IL-17 inhibits CXCL9/10-mediated recruitment of CD8+ cytotoxic T cells and regulatory T cells to colorectal tumors
The IL-17 family cytokines are potent drivers of colorectal cancer (CRC) development. We and others have shown that IL-17 mainly signals to tumor cells to promote CRC, but the underlying mechanism remains unclear. IL-17 also dampens Th1-armed anti-tumor immunity, in part by attracting myeloid cells to tumor. Whether IL-17 controls the activity of adaptive immune cells in a more direct manner, however, is unknown.
Using mouse models of sporadic or inducible colorectal cancers, we ablated IL-17RA in the whole body or specifically in colorectal tumor cells. We also performed adoptive bone marrow reconstitution to knockout CXCR3 in hematopoietic cells. Histological and immunological experimental methods were used to reveal the link among IL-17, chemokine production, and CRC development.
Loss of IL-17 signaling in mouse CRC resulted in marked increase in the recruitment of CD8+ cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs), starting from early stage CRC lesions. This is accompanied by the increased expression of anti-inflammatory cytokines IL-10 and TGF-β. IL-17 signaling also inhibits the production of T cell attracting chemokines CXCL9 and CXCL10 by tumor cells. Conversely, the inability of hematopoietic cells to respond to CXCL9/10 resulted in decreased tumor infiltration by CTLs and Tregs, decreased levels of IL-10 and TGF-β, and increased numbers of tumor lesions. Blockade of IL-17 signaling resulted in increased expression of immune checkpoint markers. On the other hand, treatment of mouse CRC with anti-CTLA-4 antibody led to increased expression of pro-tumor IL-17.
IL-17 signals to colorectal tumor cells and inhibits their production of CXCL9/10 chemokines. By doing so, IL-17 inhibits the infiltration of CD8+ CTLs and Tregs to CRC, thus promoting CRC development. Cancer immunotherapy may be benefited by the use of anti-IL-17 agents as adjuvant therapies, which serve to block both IL-17-mediated tumor promotion and T cell exclusion.
KeywordsInterleukin-17 CXCL9 CXCL10 Regulatory T cell And colorectal cancer
Cytotoxic T lymphocytes
Cytotoxic T-lymphocyte-associated protein 4
C-X-C Motif Chemokine Ligand 10
C-X-C Motif Chemokine Ligand 11
C-X-C Motif Chemokine Ligand 9
Forkhead box P3
Interleukin-17 receptor A
Myeloid-derived suppressor cells
Mesenteric lymph node
Programmed cell death-1
Programmed death-ligand 1
Programmed death-ligand 2
Type 1 T helper cells
T helper 17 cells
Tumor Necrosis Factor-α
Regulatory T cells
The IL-17 family cytokines promote tumor development in multiple organs. Using mouse models of sporadic and inducible colorectal cancers (CRC), we and others have shown that IL-17 signals to transformed colorectal epithelial cells to drive tumor development [1, 2]. This IL-17-tumor cell signaling is necessary for the survival and outgrowth of early CRC lesions, and ablation of IL-17RA, the common receptor of IL-17 family cytokines, resulted in marked reduction in tumor numbers in mouse colon [1, 3]. IL-17 also activates production of chemokines, such as CXCL1 and CXCL2 that attract myeloid cells to sites of inflammation [4, 5]. Colorectal tumor cells exhibit defective epithelial barrier function. As a result, gut commensal bacteria and their degradative products invade tumor stroma, engage tumor-infiltrating myeloid cells, and activate the production of IL-23 and its downstream cytokine IL-17 . Thus, the IL-17-myeloid cell pathway forms a self-enhancing loop that results in chronic tumor-associated inflammation. IL-17 is also known to block the effect of cytotoxic and anti-angiogenic chemotherapies against colorectal cancers [1, 6]. This correlates with the observation that loss of IL-17 signaling resulted in enhanced recruitment of CD8+ cytotoxic T lymphocytes (CTL) [1, 3, 7]. To date, it is unclear if IL-17 regulates the recruitment of adaptive immune cells to the site of CRC, and if so, what the underlying mechanism is.
