The Wnt-β-catenin-IL-10 signaling axis in intestinal antigen-presenting cells protects mice from colitis-associated colon cancer in response to gut microbiota

Abstract

Loss of immune tolerance to gut microflora is inextricably linked to chronic intestinal inflammation and colitis-associated colon cancer (CAC). The LRP5/6 signaling cascade in antigen-presenting cells (APCs) contributes to immune homeostasis in the gut but whether this pathway in APCs protects against CAC is not known. In the present study, using a mouse model of CAC, we show that the LRP5/6-β-catenin-IL-10 signaling axis in intestinal CD11c⁺ APCs protects mice from CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of LRP5/6 in CD11c⁺ APCs in mice (LRP5/6^ΔCD11c) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of pro-inflammatory factors and decreased expression of the immunosuppressive cytokine, IL-10. This condition could be improved in LRP5/6^ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor-promoting inflammatory factors in CD11c⁺ APCs via the β-catenin-IL-10 axis. Accordingly, conditional activation of β-catenin specifically in CD11c⁺ APCs or in vivo administration of IL-10 protected LRP5/6^ΔCD11c mice from CAC by suppressing the expression of inflammatory factors. In summary, here we identify a key role for the LRP5/6-β-catenin-IL-10 signaling pathway in intestinal APCs in resolving chronic intestinal inflammation and protecting against CAC in response to the commensal flora.

Introduction

Crohn’s Disease (CD) and Ulcerative Colitis (UC) are the two main clinically defined forms of inflammatory bowel disease (IBD) that afflict approximately 1 million people in the United States. Loss of immune tolerance to commensal flora results in intestinal inflammation, which subsequently leads to IBD(1–3). In IBD patients, serious long-term complication of chronic intestinal inflammation leads to colitis-associated colon carcinogenesis (2, 4). Among patients with UC and CD, the increased risk of developing colon cancer correlates with the duration, extent, and severity of the inflammatory diseases (5, 6). Antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages (MPs) play a pivotal role in suppressing intestinal inflammation and in mediating immune tolerance to commensal flora (7–10). Dysfunctional APCs play an important role in the pathogenesis of IBD and colitis-associated colorectal cancer (CAC). However, the molecular mechanisms by which intestinal APCs resolve chronic inflammation in the intestine and CAC remain largely unknown

The Wnt signaling pathway is critical for gut development and homeostasis (11). More importantly, genome-wide association studies have shown widespread abnormalities in the Wnt signaling pathways in patients suffering from IBD and IBD-associated cancer, implying a possible role for this pathway in disease pathogenesis(12–15). However, the focus of past research has been on identifying the role of this pathway in intestinal stem cell proliferation, as well as its effects on cancer initiation and progression. Recent studies have shown that the canonical Wnt signaling pathway plays a key role in shaping mucosal immune responses in the intestine (16, 17). The co-receptors LRP5 and LRP6 are critical for mediating canonical Wnt signaling in CD11c⁺ APCs (18). Our previous studies showed that conditional knockout mice that specifically lack LRP5/6 or β-catenin in CD11c⁺ APCs develop more severe acute colitis(16, 17). However, whether this pathway in APCs protects against CAC is not known. Moreover, underlying molecular mechanisms by which canonical Wnt signaling in APCs resolve chronic inflammation in the intestine and protect against CAC remain largely unknown.

Here, we demonstrate that the LRP5/6-β-catenin-IL-10 signaling axis in CD11c⁺ APCs suppresses CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Conditional deletion of the Wnt co-receptors LRP5 and LRP6 in CD11c⁺ APCs in mice results in enhanced susceptibility to CAC. This is due to increased microbiota-dependent pro-inflammatory cytokine production and decreased expression of the immunosuppressive cytokine, IL-10. The condition could be improved in LRP5/6^ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Mechanistic studies suggest that LRP5/6 regulates the expression of tumor-promoting inflammatory factors in CD11c⁺ APCs via the β-catenin-IL-10 signaling axis. Furthermore, our study demonstrates that, in LRP5/6^ΔCD11c mice, either conditional activation of β-catenin specifically in CD11c⁺ APCs or exogenous administration of rIL-10 can reduce CAC by suppressing the expression of inflammatory factors. These results reveal a mechanism by which the LRP5/6-β-catenin-IL-10 signaling axis in APCs controls chronic inflammation in the intestine and CAC in response to gut microbiota.

Materials and Methods

Mice

C57BL/6 and CD11c-cre mice were originally obtained from Jackson Laboratory and bred on-site in accordance with institutional animal care and use guidelines. LRP5-floxed (LRP5^FL) mice and LRP6-floxed (LRP6^FL) mice were originally provided by Dr. B.O. Williams (Van Andel Research Institute, Grand Rapids, MI) and were cross-bred to generate homozygous LRP5/6-floxed (LRP5/6^FL) mice as previously described (17, 19, 20). LRP5/6^FL (WT) mice were crossed to transgenic mice expressing Cre recombinase under the control of the CD11c promoter (Jackson Laboratory) to generate mice in which LRP5/6 (LRP5/6^ΔCD11c) were deficient in CD11c⁺ APCs (17, 19, 20). Successful Cre-mediated deletion was confirmed by PCR and protein expression analyses as in our previous studies (17, 19, 20). LRP5/6^FL and LRP5/6^ΔCD11c mice were caged together. LRP5/6^ΔCD11c mice expressing a constitutively-active form of β-catenin (LRP5/6^ΔCD11c/Act-β-cat^CD11c) were generated as previously described (17, 19, 20). All animals were bred and housed at the Georgia Cancer Center, Augusta University (AU) animal barrier facility under SPF conditions and controlled 12-hours, light/dark cycles. AU performs a quarterly health screening covering various bacterial, viral and parasitic organisms, all of which the mouse colony screened negative for. The animals used in the experiments presented in this manuscript had all screened negative for these pathogens. Mice were fed irradiated mouse chow (Teklad, 8904) and the cages were bedded with Teklad ¼” corncob bedding. Gender and age-matched mice (ranging from eight to 12 weeks old) were used in all experiments, with animal care protocols approved by the Institutional Animal Care and Use Committee.

Antibodies and reagents

Antibodies against mouse CD3 (145–2C11), CD4 (GK1.5), CD8a (53–6.7), CD45 (30-F11), Foxp3 (FJK-16s), IL-10 (JES5–16E3), CD11c (N418), CD11b (M1/70), I-A^b (25-9-17), CD64 (X54–5/7.1), F4/80 (BM8), CD90.1 (HIS51), IFN-γ (XMG1.2), IL-6 (MP5–20F3), TNF-α (MP6-T22), pro-IL-1β and IL17A (17B7) were purchased from eBioscience and Biolegend. LRP5, LRP6, non-phospho active β-catenin, β-catenin, and β-actin antibodies were obtained from Cell Signaling Technology. CD11c and CD11b microbeads were purchased from Miltenyi Biotec.

