation Cocktail preceded intracellular staining (ICS) for IL-17A. Live CD3⁺ IL-17⁺ cell numbers were normalized by the mass of colon tissue after cleaning and before LPL isolation (top). Percent IL-17⁺ of live CD3⁺ lymphocytes (bottom). Each symbol represents one sample, and error bars represent one standard deviation from the mean in each direction. Shown are data from two separate experiments. C) ICS for Foxp3 on distal colon LPLs from C57Bl/6 mice was performed fresh ex vivo without stimulation. Live CD3⁺ CD4⁺ Foxp3⁺ cell numbers normalized by mass of colon tissue as in B (top). Percent Foxp3⁺ of live CD3⁺ CD4⁺ lymphocytes (bottom). Each symbol represents one sample, and error bars represent one standard deviation from the mean in each direction. Shown are data from two separate experiments.

Figure 2. Treg depletion reduces microadenoma formation in Min mice

A) B6.Foxp3^DTRxMin mice were inoculated with ETBF on day 0. Either 150 μl purified sterile H₂O or 50 ng/g diphtheria toxin (DT) was administered intraperitoneal (ip) on days −1, 0, 1, 3, and every other day until sacrifice and harvest. Colons were harvested on day 13, cleaned, rolled, and fixed in 10% formalin for histology & scoring. Images are of distal colon. Microadenoma is encircled. Scale bars are 50 μm. B) Each symbol represents total inflammation score per Min mouse from Figure 2A. Data shown include 2 separate experiments with 5-12 mice per group per experiment. Bars indicate mean +/− SD. C) Microadenomas counted per colon in (B). D) B6.Foxp3^DTR mice were inoculated with sham or ETBF on day 0. Either 150 μl purified sterile H₂O or 50 ng/g diphtheria toxin (DT) was administered intraperitoneal (ip) on days −2, −1, 0, 1, 3, and 5 until sacrifice and harvest. Colons were harvested on day 7, cleaned, rolled, fixed in 10% formalin, paraffin embedded, and H&E stained for histology & scoring. Images are of distal colon. Scale bars are 50 μm. E) Each symbol represents total inflammation score per mouse from Figure 2D. Data shown include 2 separate experiments with 2-6 mice per group per experiment. Bars indicate mean +/− SD.

Figure 3. Treg depletion mitigates the Th17 response to ETBF in favor of a Th1 response

A) Colon LPLs from 1-2 B6.Foxp3^DTR mice per group were harvested on day 7 following inoculation with ETBF or Sham on day 0. Either 150 μl purified sterile H₂O or 50ng/g DT was administered ip on days −2, −1, 1, 3, & 5. Cells were stimulated ex vivo, followed by ICS. Plots show viable CD3⁺ CD4⁺ Foxp3- LPLs and are a representation of 4-5 separate experiments. B) Aggregate data from combined experiments showing percentage of viable CD3⁺ CD4⁺ Foxp3- LPLs that are IL-17A⁺. Each symbol represents 1-2 Treg-depleted B6.Foxp3^DTR mice (****, +DT) or Treg-sufficient mice (○, No DT), and error bars represent 1 standard deviation from the mean in each direction. Holm-Sidak method for multiple comparisons was used to compare No DT vs + DT groups. C) B6.Foxp3^DTR and B6.Foxp3^DTRxIFNγ^−/− mice were inoculated with ETBF on day 0. Either 150ul purified sterile H₂O or 50 ng/g DT was administered ip on days −2, −1, 1, 3, & 5. Colonic LPLs from 1-2 mice per group were harvested on day 7. Cells were stimulated ex vivo, followed by ICS. Dot plots show viable CD3⁺ CD4⁺ Foxp3- LPLs and are representative of 4-5 experiments. D) B6.Foxp3^DTR × IFNγ^−/− × Min mice were inoculated with ETBF on day 0. Either 150 μl purified sterile H₂O or 50 ng/g DT was administered ip on days −1, 0, 1, 3, and every other day until sacrifice and harvest. Colons were harvested on day 13, cleaned, rolled, and fixed in 10% formalin, paraffin embedded and H&E stained for microadenoma counting per colon. Each symbol represents 1 colon, and error bars represent 1 standard deviation from the mean in each direction. 2-5 mice per group per experiment. Combined data from 2 experiments. IFNγ^+/+ × Min microadenoma counts from Figure 2B are also shown for easier side-by-side comparison.