The chemokine CXCL9 signals through CXCR3 and mediates migration of T cells to sites of inflammation . In mouse models of transplanted tumors, CXCR3 signaling promotes CD8+ T cell infiltration that controls tumor growth [9, 10, 11]. The role of CXCL9 and its family members in sporadic CRC is unknown. Chemokine signaling through CXCR3 also mediates the recruitment of CD4+ T cells. Among them, Th17 cells promote CRC development by secreting IL-17 and IL-22 [1, 3, 12, 13], while Th1 cells have long known to inhibit tumor development . Perhaps most intriguingly, regulatory T cells (Tregs) inhibit CRC development by dampening tumor-promoting inflammation . Ablation of Treg-related cytokines IL-10 and TGF-β resulted in increased intestinal tumor burden [16, 17]. A high “Treg signature” in human CRC also indicates better prognosis . The function of the CXCR3 cascade in CRC thus depends on the immune cell types that they recruit. The unique Treg-CRC relationship also complicates the use of Treg-targeting strategies, such as anti-CTLA4 for CRC treatment .
Here we show that IL-17 signals to transformed epithelial (tumor) cells to suppress the expression of CXCL9 and CXCL10 chemokines. Signaling of CXCL9/10 through CXCR3 is required for the recruitment of CD8+ CTLs and Tregs, but not Th1 or Th17 cells, to colorectal tumors. CXCR3 signaling to hematopoietic cells is required for the expression of IL-10 and TGF-β in tumors, and for the control of CRC development. Overall, IL-17 promotes CRC development by suppressing cells responsible for anti-cancer immunity, and fostering tumor-promoting gut inflammation. This novel mechanism pinpoints gut inflammation during cancer as a barrier for tumor control through the diverting action of IL-17 on the adaptive immune system.
Il17ra−/− mice were from Amgen . C57BL/6, ApcF/F , Cd8a−/− , B2m−/− , Cdx2-Cre , Cdx2-Cre-ERT2 , and Cxcr3−/−  mice were obtained from the Jackson Laboratory. Il17raF/F mice  were obtained from Dr. Michael Karin’s laboratory at University of California, San Diego.
To generate the mouse model of sporadic CRC, Cdx2-Cre and ApcF/F mice were crossed to generate Cdx2-Cre+/ ApcF/WT mice. These mice were sacrificed around 5 months of age for tumor analyses. Mouse colon was dissected, and colorectal tumors were excised with a scissor. Tumor-adjacent colon tissues were harvested and analyzed as “normal colon tissue” for comparison.
For tamoxifen-inducible tumorigenesis, Cdx2-Cre-ERT2+/ApcF/F mice were given 75 mg/kg body weight tamoxifen (Sigma, dissolved in 5% ethanol, 95% corn oil) i.p. on a daily basis for 3 consecutive days. Mice were sacrificed 4 to 5 weeks after the last dose of tamoxifen for tumor statistics and analysis. Mouse colon was dissected, and visible colorectal tumors (typically 1–2 mm in diameter) were excised with a scissor.
All mice were maintained in filter-topped cages on autoclaved food and water at UConn Health. All experiments used co-housed, gender matched littermates to ensure consistency of common microflora. Both male and female mice were used for all experiments.
Bone marrow transplantation
Six- to eight-week-old recipient mice were irradiated twice during 1 day to achieve a lethal dose (2 × 600 rad) and intravenously injected with single-cell suspension of 107 donor bone marrow cells. Recipients were co-housed littermates, which were transplanted with both gene-deficient and wild-type bone marrow for comparison. After transplantation the recipients were placed on sulphamethoxazole and trimethoprime in drinking water for 2 weeks, followed by regular water. Mice were sacrificed and analyzed for tumor development 4–5 months after transplantation.
Antibody treatment in mice
For sporadic CRC model (Cdx2-Cre+/ApcF/WT mice), IL-17A, CTLA-4, and PD-1 neutralizing antibodies or isotype control antibodies (Bio X Cell) were i.p. injected at a dose of 100 μg per mouse every 3 days until sacrifice.