Induction of colitis-associated colon cancer

Inflammation-associated colon cancer was induced as previously described (21). Briefly, mice were injected intraperitoneally with azoxymethane (10 mg/kg body weight; Sigma-Aldrich) and then subjected to three cycles of DSS treatment, wherein one cycle constituted mice being given 2.0% DSS (36–50 kDa; MP Biomedicals) in their drinking water (the regimen followed for each experiment is indicated in Results) for 7 days followed by 14 days of normal drinking water. Some mice were treated with antibiotics and transplanted with fecal microbiota before DSS treatment. In some experiments, mice were also injected intraperitoneally with rIL-10 (100 ng/mouse/injection in 0.1ml PBS; Peprotech Inc.) weekly during the entire course of DSS treatment. Mice were monitored for weight change, diarrhea and rectal bleeding as previously described (17, 21, 22). Diarrhea was scored as (0) normal stool; (1) soft but formed pellet; (2) very soft pellet; (3) diarrhea (no pellet); or (4) dysenteric diarrhea. Rectal bleeding was recorded as (0) no bleeding; (2) presence of occult blood in the stool, or; (4) gross macroscopic bleeding. Following the final cycle, mice were allowed to recover for two weeks before sacrifice at approximately 62 days. Colons were removed and processed to quantify colon length, polyp number, and polyp size. Colons were cut longitudinally and cleaned by vigorous shaking in ice-cold PBS. Colons were then flattened against a lightbox and moistened by cold PBS while quantification and images were gathered. Portions of the colon from each genotype were then prepared for histology, RNA, protein, or single-cell isolation.

Antibiotic-treatment of mice

Antibiotic-treatment of mice was performed as described in our previous studies (16, 17, 22). In brief, WT and LRP5/6^ΔCD11c mice were fed with an antibiotic cocktail (1 g/L ampicillin, 1 g/L metronidazole, 1 g/L neomycin sulfate and 0.5 g/L vancomycin) in drinking water for two weeks before DSS treatment. All antibiotics were purchased from Sigma-Aldrich.

Fecal microbiota transplantation (FMT)

Fresh fecal samples were collected directly from the rectums of 10–12 weeks old WT and LRP5/6^ΔCD11c mice. Samples collected were then homogenized immediately in sterile PBS and then filtered through a sterile 70μm strainer to remove large particulate and fibrous matter. For the colonization of antibiotic-treated WT and LRP5/6^ΔCD11c mice, each mouse was administered 0.1 ml of fecal suspension containing 20 mg feces by oral gavage once every two days before DSS treatment. WT and LRP5/6^ΔCD11c mice were caged separately.

Ex vivo colon culture and ELISAs

Ex vivo colon culture was performed as described in our previous studies (17). In brief, biological triplicates of 1 cm-long section of the ascending colon were excised, feces flushed out using sterile HBSS, longitudinally opened, and washed three times with sterile HBSS, as previously described (23). One cm² colon sections were then placed into culture in complete RPMI-1640 media (supplemented with L-glutamine, penicillin, streptomycin, tetracycline and 2% FBS) and cultured for two days at 37°C in a 95% air/5% CO₂ atmosphere. Supernatants were then collected and the cytokines IL-17, IL-6, IL-10, TNF-α, IFN-γ and IL-1β quantitated using ELISA kits from BD Biosciences.

Colonic antigen-presenting cell sorting

Leukocytes from colonic lamina propria (LP) were isolated as described in our previous study (16). CD11c⁺ APCs were enriched from the leukocyte preparation by elution via MACS LS columns (Miltenyi Biotec) and then FACS sorted for CD11c⁺ APCs (CD45⁺I-A^b+CD11c⁺) (17, 24). After sorting, APCs (10⁵) were cultured in 0.2 ml RPMI 1640 complete medium in 96-well round-bottom plates. After 48 hr incubation, cell culture supernatants were used to quantify cytokines (indicated in Results) by ELISA.

Bacterial DNA extraction

Quantification of indicated bacterial groups in feces of WT and LRP5/6^ΔCD11c mice was performed by qPCR as described previously (17, 21, 22). Briefly, fecal pellets were collected from mice and bacterial DNA was extracted with the QIAamp DNA Stool Kit (QIAGEN). Quantitative PCR for the 16S rRNA gene was performed with SYBR Green (Bio-Rad). Amounts of indicated bacterial groups were first normalized to that of total bacterial DNA. Reactions were run with the MyiQ5 ICycler Real-Time PCR Detection System (Bio-Rad). Primers used in this study have been previously described (21, 22).

Leukocyte preparation and flow cytometry

Lamina propria (LP) leukocytes from colons were isolated at the end of AOM-DSS treatment as described in our previous study (16). Isolated LP leukocytes were collected, washed, and stained with antibodies specific for mouse CD11c (CD45⁺ I-A^b+ CD11c⁺) followed by intracellular staining non-phospho (Active) β-Catenin or phospho-p38 MAPK (Cell Signaling Technology) and analyzed by FACS. Briefly, single-cell suspensions from LP were re-suspended in PBS containing 5% FBS. After incubation for 15 min at 4°C with the blocking Ab 2.4G2 (anti-FcγRIII/I), the cells were stained with the appropriately labeled Abs. Samples were then washed twice in PBS containing 5% FBS. In some experiments, mononuclear cells from colonic LP were cultured with phorbol myristate acetate (50 ng/mL) plus ionomycin (750 ng/mL) in the presence of GolgiStop and GolgiPlug for 6 hr. The cells were then stained for CD11c (CD45⁺ I-A^b+ CD11c⁺) or CD4 (CD45⁺ CD4⁺) followed by intracellular staining of IL-6, pro-IL-1β, TNF-α IFN-γ, IL-17A, and IL-10.

Real-time PCR

Total mRNA was isolated from colon or cells using the Omega Total RNA Kit according to the manufacturer’s protocol. cDNA was then generated using an RNA to cDNA Ecodry Premix Kit (Clontech) according to the manufacturer’s protocol. Subsequently, quantitative real-time PCR was done using SYBR Green Master Mix (Roche) with gene-specific primers (19) and a MyiQ5 ICycler (BioRad), and with gene expression normalized relative to Gapdh.