Figure 4. Mixed chimeras reveal a cell extrinsic requirement for Foxp3 in Th17 differentiation

A) Schematic of the experiment designed to determine whether Foxp3 expression is a cell extrinsic or intrinsic requirement for Th17 differentiation. B) 10⁷ total bone marrow cells from B6.Foxp3^DTR CD45.2 mice only (DT control), or mixed 1:1 with bone marrow cells from B6.Foxp3^WT CD45.1 mice, were transferred retro-orbitally into Rag1^−/− recipients that had received 300 rads irradiation 5-6 hours prior. Following 6 weeks for hematopoietic cell reconstitution, mice were inoculated with ETBF or sham on day 0, and either 150 μl purified sterile H₂O or 50 ng/g DT was administered ip on days −2, −1, 1, 3, & 5. Colonic LPLs from 1-2 mice were harvested on day 7 following ETBF or Sham inoculation. Cells were stimulated ex vivo followed by ICS. Dot plots show viable CD3⁺ CD4⁺ LPLs and are representative of 2 experiments. C) Colon LPLs from 1 B6.Foxp3^DTR mouse per group were harvested on day 7 following inoculation with ETBF or sham on day 0. For no depletion and early depletion, purified sterile H₂O or DT, respectively, was administered ip on days −2, −1, 1, 3, & 5. For late depletion, DT was administered ip on days 1, 3, & 5. Cells were stimulated ex vivo, followed by ICS. Plots show viable CD3⁺ CD4⁺ Foxp3- LPLs and are representative of 2 mice per group.

Figure 5. Anti-IL-2 restores the Th17 response to ETBF in the absence of Foxp3⁺ Tregs

A) 4-9 B6.Foxp3DTR mice per group were inoculated with ETBF. Either 150 μl purified sterile H₂O or 50 ng/g DT was administered ip on days −2, −1, 1, 3, & 5, and either rat anti-mouse IL-2 (S4B6-1) or rat IgG2a isotype (JES3-19F) was delivered ip daily (day −2 through day 6). Colonic LPLs from each mouse were harvested on day 7. Cells were stimulated ex vivo, followed by ICS. Representative dot plots show viable CD3⁺ CD4⁺ Foxp3- LPLs from 1 mouse per group. aIL-2, anti-mouse IL-2. B) Aggregate data from 2-3 mice per group showing percentage of viable CD3⁺ CD4⁺ Foxp3- LPLs that are IL-17A⁺. Each symbol represents one B6.Foxp3^DTR mouse, and error bars represent 1 standard deviation from the mean in each direction. Isotype-treated animals were not different from untreated animals, so these two animal groups were combined. C) Tissue for RNA isolation was taken from the middle colon from each mouse in Figure 5B and Taqman qRT-PCR was performed for IL-17A. ^Ct was calculated by subtracting Ct of Gapdh from Ct of IL-17A and averaging 2 technical replicates. Each symbol represents one mouse, and error bars represent 1 standard deviation from the mean in each direction. D) 6-12 B6.Foxp3^DTR mice per group were inoculated with ETBF on day 0. Either 150 μl purified sterile H₂O or 50 ng/g DT was administered ip on days −2, −1, 1, 3, & 5. Colonic LPLs from 1-3 mice per group were harvested on day 7 for fluorescence-associated cell sorting. Effector T cells (CD11b-, Foxp3-, CD4+ or CD8+) were sorted from Treg-depleted mice (****, +DT) and from Treg-sufficient mice ((, No DT). RNA was isolated from Trizol and Taqman qRT-PCR was performed for indicated genes. Gapdh was used as housekeeping control for total RNA quantity (^Ct), and average of 2 technical replicates were used for each sample. CD4+ Foxp3+ Tregs were sorted from Treg-sufficient mice as the reference sample for calculating ^^Ct. Fold change = 2^-(^^Ct). Each symbol represents one mouse (or pooled group), and error bars represent 1 standard deviation from the mean in each direction. Holm-Sidak method for multiple comparisons was used to compare No DT vs + DT groups. E) B6.Foxp3^DTRxMin mice were inoculated with ETBF on day 0. Either 150 μl purified sterile H₂O or 50 ng/g DT was administered ip on days −1, 0, 1, 3, and every other day until sacrifice and harvest. Either rat anti-mouse IL-2 (S4B6-1) or rat IgG2a isotype (JES3-19F) was delivered ip daily (day −1 through day 13). Colons were harvested on day 13, cleaned, rolled, and fixed in 10% formalin for histology & scoring. Microadenomas were counted per colon and include 2 separate experiments with 3-12 mice per group per experiment. DT animals treated with isotype Ab (N=10) were not different from DT only-treated animals (N=18), so DT animals treated with or without isotype Ab were combined. Each symbol represents one mouse, and error bars represent 1 standard deviation from the mean in each direction. IFNγ^−/− × Min + DT microadenoma counts from Figure 3D are also shown for easier side-by-side comparison. F) Inflammation scores per colon in (E).