For the tamoxifen inducible model of tumorigenesis, antibodies (100 μg per mouse, every 3 days) were injected 1 day after the dose of tamoxifen until sacrifice.
Immunofluorescent staining and ELISA
Immunofluorescent staining was performed on cryosectioned colorectal tumors with antibody against CD8α (Thermo Fisher), followed with Alexa-488-conjugated secondary antibody (Life Technology). Sections were further stained with DAPI and imaged under a confocal microscopy. For ELISA analysis of CXCL9 (Biolegend) and CXCL10 (R&D Systems), colonic tumors were cultured in opti-MEM containing 1% Antibiotic-Antimycotic (Life Technologies) for 24 h. Tissue culture supernatant was analyzed by ELISA. Concentrations of chemokines were normalized to the weight of tumors in each well.
Cell culture and cytokine treatment
Primary CRC tumor sphere culture was previously described . Briefly, tumor cells were isolated from colorectal tumors of Cdx2-Cre-ERT2+/ApcF/F mice 4 weeks after tamoxifen injection. Cells were plated in Matrigel (BD Bioscience) and maintained in DMEM/F12 media (Life Technologies) containing B27 and N2 supplements (Life Technologies), 1.25 mM N-acetyl L-cysteine (Sigma), 100 ng/ml noggin (Peprotech), 50 ng/ml mEGF (Biosource), and 10% Rspo1-Fc-conditioned medium. To study IL-17 signaling in vitro, tumor spheres were replenished with serum and growth factor free medium for 24 h, and treated with 100 ng/ml recombinant human IL-17A, C or F for another 24 h.
Flow cytometry and cell sorting
Colorectal tumors were minced with scissors and digested with 1 mg/kg collagenase IV (Sigma Aldrich) for 20 min. Cells were filtered with 70-μm cell sieve, and stained with Live/Dead fixable exclusion dye (Tonbo Bioscience), followed by fluorochrome-conjugated antibodies in PBS with 2% fetal bovine serum (FBS) and 1 mM EDTA. Anti-CD3 (Cat # 100206), anti-CD4 (Cat # 100536), anti-CD45 (Cat # 103138), anti-CD19 (Cat # 152408), anti-CD11b (Cat # 101224), anti-F4/80 (Cat # 123108), anti-Gr-1 (Cat # 108428), anti-Ly6C (Cat # 128016), anti-Ly6G (Cat # 127641), anti-PD-1 (Cat # 135216), anti-Ep-CAM (Cat # 118216), anti-IL-10 (Cat # 505008), anti-IL-17A (Cat # 506904), anti-IFNγ (Cat # 505806), and anti-TNF-α (Cat # 506306) antibodies were from Biolegend. Anti-CD44 (Cat # 12–0441-82), anti-CD62L (Cat # 47–0629-42), anti-Foxp3 (Cat # 11–5773-82), and anti-Ki-67 (Cat # 11–5698-82) antibodies were from eBioscience. Anti-CD25 (Cat # 20–0251) and anti-CD3 (Cat # 20–0032) antibodies were from Tonbo Biosciences. Anti-CD8α antibody (Cat # 558106) was from BD Bioscience. For intracellular cytokine staining, cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 4 h, followed with fixation and staining with Foxp3/transcription factor staining buffer set (eBioscience). Flow cytometry analyses were performed on a BD LSRII flow cytometer. Cell sorting was performed on a BD FACS ARIA II high speed cell sorter. Data was analyzed with FlowJo software.
Total RNA was extracted with RNeasy Plus kit (Qiagen) and reverse transcribed using an IScript kit (Biorad). Q-RT-PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Biorad) on a Biorad CFX96 machine. Expression data were normalized to RPL32 mRNA levels. The data were calculated as 2(Ct(RPL32–gene of interest)) to compare experimental groups to controls, and are presented in arbitrary units. Primer sequences are listed in Additional file 1: Table S1. Whenever possible, primers were intron-spanning, such that amplification is feasible on complementary DNA.