Histopathology analysis and Immunohistochemistry

Sections (6 μm thick) from formalin-fixed and paraffin-embedded colons were placed onto glass slides. H&E-stained sections were blindly scored for the severity of colonic inflammation and damage as described previously (21, 22). The degree of inflammation and mucosal damage was scored as follows: (0) no inflammation, (1) mild inflammation or prominent lymphoid aggregates, (2) moderate inflammation, (3) moderate inflammation associated with crypt loss, and (4) severe inflammation with crypt loss and ulceration. Crypt destruction was graded as follows: (0) no destruction, (1) 1%–33% of crypts destroyed, (2) 34%–66% of crypts destroyed, and (3) 67%–100% of crypts destroyed. The individual scores from inflammation and crypt damage were summed to derive a histological score for colonic inflammation (maximum score 7). Immunohistochemistry of sections was additionally performed, with sections stained for Ki67 as previously described (Singh et al., 2014). Ki67-stained sections were analyzed for cell proliferation, and Ki67⁺ cells were manually counted to note the extent of proliferation.

Plasmids, cell culture, transient transfection, and reporter assay

Wild type β-catenin, dominant-negative β-catenin and constitutively-active β-catenin plasmids were provided by Dr. Zuoming Sun (City of Hope). TCF4 and 1.5 kb IL-10 promoter-luciferase constructs were purchased from Addgene (25). 293T cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells (2–3×10⁵ in each well of a 24 well-plate) were transfected with the IL-10 promoter-reporter plasmid (100 ng) and expression vectors (500 ng) by the Lipofectamine method (Invitrogen). The total amount of transfected DNA was kept constant by adjusting the amount of the empty vector. Transfected cells were then treated with or without Wnt3a (20 ng/ml; PeproTech Inc.). After 24 h, cells were lysed in 200 μl reporter lysis buffer and luciferase activities were measured by the Luciferase system, according to the manufacturer’s instructions (Promega), and normalized against Renilla luciferase activities. “Fold Induction” represents normalized luciferase activity divided by activity measured in reporter-only groups.

Statistical analyses

Statistical analyses were performed using GraphPad Prism software. An unpaired one-tailed Student’s t test was used to determine statistical significance between different groups for mRNA expression levels, Treg percentages and cytokines released by various cell types. A P value less than 0.05 (*) was considered to be significant, a P value less than 0.01 (**) was considered to be very significant, and a P value less than 0.001 (***) was considered to be extremely significant.

Results

LRP5/6 deficiency in CD11c⁺ APCs exacerbates AOM-DSS-induced colon cancer in mice.

To study the potential role of LRP5/6 signaling in CD11c⁺ APCs on inflammation-induced colorectal cancer, we employed the very well characterized azoxymethane/dextran sodium sulfate (AOM-DSS) mouse model in which DSS-mediated injury induces inflammation that contributes to colon carcinogenesis caused by azoxymethane (AOM). Also, we utilized conditional knockout mice that specifically lack LRP5 and LRP6 in CD11c⁺ APCs (LRP5/6^ΔCD11c) (17, 19). We initially treated WT (LRP5/6^FL) and LRP5/6^ΔCD11c mice with AOM and three cycles of DSS (2.0%) treatment (Supplementary Figure 1A). Both WT and LRP5/6^ΔCD11c mice survived the AOM-DSS treatment, but LRP5/6^ΔCD11c mice showed a marked increase in weight loss and a significant reduction in colon length compared to WT mice (Figs. 1A & B). Next, we assessed the tumor burden in the colons of AOM-DSS-treated mice. As expected, WT mice had tumors in the distal colon in response to AOM-DSS treatment (Fig. 1C). In contrast, LRP5/6^ΔCD11c mice displayed a higher tumor burden with many tumors arising further towards the proximal colon (Fig. 1C). This was associated with a significant increase in tumor numbers and tumor load in LRP5/6^ΔCD11c mice compared to WT mice (Fig. 1D, 1E). Histopathological analysis of colonic sections from AOM-DSS-treated LRP5/6^ΔCD11c mice revealed markedly increased damage to the mucosa with epithelial erosion, frequent ulceration, and loss of crypt structure (Fig. 1F). Immunohistochemistry with Ki67 further revealed that increased tumor numbers in LRP5/6^ΔCD11c mice is associated with markedly increased cell proliferation (Figs. 1G & H). Together, the clinical and histopathology data indicate an important role for LRP5/6 signaling in CD11c⁺ APCs in suppressing colitis-associated tumorigenesis.

Figure 1. LRP5/6 deficiency in CD11c⁺ APCs exacerbates AOM-DSS-induced colon cancer.

WT (LRP5/6^FL/FL) and LRP5/6^ΔCD11c mice were treated with AOM+DSS (36–50 kDa) in their drinking water before returning to normal water and then analyzed for the indicated inflammation parameters and inflammation-associated carcinogenesis as described in the Methods (see also Supplementary Figure 1A). (A, B) Change in body weight and colon length of WT and LRP5/6^ΔCD11c mice (n ≥ 5). (C) Representative images of colon polyps as induced by AOM-DSS in WT and LRP5/6^ΔCD11c mice (n ≥ 5). (D) Average polyp numbers and (E) polyp size (mm) from WT and LRP5/6^ΔCD11c mice. (n ≥ 5). (F, G) Representative colon sections from mice treated with AOM-DSS were microscopically analyzed for mucosal damage after staining with hematoxylin and eosin (F) or for proliferating cells after staining with Ki67 antibody (G). Scale bars (blue), 200 μm. (H) Ki67-positive cells were manually counted in WT and LRP5/6^ΔCD11c mice treated with AOM-DSS (n ≥ 5). Values are mean ± SEM and are representative of at least 2 independent experiments. **P < 0.01; ***P < 0.001

LRP5/6 signaling in intestinal CD11c⁺ APCs regulates inflammatory factors that promote CAC.

The cytokine milieu present in the gut microenvironment can either suppress or promote colonic inflammation and CRC, and accumulating evidence also supports an important role for chemokines (4). Inflammatory cytokines such as IL-1β, IL-6 and TNF-α promotes, while IL-10 suppresses, inflammation-driven colon tumorigenesis (4, 26). Thus, we analyzed the cytokine and chemokine profiles in the colons of WT and LRP5/6^ΔCD11c mice before and after AOM-DSS-treatment. As evidenced in Fig. 2A and Fig. 2B, the colons from LRP5/6^ΔCD11c mice expressed significantly higher levels of inflammatory cytokines such as IL-1β, IL-6 and TNF-α compared to the colons from WT mice treated with AOM-DSS. In contrast, the colons from LRP5/6^ΔCD11c mice expressed markedly reduced levels of anti-inflammatory cytokine IL-10 compared to the colons from WT mice treated with AOM-DSS (Figs. 2A & B). Also, the colons from LRP5/6^ΔCD11c mice expressed significantly higher levels of chemokines such as CXCL1, CXCL2 and CCL2 compared to the colons from WT mice after AOM-DSS-treatment (Fig. 2C). Next, we analyzed the expression levels of IL-1β, IL-6, and TNF-α in colonic CD11c⁺ APCs isolated from WT and LRP5/6^ΔCD11c mice after AOM-DSS treatment. LRP-5/6-deficient colonic CD11c⁺ APCs expressed markedly higher levels of IL-1β, IL-6, and TNF-α and lower levels of IL-10 compared to WT colonic CD11c⁺ APCs in response to AOM+DSS treatment (Figs. 2D, E, F & G).

