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. 2025 Aug;11(31):eads3530.
doi: 10.1126/sciadv.ads3530. Epub 2025 Aug 1.

TH17 cells regulate chemokine expression in epithelial cells through C/EBPβ and dictate host sensitivity to colitis and cancer immunity

Changsheng Xing  1   2 , Tianhao Duan  1   2 , Linfeng Li  2   3 , Lang Chen  2   4 , Pengfei Zhang  5 , Yang Du  1   2 , Siyao Liu  5 , Nihal Annaparthi  1 , Shuo Wang  5 , Chen Qian  1   2 , Helen Y Wang  1   2   6 , Rong-Fu Wang  1   2   6   7
Affiliations

TH17 cells regulate chemokine expression in epithelial cells through C/EBPβ and dictate host sensitivity to colitis and cancer immunity

Changsheng Xing et al. Sci Adv. 2025 Aug.

Abstract

TH17 cells play a critical role in inflammation, cancer development, and antitumor immunity in a context-dependent manner, but detailed mechanisms and their downstream signaling events remain poorly understood. Here, we describe that TH17 cytokines strongly inhibit expression of critical chemokines in epithelial tissues, which leads to blocking infiltration of proinflammatory immune cells into the colon, rendering resistance to DSS-induced colitis and colon cancer. We show that key chemokine expression dictates the sensitivity of WT mice to DSS treatment. Mechanistically, we identified C/EBPβ and STAT3 as negative regulators of key chemokine expression following IL-17 and IL-22 stimulation. Knockout of either C/EBPβ or STAT3 in mouse epithelial cells abolished the protective function of TH17 cytokines and converted resistant to sensitive phenotype. C/EBPβ ablation in cancer cells markedly enhanced chemokine expression, thus sensitizing cancer cells for anti-PD-1 immunotherapy. Overall, our findings have identified a previously unrecognized critical gap between TH17 cytokines, epithelial chemokine expression, and immune cell infiltration through a C/EBPβ-mediated pathway.