Data are presented as averages +/− S.E.M. and were analyzed by the Students’ t test. P values less than 0.05 were considered significant.
IL-17 inhibits the infiltration of tumor-associated CD4+ T cells and the production of IL-10 and TGF-β
IL-17 inhibits the infiltration of CTLs in early stage CRC
IL-17 suppresses the expression of CXCL9, 10, and 11
IL-17 signals to transformed colonic epithelial cells to suppress the production of CXCL9, 10, and 11
CXCR3 signaling attracts CTLs and Treg cells to inhibit CRC development
IL-17 blockade upregulates the expression of immune checkpoint markers
IL-17 is known to promote neutrophil infiltration by activating the production of their attracting chemokines. In mouse model of CRC, ablation of IL-17 resulted in reduced levels of CXCL1 and CXCL2, which correlates with decreased numbers of tumor infiltrating myeloid cells [1, 4, 5, 44]. We also showed that these tumor-infiltrating myeloid cells respond to bacterial products that pass through defective surface barrier due to tumorigenesis, and produce IL-23 . IL-23 in turn promotes the production of IL-17 by T cells and innate lymphoid cells . In this way, IL-17 and tumor-infiltrating myeloid cells form an auto-enhancing loop to promote tumor-associated inflammation. Combined with our new finding that IL-17 inhibits T cell infiltration through the downregulation of CXCL9/10, it is now clear that IL-17 skews tumor immune environment towards an innate cell-dominant, tumor-promoting inflammation. In different settings, IL-17 has also been shown to promote the infiltration and develop of myeloid-derived suppressor cells (MDSC), which inhibit the activity of CTLs and thus promotes tumor development [31, 45]. The contribution of MDSC to T cell inactivation in sporadic CRC is unknown, but may represent an alternative pathway by which IL-17 indirectly inhibits CD8+ CTL activity. It is therefore possible that tumor-infiltrating myeloid cells play dual roles in CRC: 1) these cells respond to commensal bacteria and promote tumor-associated inflammation (such as the production of IL-23 and IL-17), which subsequently leads to reduced CXCL9/10 production and T cell attraction; 2) these cells may serve as suppressors of anti-tumor immunity. Additional research is required to dissect the inflammation-promoting v.s. T cell inactivating roles of myeloid cells in tumors. For example, one may employ myeloid-specific ablation of effector molecules (such as arginase ) to examine the effect of MDSC in sporadic CRC.
Chemokine signaling through CXCR3 has been shown to inhibit tumor growth in several transplantable tumor models [10, 11, 46]. This anti-tumor function of CXCR3 and its cognate ligands were attributed to the recruitment of CD8+ CTLs into tumors. Consistently, in human CRC, a high CXCL10 level correlates with CD8+ T cell infiltration . In our study, we also observed reduced CTL number in colorectal tumors upon ablation of CXCR3 in hematopoietic cells. In contrast, CXCR3 signaling was dispensable for Th1 and Th17 cell infiltration. Intriguingly, we found that CXCR3 functions to recruit Treg cells to CRC tumors, and CXCR3 loss results in marked decreases in the levels of IL-10 and TGF-β. Given the anti-tumor roles of IL-10 and TGF-β in early stage colon cancer development, we concluded that CXCR3 inhibits early stage colorectal tumorigenesis by attracting both CTLs and Treg cells. This conclusion was supported by the observation that loss of CXCR3 in blood cells resulted in increased tumor incidence in mouse colon, but no changes in tumor size. It is also in agreement with the known role of IL-17 in promoting early stage CRC development .
In this study, we report a novel mechanism by which IL-17 inhibits the recruitment of CD8+ CTLs and Treg cells by downregulating the production of CXCL9/10 chemokines. Such knowledge will demonstrate the feasibility of interfering with IL-17-Treg interaction for CRC prevention and immunotherapy. For instance, blockade of IL-17 signaling may be useful for the prevention of CRC in genetically susceptible populations, such as FAP (familial adenomatous polyposis) patients that harbor germline mutations in the Apc tumor suppressor gene. Given the known role of IL-17 in promoting early stage CRC development , and its negative impact in the inhibition of CD8+ CTLs and Tregs, blocking IL-17 may suppress tumor-promoting inflammation, activate tumor immunosurveillance, and reduce the rate of tumorigenesis in this genetically predisposed population.