Figure 2. LRP5/6 in intestinal CD11c⁺ APCs regulates inflammatory factors that promote colitis-associated colon cancer.

(A, C) RNA was extracted from the colons of untreated and AOM-DSS-treated WT and LRP5/6^ΔCD11c mice. Expression of indicated genes was quantified by qPCR (n >5). (B) Excised colon samples of untreated and AOM-DSS-treated WT-FL and LRP5/6^ΔCD11c mice were cultured for 48h ex vivo, and the cytokine levels in the culture supernatants were quantified by ELISA (n >5). (D) Quantitative real-time PCR analysis of inflammatory factors levels in colonic CD11c⁺ APCs isolated from AOM-DSS-treated WT and LRP5/6^ΔCD11c mice (n>5). (E) Representative histograms showing the pro-IL-1β levels in colonic CD11c⁺ APCs isolated from AOM-DSS-treated WT and LRP5/6^ΔCD11c mice. (F, G) FACS plots representing percentages or cumulative frequencies of CD11c⁺ APCs positive for IL-6, TNF-α or IL-10 isolated from colons of AOM-DSS-treated WT and LRP5/6^ΔCD11c mice. (n>5). (H) Representative histogram showing the phosphorylated (Thr180/Tyr182) form of p38 MAPK in colonic CD11c⁺ APCs isolated from WT-FL and LRP5/6^ΔCD11c mice treated without or with AOM-DSS. (I, J) FACS plots representing percentages or cumulative frequencies of CD4⁺ T cells positive for IL-17A, IFN-γ or IL-10 isolated from colons of WT and LRP5/6^ΔCD11c mice treated with AOM-DSS (n>5). Values are mean ± SEM and are representative of at least 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

The p38α MAPK signaling pathway is critical for the expression of inflammatory factors and has been shown to play a key role in the pathogenesis of IBD or inflammation-associated colorectal cancer (27, 28). Thus, we assessed whether increased expression of inflammatory factors in LRP-5/6-deficient colonic CD11c⁺ APCs is associated with the p38α MAPK signaling pathway. LRP5/6-deficiency in colonic CD11c⁺ APCs resulted in a marked increase in the phosphorylated (active) form of p38 MAPK under homeostatic conditions as well as upon AOM-DSS treatment (Fig. 2H). These results show that LRP5/6-meditated signaling is critical for regulating the activity of p38 MAPK in colonic CD11c⁺ APCs.

IL-17A-producing CD4⁺ T (Th17) cells play an important role in the pathogenesis of IBD and CAC(29, 30). IL-6 and IL-1β induce the differentiation of naïve CD4+ T cells to Th17 cells. Since LRP5/6^ΔCD11c mice contain higher levels of IL-1β and IL-6 in the colon after AOM-DSS treatment, we asked whether this also leads to alterations in Th1/Th17/ regulatory T cells. Thus, we quantified the frequency of Th1/Th17/ regulatory T cells in the colon of WT and LRP5/6^ΔCD11c mice after AOM-DSS treatment. Consistent with increased levels of IL-1β and IL-6, LRP5/6^ΔCD11c mice displayed higher frequencies of CD4⁺ cells producing IL-17A in the colon compared to WT mice (Figs. 2I & J). In contrast, LRP5/6^ΔCD11c mice contained lower frequencies of CD4⁺ cells producing IL-10 in the colon compared to WT mice after treatment (Figs. 2I & J). Collectively, these results indicate an important role for LRP5/6-signalling in CD11c⁺ APCs in regulating inflammatory factors that promote CAC.

The depletion of gut microbiota in LRP5/6^ΔCD11c mice decreases AOM-DSS-induced colon cancer.

The composition of gut bacterial communities plays a critical role in either suppressing or promoting intestinal inflammation and CAC (31). Genetic modification of the host can lead to microbial dysbiosis resulting in host susceptibility to colonic inflammation and CAC (21, 22, 32–34). Our previous study has shown that LRP5/6^ΔCD11c mice harbor altered commensal microflora with the increased presence of SFB, Prevotellaceae and TM7 groups of commensal bacteria(17). Thus, we investigated whether the increased inflammation and tumor burden in LRP5/6^ΔCD11c mice after AOM-DSS treatment is associated with commensal dysbiosis. We performed microbiota depletion studies in LRP5/6^ΔCD11c mice using an antibiotic cocktail treatment and then subjected these mice to AOM-DSS treatment (Supplementary Fig. 1B). The depletion of gut microbiota markedly reduced tumor burden in the colons of LRP5/6^ΔCD11c mice after AOM-DSS treatment (Fig. 3A). This was associated with a significant decrease in tumor numbers and tumor load in LRP5/6^ΔCD11c mice compared to WT mice (Fig. 3B, C). Ki67 staining of colonic sections from commensal-depleted LRP5/6^ΔCD11c mice revealed that decreased tumor number is associated with markedly decreased cell proliferation (Figs. 3D & E). Consistent with reduced tumor numbers, we found that commensal depletion attenuated the levels of IL-6, IL-1β and TNF-α in the colons of LRP5/6^ΔCD11c mice after AOM-DSS treatment (Fig. 3F). Further analysis showed that colonic CD11c⁺ APCs from antibiotic-treated LRP5/6^ΔCD11c mice expressed markedly lower levels of IL-1β, IL-6, and TNF-α compared to colonic CD11c⁺ APCs from untreated mice (Figs. 3G & H). Besides, antibiotic treatment resulted in a significant reduction in IL-17A or IFNγ producing CD4⁺ T cells in the colons of LRP5/6^ΔCD11c mice (Fig. 3I).

Figure 3. The depletion of gut microbiota in LRP5/6^ΔCD11c mice decreases AOM-DSS-induced colon cancer.