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Figures

Fig. 1.
Fig. 1.. Infiltration of immune cells into colon tissues is closely linked to the sensitivity of mice to DSS treatment.
(A and B) IHC staining of CD4, CD8, CD68 (macrophage/monocyte), and MPO (neutrophil) (40×) on colon sections from WT, Tak1ΔM/ΔM, and DKO mice with or without DSS treatment (scale bars, 100 μm), and the analyses of positive cell percentage. Representative data from three independent experiments, means ± SEM. (C and D) Intestinal LPLs were purified from Tak1ΔM/ΔM;Il17-GFP+ mice. Control CD4+ (CD4+/GFP−) and TH17 cells (CD4+/GFP+) were sorted and injected intraperitoneally to WT mice. Mice were then treated with 5% DSS for 5 days. (C) IHC staining of MPO and CD8 (40×) on colon sections collected on day 5 (scale bars, 100 μm). (D) Analyses of positive cell percentage. Combined data from two independent experiments, means ± SEM. (E and F) Tak1ΔM/ΔM and Tak1ΔM/ΔM;Rorc−/− mice were treated with 5% DSS for 5 days. (E) IHC staining of MPO and CD8 (40×) on colon sections collected on day 5 (scale bars, 100 μm). (F) Analyses of positive cell percentage. Representative data from two independent experiments, means ± SEM. Statistical analyses: Student’s unpaired t test (B, D, and F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant; Ctrl, control.
Fig. 2.
Fig. 2.. Chemokine expression is inhibited in the colon tissues of colitis-resistant mice.
(A) WT and Tak1ΔM/ΔM mice were treated with water control or 2.5% DSS for 5 days. On day 8, chemokine expression in colon was measured by qPCR. Representative data from three independent experiments, means ± SEM. (B) IHC staining of CCL5, CXCL5, and CXCL10 (40×) on colon sections collected on day 5 after 5% DSS treatment (scale bars, 100 μm). Representative data from three independent experiments. (C) WT, Tak1ΔM/ΔM, Tak1ΔM/ΔM;Il1r1−/− and Tak1ΔM/ΔM;Il6−/− mice were treated with water control or 2.5% DSS for 5 days. On day 8, chemokine expression in colon was measured by qPCR. Representative data from three independent experiments, means ± SEM. (D) IHC staining of CCL5, CXCL5, and CXCL10 (40×) on colon sections from WT, Tak1ΔM/ΔM, Tak1ΔM/ΔM;Il1r1−/−, and Tak1ΔM/ΔM;Il6−/− mice collected on day 5 after 5% DSS treatment (scale bars, 100 μm). Representative data from three independent experiments. Statistical analyses: Student’s unpaired t test (A and C). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant; Ctrl, control.
Fig. 3.
Fig. 3.. TH17 cells and their cytokines inhibit key chemokine expression in the colon after DSS treatment.
(A to C) IHC staining of CCL5, CXCL5, and CXCL10 (40×) on colon sections from control CD4+ or TH17 cell–injected WT mice (A), control immunoglobulin G or CD4-depleting Ab–injected Tak1ΔM/ΔM mice (B), or Tak1ΔM/ΔM;Rorc−/− mice (C). Tissues were collected on day 5 after 5% DSS treatment (scale bars, 100 μm). Representative data from two or three independent experiments. (D) HCT116 cells were pretreated with BSA control, recombinant IL-17A, or IL-22, followed by overnight 2% DSS treatment. Control buffer and cytokines were added when replacing the medium. Chemokine expression was tested by qPCR on day 4. Representative data from two independent experiments, means ± SEM. (E) WT mice were intraperitoneally injected with BSA buffer as control (n = 8) or recombinant IL-17A (n = 14) and IL-22 (n = 10) and treated with 5% DSS for 5 days. Survival curves were observed. Combined data from two independent experiments. (F) IHC staining of CCL5, CXCL5, CXCL10, CLDN3, MPO, and CD8 (40×) on colon sections collected on day 5 after DSS treatment (scale bars, 50 μm). Representative data from two independent experiments. Statistical analyses: Student’s unpaired t test (D) and Mantel-Cox log-rank test (E). *P < 0.05; **P < 0.01; ***P < 0.001. Ctrl, control.
Fig. 4.
Fig. 4.. Key chemokines are required for immune cell infiltration to promote colitis and colon cancer.
(A and B) Survival curves and body weights (BW) of WT (n = 7), Ccl5−/− (n = 6), and Cxcl10−/− (n = 7) mice with 5% DSS treatment for 5 days. Combined data from two independent experiments. (C and D) Hematoxylin and eosin (H&E) staining and IHC staining of CLDN3, MPO, and CD8 on colon sections collected on day 5 (scale bars, 50 μm), and the analyses of positive immune cell percentage. Representative data from two independent experiments, means ± SEM. (E) Colon tumors in WT (n = 5), Ccl5−/− (n = 5), and Cxcl10−/− (n = 4) mice on day 80 after AOM/DSS treatment (left), and the analysis of tumor numbers (right). Representative data from two independent experiments, means ± SEM. (F) IHC staining of Ki-67 (40×) on colon sections collected on day 80 after AOM/DSS treatment (left; scale bars, 100 μm), and the analysis of positive cell percentage (right). Representative data from two independent experiments, means ± SEM. Statistical analyses: Mantel-Cox log-rank test (A), ANOVA and student’s unpaired t test (B), Student’s unpaired t test (D; E, right; and F, right). *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 5.
Fig. 5.. TH17 cytokines regulate chemokine expression through C/EBPβ and STAT3 signaling.