Immunotherapy against human CRC has shown limited success, as it is effective only in microsatellite instable (MSI) cases [37, 38]. For the 85% of microsatellite stable CRC, checkpoint inhibition largely does not work. Our mouse models of CRC are based on allelic inactivation of the Apc tumor suppressor gene [24, 25, 27], and do not carry MSI lesions. Yet, in both sporadic and early stage CRC models, ablation of IL-17 signaling resulted in increased recruitment of anti-tumor CD8+ CTLs via upregulation of CXCL9 family chemokines, without the requirement of MSI. It is possible that in human CRC that are microsatellite stable, blockade of IL-17 can also result in increased production of CXCL9 family chemokines and enhanced infiltration of CD8+ T cells to tumors, which is a desirable trait for cancer immunotherapy. Upregulation of IL-17 in mouse models of CRC stems from the loss of surface barrier function in the process of epithelium transformation. In this regard, it remains to be tested if IL-17 plays a similar role in limiting T cell infiltration in MSI tumors.
While IL-17 blockade may also increase the number of Tregs in human CRC, blockade of immune checkpoints should be sufficient to neutralize their inhibition on anti-cancer immunity. In this regard, neutralizing antibodies against IL-17A and IL-17RA, which have been tested safe and effective for the treatment of autoimmunity in humans , may be tested as adjuvant therapies that accompany current cancer immunotherapies. IL-17 production is restricted to the CRC tumor site, and its blockade should result in selective upregulation of CXCL9 family chemokines in tumors. In this perspective, IL-17 blockade should be effective in attracting T cells to tumors, and poses less risk of systemic immune activation.
Our data show a novel role of IL-17 in inhibiting the infiltration of CD8+ CTLs and Tregs to CRC. This is mediated by IL-17’s signaling to colorectal tumor cells, which leads to the reduced production of CXCL9/10 chemokines. CXCL9/10 chemokines, signaling through their cognate receptor CXCR3, recruit CD8+ CTLs and Tregs to CRC, but are dispensable for the recruitment or activation of other T cells and myeloid cells. By excluding Tregs and CTLs from CRC, IL-17 fosters the dominance of tumor-promoting inflammation. To this end, cancer immunotherapy may be benefited by the use of anti-IL-17 agents, as blockade of IL-17 reduces the rate of tumor growth and increases the infiltration of CTLs that are vital for effective cancer treatment.
We thank Dr. Michael Karin at University of California, San Diego for the Il17raF/F mice, and Ms. Li Zhu at the UConn Health Flow Cytometry Core Facility for technical assistance.
JC and KW conceived the project. JC, XY, EP, ML, EJ, and KW performed the experiments and analyzed the data. JW, AA and AV provided conceptual advice. JC and KW wrote the manuscript, with all authors contributing to the writing, editing, and providing key advice. All authors read and approved the final manuscript.
All work was conducted at UConn Health and supported by institutional startup funds to K.W. Salary support for J.C. was provided by UConn Health; Salary support for X.Y. was provided by UConn Health and Shenzhen PKU-HKUST Medical Center; Salary support for E.P. was provided by NIH T90 training grant (T90-DE022526) under the support of Dr. Mina Mina, and UConn Health; Salary support for M.L. was provided by UConn Health, Beijing University of Chinese Medicine, and the China Scholarship Council. J.W. was supported by the National Natural Science Foundation of China (81571043), Natural Science Foundation of Guangdong Province (2016A030312016), and Shenzhen Basic Research Grants (JCYJ20170306161450254 and JCYJ20170815153617033).
All animal experiments were approved by the IACUC of UConn Health, and performed in accordance with the university and NIH guidelines and regulations.
Consent for publication
The work does not contain data from any individual person, and therefore consent for publication is not applicable.
The authors declare that they have no competing interests.
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