(A-I) LRP5/6^ΔCD11c mice were treated with (+ABX) or without antibiotics (–ABX) in addition to receiving AOM-DSS as described in the Methods (see also Supplementary Figure 1B) (A) Representative images of colon polyps from LRP5/6^ΔCD11c mice treated with AOM-DSS and with or without antibiotics (n ≥ 5). (B) Average polyp numbers and (C) polyp size (mm) in the colons of LRP5/6^ΔCD11c mice treated with AOM-DSS and with or without antibiotics (n ≥ 5). (D) Representative colon sections stained with Ki67 antibody from mice treated with AOM-DSS and with or without antibiotics. Scale bars (blue), 200 μm. (E) Ki67 positive cells were manually counted in WT and LRP5/6^ΔCD11c mice treated with AOM-DSS and antibiotics (n ≥ 5). (F) Excised colon samples of (AOM+DSS)-treated LRP5/6^ΔCD11c mice additionally treated with or without antibiotics were cultured for 48h ex vivo, and the cytokine levels in the culture supernatants were quantified by ELISA (n ≥ 5). (G) Representative histograms showing the pro-IL-1β levels in colonic CD11c⁺ APCs isolated from LRP5/6^ΔCD11c mice treated with AOM+DSS and with or without antibiotics (n ≥ 5). (H) Percentages or cumulative frequencies of CD11c⁺ APCs positive for IL-6, TNF-α or IL-10 isolated from colons of LRP5/6^ΔCD11c mice treated with AOM+DSS and with or without antibiotics (n ≥ 5). (I) Percentages or cumulative frequencies of CD4⁺ T cells positive for IL-17A or IFN-γ isolated from colons of LRP5/6^ΔCD11c mice treated with AOM+DSS and with or without antibiotics (n ≥ 5). (J-M) In addition to receiving AOM-DSS, WT and LRP5/6^ΔCD11c mice were treated with antibiotics and then transplanted with fecal microbiota (FMT) from either WT or LRP5/6^ΔCD11c mice as described in the Methods (see also Supplementary Figure 1C). (J) Representative macroscopic appearance of colon, (K) polyp numbers and (L) average poly size in the colons of WT and LRP5/6^ΔCD11c mice after FMT and AOM-DSS treatment (n > 5). Values are mean ± SEM and are representative of at least 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Increased representation of SFB, Prevotellaceae and TM7 groups of bacteria is associated with enhanced risk of colitis-associated cancer in mice (21, 32, 35). To elucidate the possible role of altered gut microbiota in tumor susceptibility in LRP5/6^ΔCD11c mice, we first depleted gut microbiota with antibiotics and then transplanted mice with FMT from either WT or LRP5/6^ΔCD11c mice (Supplementary Fig. 1C). Quantification of the relative levels of different bacterial species in the feces of microbiota-transplanted mice showed markedly decreased representation of SFB, Prevotellaceae and TM7 groups of bacteria in LRP5/6^ΔCD11c mice transplanted with WT fecal microbiota compared to LRP5/6^ΔCD11c mice transplanted with LRP5/6^ΔCD11c fecal microbiota (Supplementary Fig. 1D). Although both groups did exhibit much greater weight loss compared to WT mice transplanted with WT fecal microbiota (Supplementary Fig. 1E). In line with these observations, LRP5/6^ΔCD11c mice transplanted with WT mouse fecal microbiota showed lower tumor numbers and tumor load in the colon compared to such mice transplanted with LRP5/6^ΔCD11c mouse fecal microbiota instead (Figs. 3J, K, L). However, LRP5/6^ΔCD11c mice transplanted with either WT fecal microbiota exhibited much greater weight loss and increased tumor numbers in the colon compared to WT mice transplanted with WT fecal microbiota (Supplementary Fig. 1E; Figs. 3J, K, L). Collectively, these results suggest that both altered gut microbiota and loss of immune tolerance to gut flora contribute to CAC in LRP5/6^ΔCD11c mice.

LRP5/6-β-catenin signaling regulates the expression of inflammatory factors through the autocrine effects of IL-10 on colonic CD11c⁺ APCs.

IL-10 is a key anti-inflammatory cytokine that is critical for suppressing intestinal inflammation and colitis-associated colon cancer (36–39). IL-10-deficient mice are more susceptible to colitis and CAC. Multiple immune cells produce IL-10 and intestinal APCs are the major producers. Given the reduced expression of IL-10 in the colons of LRP5/6^ΔCD11c mice treated with AOM-DSS, we investigated whether LRP5/6 signaling is critical for the induction of IL-10 in CD11c⁺ APCs in the colon. LRP5/6-deficient colonic CD11c⁺ APCs cultured ex vivo produced markedly lower levels of IL-10 compared to colonic WT CD11c⁺ APCs without or with AOM-DSS treatment (Fig. 4A). Suppressors of cytokine signaling 1 (SOCS1) and SOCS3 play a critical role in regulating intestinal inflammation (40, 41). Our prior study has shown that IL-10 produced by APCs exerts autocrine effects to suppress the expression of inflammatory factors by inducing SOCS1 and SOCS3 genes (42). Thus, we quantified the expression levels of SOCS1 and SOCS3 in colonic CD11c⁺ APCs. Consistent with decreased IL-10 production, CD11c⁺ APCs-deficient in LRP5/6 expressed markedly lower levels of SOCS1 and SOCS3 compared to WT CD11c⁺ APCs isolated from the colon before and after AOM-DSS treatment (Figs. 4B & 4C). Next, we investigated whether the addition of exogenous IL-10 to LRP5/6-deficient CD11c⁺ APCs could restore the expression of SOCS1 and SOCS3. Thus, we cultured colonic CD11c⁺ APCs deficient in LRP5/6 in the presence or absence of IL-10 and determined the expression of the two SOCS genes and quantitated the pro-inflammatory cytokines produced. As can be discerned from the data presented in Figs. 4D and 4E, incubation of LRP5/6-deficient CD11c⁺ APCs with rIL-10 significantly stimulated the expression of SOCS1 and SOCS3 and, as expected, significantly decreased the production of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. These results demonstrate that IL-10 produced in response to LRP5/6 signaling by intestinal APCs can act in an autocrine manner to suppress the production of pro-inflammatory cytokines.

Figure 4. Wnt3A-LRP5/6-β-catenin signaling induces IL-10 and SOCS3 to suppress inflammatory factors in intestinal CD11c⁺ APCs.

(A) Colonic CD11c⁺ APCs isolated from untreated and AOM-DSS-treated WT and LRP5/6^ΔCD11c mice were cultured for 2 days ex vivo and IL-10 levels in the culture supernatants were quantified by ELISA (n>4). (B, C) Quantitative real-time PCR analysis of the mRNA of SOCS1 and SOCS3 genes in colonic CD11c⁺ APCs isolated from untreated and AOM-DSS-treated WT and LRP5/6^ΔCD11c mice. (n=5). (D) Colonic CD11c⁺ APCs isolated from AOM-DSS-treated LRP5/6^ΔCD11c mice were cultured for 2 days ex vivo in the presence or absence of rIL-10 (5 ng/ml). Inflammatory cytokine amounts in the culture supernatants were quantified by ELISA (n>4). (E) Quantitative real-time PCR analysis of the mRNA of SOCS1 and SOCS3 genes in colonic CD11c⁺ APCs isolated from AOM-DSS-treated LRP5/6^ΔCD11c mice cultured with or without rIL-10 (n>5). (F) Quantitative real-time PCR analysis of Wnt3A mRNA levels in the colons of untreated and AOM-DSS-treated WT mice. (n ≥ 5). (G) Representative histograms showing the active form of β-catenin levels in colonic CD11c⁺ APCs isolated from untreated and AOM-DSS-treated WT and LRP5/6^ΔCD11c mice. (H) IL-10 promoter activity in 293T cells after transfection of reporter plasmid alone or co-transfected with WT-β-catenin (WT-βcat), dominant-negative β-catenin (DN-βcat), constitutively active β-catenin (CA-βcat), or WT-TCF4 expression vectors and cultured in the presence or absence of Wnt3A (20 ng/ml). The results are represented as fold activation relative to IL-10 promoter vector transfection control alone (n>4). Values are mean ± SEM and are representative of at least 2 independent experiments. **P < 0.01; ***P < 0.001.

Next, we explored the potential molecular mechanisms by which LRP5/6 signaling in intestinal CD11c⁺ APCs regulate IL-10 expression. The transcriptional co-factor β-catenin and the TCF family of transcription factors are the main downstream mediators of canonical Wnt-signaling (18, 43). Previous studies have shown that Wnt3A activates the canonical Wnt pathway in DCs (19). Thus, we quantified the expression levels of Wnt3A in the colon of WT mice before and after AOM-DSS treatment. WT mice had markedly higher transcript levels of Wnt3A in colon tissues after AOM-DSS treatment (Fig. 4F). Next, we examined the activation of β-catenin in colonic CD11c⁺ APCs isolated from WT and LRP5/6^ΔCD11c mice treated with AOM-DSS. Colonic CD11c⁺ APCs deficient in LRP5/6 showed markedly reduced activation of β-catenin compared to WT colonic CD11c⁺ APCs without and with AOM-DSS treatment (Fig. 4G). Next, we investigated whether canonical Wnt signaling directly regulates IL-10 expression. Promoter analysis using consensus TCF binding sites showed several putative TCF sites in the IL-10 promoter. To further investigate transcriptional regulation of IL-10 by β-catenin/TCF4, 293T cells were transfected with a 1.5 kbp IL-10 promoter-luciferase reporter plasmid in combination with wild type or mutant β-catenin or TCF-4 and then treated with or without Wnt3A (Fig. 4H). Co-transfection with WT β-catenin or WT-TCF4 resulted in a 3–4-fold increase in IL-10 promoter activity without any treatment, and this result was even further increased with Wnt3A treatment (Fig. 4H). The importance of β-catenin in this stimulation of IL-10 promoter activity was further tested using the expression of dominant-negative (DN-βcat) and constitutively active (CA-βcat) mutants of β-catenin along with WT-TCF4. Expression of mutant DN-βcat markedly decreased while expression of CA-βcat markedly increased IL-10 promoter activity in response to Wnt3A (Fig. 4H). These results demonstrate that LRP5/6 signaling promotes transcriptional activation of IL-10 gene expression via the β-catenin/TCF pathway.

LRP5/6 signaling in intestinal CD11c⁺ APCs regulates colitis-associated colon cancer in a β-catenin-dependent manner.

The reduced activation of β-catenin in colonic CD11c⁺ APCs of LRP5/6^ΔCD11c mice prompted us to investigate whether conditional activation of β-catenin in LRP5/6^ΔCD11c mice can suppress CAC. For this, we treated LRP5/6^ΔCD11c mice expressing an active form of β-catenin specifically in CD11c⁺ APCs (Act-βcat^CD11c) with AOM-DSS (17, 19). β-catenin activation in CD11c⁺ APCs markedly reduced tumor numbers and tumor load in the colons of LRP5/6^ΔCD11c / Act-βcat^CD11c mice compared to control LRP5/6^ΔCD11c mice after AOM-DSS treatment (Fig. 5A, 5B, C). Histopathological analysis of colonic sections from LRP5/6^ΔCD11c / Act-βcat^CD11c mice revealed markedly reduced damage to mucosa when compared to LRP5/6^ΔCD11c mice treated with AOM-DSS (Fig. 5D). Further analysis of Ki67 staining of colonic sections from LRP5/6^ΔCD11c / Act-βcat^CD11c mice revealed markedly decreased cell proliferation when compared to LRP5/6^ΔCD11c mice (Figs. 5E & F). Consistent with reduced inflammation and tumor burden, the colons of LRP5/6^ΔCD11c /Act-βcat^CD11c mice expressed higher levels of IL-10 and lower levels of inflammatory cytokines such as IL-1β, IL-6 and TNF-α compared to colons of LRP5/6^ΔCD11c mice treated with AOM-DSS (Figs. 5G & H). Further characterization of colons of LRP5/6^ΔCD11c /Act-βcat^CD11c mice showed markedly reduced frequencies of CD11c⁺ APCs expressing IL-6, and TNF-α and markedly increased frequencies of CD11c⁺ APCs expressing IL-10 after AOM-DSS treatment (Fig. 5I). In line with these observations, LRP5/6^ΔCD11c /Act-βcat^CD11c mice displayed lower frequencies of CD4⁺ cells producing IL-17A and higher frequencies of CD4⁺ cells producing IL-10 in the colon when compared to LRP5/6^ΔCD11c mice after AOM-DSS treatment (Fig. 5J). Together, these results support an important role for LRP5/6-β-catenin signaling in CD11c⁺ APCs in suppressing colitis-associated tumorigenesis.

Figure 5. Conditional activation of β-catenin specifically in CD11c⁺ APCs reduces AOM-DSS-induced colon cancer in LRP5/6^ΔCD11c mice.

(A) Representative images of colon polyps from LRP5/6^ΔCD11c and LRP5/6^ΔCD11c/Act-βcat^CD11c mice treated with AOM-DSS. (B) Average polyp numbers and (C) polyp size (mm) in the colons of LRP5/6^ΔCD11c and LRP5/6^ΔCD11c/Act-βcat^CD11c mice after AOM-DSS treatment (n > 5). (D, E) Representative colon sections from mice treated with AOM-DSS were microscopically analyzed for mucosal damage after staining with hematoxylin and eosin (D) or for proliferating cells after staining with Ki67 antibody (E). Scale bars (blue), 200 μm. (F) Ki67 positive cells were manually counted in LRP5/6^ΔCD11c and LRP5/6^ΔCD11c/Act-βcat^CD11c mice after AOM-DSS treatment (n ≥ 5). (G) RNA was extracted from the colons of AOM-DSS-treated LRP5/6^ΔCD11c and LRP5/6^ΔCD11c/Act-βcat^CD11c mice. Expression of indicated genes was quantified by qPCR (n>5). (H) Excised colon samples of AOM-DSS-treated LRP5/6^ΔCD11c and LRP5/6^ΔCD11c/Act-βcat^CD11c mice were cultured for 48h ex vivo, and the cytokine levels in the culture supernatants were quantified by ELISA. (I) Cumulative frequencies of CD11c⁺ APCs positive for IL-6, TNF-α or IL-10 isolated from colons of AOM-DSS-treated LRP5/6^ΔCD11c and LRP5/6^ΔCD11c/Act-βcat^CD11c mice. (n > 5). (J) Cumulative frequencies of CD4⁺ T cells positive for IL-17A, IFN-γ or IL-10 isolated from colons of AOM-DSS-treated LRP5/6^ΔCD11c and LRP5/6^ΔCD11c/Act-βcat^CD11c mice. (n > 5). Values are mean ± SEM and are representative of at least 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

LRP5/6 signaling in intestinal CD11c⁺ APCs regulates colitis-associated colon cancer in an IL-10-dependent manner.

The above results show that IL-10 is a key downstream mediator of LRP5/6-β-catenin signaling in colonic CD11c⁺ APCs and is critical for suppressing the expression of inflammatory factors. Thus, we asked whether the administration of IL-10 into LRP5/6^ΔCD11c mice could suppress chronic intestinal inflammation and CAC. IL-10 treatment markedly reduced the tumor numbers and tumor load in the colon of LRP5/6^ΔCD11c mice compared to the untreated control mice in response to AOM-DSS. (Figs. 6A, B & C). Histopathological analysis of colonic sections from IL-10-treated LRP5/6^ΔCD11c mice revealed markedly reduced damage to mucosa when compared to the control mice in response to AOM-DSS (Fig. 6D). Further analysis of Ki67 staining of colonic sections from IL-10-treated LRP5/6^ΔCD11c mice showed markedly decreased cell proliferation compared to the control mice (Figs. 6E & F). Consistent with reduced inflammation and tumor numbers, the colon of IL-10-treated LRP5/6^ΔCD11c mice had lower transcript levels of IL-1β, IL-6 and TNF-α compared to the colon of untreated LRP5/6^ΔCD11c mice (Fig. 6G,). In line with mRNA expression, the secreted pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF-α were significantly lower in the colon of IL-10-treated LRP5/6^ΔCD11c mice compared to the colon of untreated LRP5/6^ΔCD11c mice (Fig. 6H). Together, these results support an important role for the LRP5/6-β-catenin-IL-10 axis in CD11c⁺ APCs in suppressing CAC.

Figure 6. LRP5/6 in intestinal CD11c⁺ APCs regulates colitis-associated colon cancer in an IL-10-dependent manner.

(A) Representative images of colon polyps from LRP5/6^ΔCD11c mice treated with AOM-DSS and with or without rIL-10 treatment. (B) Average polyp numbers and (C) polyp size (mm) in the colons of LRP5/6^ΔCD11c mice after AOM-DSS treatment and with or without rIL-10 treatment (n > 5). (D, E) Representative colon sections from LRP5/6^ΔCD11c mice treated with AOM-DSS and with or without rIL-10 were microscopically analyzed for mucosal damage after staining with hematoxylin and eosin (D) or for proliferating cells after staining with Ki67 antibody (E). Scale bars (blue), 200 μm. (F) Ki67 positive cells were manually counted in LRP5/6^ΔCD11c mice after AOM-DSS and with or without rIL-10-treatment (n ≥ 5). (G) RNA was extracted from the colons of LRP5/6^ΔCD11c mice after AOM-DSS treatment and with or without rIL-10 treatment. Expression of indicated genes was quantified by qPCR (n>5). (H) Excised colon samples of (AOM+DSS)-treated LRP5/6^ΔCD11c mice either with or without having received administration of rIL-10 were cultured for 48h ex vivo, and cytokine levels in the culture supernatants were quantified by ELISA (n > 5). The error bars indicate mean ±SEM and are representative of at least 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Discussion

The current study defines an essential role for the LRP5/6-β-catenin-IL-10 signaling pathway in CD11c⁺ APCs in controlling chronic intestinal inflammation and CAC in response to gut microbiota. Accordingly, mice that are deficient in the co-receptors LRP5 and LRP6 specifically in CD11c⁺ APCs were more susceptible to CAC. The exacerbated colonic inflammation and increased tumor burden in LRP5/6^ΔCD11c mice were due to a loss of tolerance to commensal flora resulting in increased p38 activation and higher levels of tumor-promoting pro-inflammatory factors. Also, we show that LRP5/6-β-catenin signaling regulates the expression of inflammatory factors through the autocrine effects of IL-10 on colonic CD11c⁺ APCs. Accordingly, conditional activation of β-catenin specifically in CD11c⁺ APCs or in vivo administration of IL-10 in LRP5/6^ΔCD11c mice markedly reduced intestinal inflammation and CAC. Collectively, these findings support the hypothesis that the LRP5/6-β-catenin-IL-10 signaling pathway in colonic APCs is critical for suppressing chronic intestinal inflammation and CAC in response to microbiota. Hence, this pathway constitutes a new therapeutic target for the treatment of chronic intestinal inflammation and CAC.

Aberrant Wnt signaling is associated with many cancers, including colon cancer (18). However, the focus of most research has been on the effects of this pathway on intestinal stem cell proliferation, as well as its effects on cancer initiation and progression. Recent studies have shown that Wnt signaling plays an essential role in mucosal regeneration following intestinal injury (44, 45). Moreover, our prior studies have shown that Wnt ligands in the gut environment also act on APCs in shaping immune responses to gut microbiota (16, 17). Chronic intestinal inflammation is a key driving force for the initiation and progression of tumors in the colon (4). Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt-signaling in APCs is critical for suppressing DSS-induced acute inflammation (16, 17), but whether this pathway in APCs protects against CAC is not known. The present study shows that genetic deletion of LRP5/6 specifically in CD11c⁺ APCs in mice results in enhanced intestinal inflammation with increased tumor size and numbers in the colon. Patients with UC, CD and CAC had elevated levels of IL-6, IL-1β and TNF-α in the gut microenvironment (4, 26, 46). In this study, we show that increased tumor burden in the colon of LRP5/6^ΔCD11c mice is due to markedly higher levels of IL-6, IL-1β and TNF-α. Moreover, colonic CD11c⁺ APCs that are deficient in LRP5/6 express markedly higher levels of these inflammatory factors under homeostatic conditions and upon AOM-DSS treatment. This is associated with increased activation of the p38 MAPK signaling pathway. The p38α MAPK signaling pathway has been shown to play a key role in the pathogenesis of IBD or inflammation-associated colorectal cancer(27, 28). Accordingly, mice that specifically lack p38 in DCs are highly susceptible to intestinal inflammation and CAC(47–49).

IL-10 is a key immunosuppressive cytokine that is critical for suppressing intestinal inflammation (36–39). IL-10-deficient mice are more susceptible to CAC. Additionally, loss of IL-10 or blocking IL-10R signaling induces spontaneous colitis and promotes tumor development in mice (36–39). IL-10 or IL-10 receptor polymorphisms are linked to an increased incidence of IBD (50, 51). Intestinal APCs are the major producers of IL-10, but the underlying molecular mechanisms are unknown. The present study shows that the LRP5/6-β-catenin signaling pathway in intestinal APCs is critical for IL-10 production. Accordingly, colonic CD11c⁺ APCs that are deficient in LRP5/6 express markedly lower levels of IL-10 and IL-10 target genes SOCS1 and SOCS3 under homeostatic conditions and in response to AOM-DSS treatment. Our studies also show that attenuated IL-10 levels in the colon of LRP5/6^ΔCD11c mice lead to markedly higher levels of pro-inflammatory factors. Furthermore, our mechanistic studies show that LRP5/6- β-catenin signaling regulates the expression of inflammatory factors through the autocrine effects of IL-10 on colonic CD11c⁺ APCs. Accordingly, conditional activation of β-catenin specifically in CD11c⁺ APCs or in vivo administration of IL-10 in LRP5/6^ΔCD11c mice markedly reduced levels of pro-inflammatory factors and tumor burden in the colon. Other regulatory factors such as Indoleamine 2,3-dioxygenase (IDO) and retinoic acid (RA), though not analyzed in the present study, may play a role in regulating colitis-associated, inflammation-induced CRC through the LRP5/6-β-catenin axis (16, 17, 52). The present study was focused on IL-10 because the exogenous addition of IL-10 suppressed the expression of inflammatory factors associated with colon cancer. Furthermore, the administration of exogenous IL-10 in LRP5/6^ΔCD11c mice ameliorates intestinal inflammation and CRC. Thus, it is quite possible that IL-10 signaling could also regulate CAC by modulating the expression of LRP5/6 or β-catenin activation in intestinal APCs.

A delicate balance between regulatory T cells versus pathological effector T cells underlies disease progression in many inflammatory diseases, including IBD and CAC. APCs control both T cell differentiation and expansion through the secretion of various inflammatory and anti-inflammatory cytokines. Intestinal APCs promotes immune tolerance through induction of regulatory T cells while limiting the differentiation of pathological Th1/Th17 cells in the gut (9, 53, 54). Accumulating evidence suggests that IL-17A-producing CD4⁺ T (Th17) cells play an important role in the pathogenesis of IBD and CAC (29). In contrast, regulatory T cells such as Foxp3⁺ Tregs and IL-10 producing Tr1 cells play a pivotal role in controlling inflammatory conditions and maintaining immune tolerance in the intestine (49, 55). IL-6 and IL-1β induce the differentiation of naïve CD4⁺ T cells to Th17 cells. Our studies show that the LRP5/6- β-catenin-IL-10 signaling axis in APCs suppresses Th17 cell differentiation while promoting the Tr1 cell differentiation in the colon. Thus, LRP5/6-deficiency in CD11c⁺ APCs in mice resulted in a marked increase in the frequency of Th17 cells with a concomitant decrease in Tr1 cells in the colon after AOM-DSS treatment. Though not analyzed in the present study, LRP5/6-mediated signals in intestinal APCs might modulate CAC by regulating differentiation and the suppressive function of Foxp3⁺ Tregs.

The gut microbiota plays a pivotal role in the pathogenesis of IBD and inflammation-induced CRC (56–59). Loss of immune tolerance to commensal microflora results in host susceptibility to colonic inflammation and inflammation-associated CRC (8, 60), and genetic modification of the host leads to microbial dysbiosis, resulting in host susceptibility to colonic inflammation and CRC (21, 22, 32–34). Colon cancer incidence is markedly reduced in germ-free mice and antibiotics-treated mice (61, 62). Furthermore, the depletion of gut microbiota ameliorates intestinal inflammation and CAC in mouse models of spontaneous colitis (59, 61, 63, 64). Our results show that LRP5/6^ΔCD11c mice harbor altered microbiota with increased representation of SFB, Prevotellaceae and TM7 groups of bacteria that are associated with enhanced risk of colitis and CAC in mice (21, 32, 35). This condition in LRP5/6^ΔCD11c mice could be improved by treatment with antibiotics or transplantation with fecal microbiota from WT mice. Furthermore, our results show that conditional activation of β-catenin in LRP5/6^ΔCD11c mice resulted in a significant decrease in the representation of SFB, Prevotellaceae and TM7 groups of bacteria with a concomitant decrease in tumor load in the colon (Supplementary Figure 1F). Further studies are warranted to understand the mechanisms by which LRP5/6-β-catenin signaling regulates commensal homeostasis in the intestine. IL-22 deficiency is associated with commensal dysbiosis and aberrant expansion of SFB, Prevotellaceae, and TM7 groups of commensal bacteria. Our previous study has shown that the deletion of LRP5/6 in CD11c⁺ APCs resulted in markedly lower levels of IL-22 whereas the activation of β-catenin in LRP5/6^ΔCD11c mice resulted in a significant increase in IL-22 in the colon. These observations suggest that LRP5/6-β-catenin signaling in APCs can regulate commensal homeostasis and CAC through IL-22. Though not analyzed in the present study, recent studies have shown an important role for the tissue-associated microbiota in the pathogenesis of CAC (65, 66). Since the chemical-induced colitis-associated colon cancer models cannot completely mimic the natural course and type of inflammation in human IBD and IBD-associated CRC, further studies are warranted to confirm the role of this regulatory pathway in other animal models of spontaneous colorectal cancer and may need to be validated in clinical specimens.

In summary, our study reveals a novel role for the LRP5/6-β-catenin-IL-10 signaling axis in intestinal APCs in exerting a protective effect on chronic intestinal inflammation and tumorigenesis by regulating inflammatory mediators and by altering the representation of inflammation-associated intestinal bacteria. Therefore, targeted activation of the LRP5/6-β-catenin-IL-10 pathway in intestinal APCs may feasibly be a promising biological strategy to inhibit inflammatory symptoms associated with human IBD and attenuate the progression of CAC.

Key points.

The canonical Wnt pathway in intestinal APCs protects against CAC. LRP5/6-β-catenin-IL-10 signaling in APCs suppresses chronic intestinal inflammation. This pathway plays an important role in regulating intestinal commensal homeostasis.