(A) Survival curves and body weight (BW) changes of WT (n = 15), Il17rc−/− (n = 12), Tak1ΔM/ΔM (n = 8), and Tak1ΔM/ΔM;Il17rc−/− (n = 15) mice with 5% DSS treatment for 5 days. Combined data from three independent experiments, means ± SEM. BW, (B) HCT116 cells (WT, C/EBPβ KO, and STAT3 KO) were pretreated with BSA control, recombinant IL-17A, or IL-22, followed by overnight 2% DSS treatment. Control buffer and cytokines were added when replacing the medium. Chemokine expression was tested by qPCR on day 4. Representative data from two independent experiments, means ± SEM. (C) WT HCT116 cells were treated with recombinant IL-17A or IL-22, and cell lysates were harvested at different time points. Western blotting was performed to determine the expression and activation of C/EBPβ and STAT3. Representative data from three independent experiments. (D) HCT116 cells (WT, C/EBPβ KO, and STAT3 KO) were treated with recombinant IL-22, and cell lysates were harvested at different time points. Western blotting was performed to determine the expression and activation of C/EBPβ and STAT3 in the KO cells. Representative data from three independent experiments. (E) A diagram showing the putative downstream signaling and key factors for IL-17A and IL-22 to regulate the chemokine expression in colon epithelial cells. Statistical analyses: ANOVA and Student’s unpaired t test (A, left), Mantel-Cox log-rank test (A, right), and Student’s unpaired t test (B). *P < 0.05; ****P < 0.0001. Ctrl, control; h, hour.
Fig. 6.
Fig. 6.. TH17 cytokines fail to protect C/EBPβ or STAT3 KO mice against colitis.
(A) WT and CebpbF/F;Vil-Cre+/+ mice were intraperitoneally injected with BSA buffer as control (WT, n = 12; KO, n = 12) or recombinant IL-17A (WT, n = 12; KO, n = 13) and treated with 5% DSS for 5 days. Survival curves were observed. Combined data from three independent experiments. (B) WT and CebpbF/F;Vil-Cre+/+ mice were intraperitoneally injected with BSA buffer as control or recombinant IL-22 and treated with 5% DSS for 5 days (n = 10 for all groups). Survival curves were observed. Combined data from two independent experiments. (C and D) IHC staining of CCL5 and CXCL10 (10×) on colon sections collected on day 7 after 2.5% DSS treatment for 5 days (scale bars, 100 μm), with the quantification and statistical analyses of chemokine levels by the H-score of the IHC images. Representative data from two independent experiments, means ± SEM. (E and F) WT and Stat3F/F;Vil-Cre+/+ mice were intraperitoneally injected with BSA buffer as control (WT, n = 8; KO, n = 12) or recombinant IL-17A (WT, n = 8; KO, n = 11) or IL-22 (WT, n = 7; KO, n = 12) and treated with 5% DSS for 5 days. Survival curves were observed. Combined data from two independent experiments. Statistical analyses: Mantel-Cox log-rank test (A, B, E, and F) and Student’s unpaired t test (D). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant.
Fig. 7.
Fig. 7.. C/EBPβ KO triggers chemokine expression in cancer cells and promotes the efficacy of cancer immunotherapy.
(A and B) C/EBPβ was knocked out in MDA-MB-231 and EO771 cells. The chemokine expression on RNA levels was detected in WT and KO cells by qPCR. Representative data from three independent experiments, means ± SD. (C) MTT assay for the in vitro growth curves of WT and C/EBPβ KO EO771 cells [read daily at optical density (OD) at 600 nm]. Representative data from two independent experiments, means ± SEM. (D) WT and C/EBPβ KO EO771 cells were injected into the mammary fat pad of NSG mice (n = 3). Tumor sizes were observed every 4 to 5 days. Representative data from two independent experiments, means ± SD. (E) WT and C/EBPβ KO EO771 cells were injected into the mammary fat pad of WT B6 mice (n = 5). Isotype control or PD-1 Ab was intraperitoneally injected twice a week since day 5. Tumor sizes were observed twice a week. The four study groups were conducted in parallel. Individual growth curves for each mouse were shown in fig. S7I. Representative data from two independent experiments, means ± SD. (F and G) IHC staining of CCL5, CXCL10, and tumor-infiltrated T cells (CD4 and CD8) on WT and C/EBPβ KO EO771 tumor sections from WT B6 mice with or without PD-1 Ab treatment (40×; scale bars, 100 μm). Representative data from two independent experiments. (H) Analyses of tumor-infiltrated T cells (positive cell number per 10× field) in WT and C/EBPβ KO EO771 tumors with or without PD-1 Ab treatment. Representative data from two independent experiments, means ± SEM. Statistical analyses: Student’s unpaired t test (A, B, and H) and ANOVA and Student’s unpaired t test (C to E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant.
Fig. 8.
Fig. 8.. C/EBPβ expression is negatively associated with CCL5 expression, CD8+ T cell infiltration, and patient survival across multiple cancer types.
(A) C/EBPβ expression shows a negative correlation with CCL5 across multiple human tissues. (B) C/EBPβ expression negatively correlates with CD8+ T cell infiltration across multiple cancer types. (C) High C/EBPβ expression is associated with poorer OS and disease-free survival across multiple cancer types. (D) High C/EBPβ expression is a risk factor for OS and disease-free survival in pan-cancer analysis. (E) C/EBPβ expression is negatively associated with CD8+ T cell infiltration and patient survival in patients with metastasis urothelial carcinoma receiving anti–PD-L1 immunotherapy. LGG, lower-grade glioma; GBM, glioblastoma multiforme; KIRP, kidney renal papillary cell carcinoma; THYM, thymoma; UVM, uveal melanoma; mUC, metastatic urothelial carcinoma; HR, hazard ratio.
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References

    1. Dong C., TH17 cells in development: An updated view of their molecular identity and genetic programming. Nat. Rev. Immunol. 8, 337–348 (2008). - PubMed
    1. Esplugues E., Huber S., Gagliani N., Hauser A. E., Town T., Wan Y. Y., O’Connor W., Rongvaux A., Van Rooijen N., Haberman A. M., Iwakura Y., Kuchroo V. K., Kolls J. K., Bluestone J. A., Herold K. C., Flavell R. A., Control of TH17 cells occurs in the small intestine. Nature 475, 514–518 (2011). - PMC - PubMed
    1. Xing C., Du Y., Duan T., Nim K., Chu J., Wang H. Y., Wang R. F., Interaction between microbiota and immunity and its implication in colorectal cancer. Front. Immunol. 13, 963819 (2022). - PMC - PubMed
    1. Ghoreschi K., Laurence A., Yang X.-P., Tato C. M., McGeachy M. J., Konkel J. E., Ramos H. L., Wei L., Davidson T. S., Bouladoux N., Grainger J. R., Chen Q., Kanno Y., Watford W. T., Sun H.-W., Eberl G., Shevach E. M., Belkaid Y., Cua D. J., Chen W., O’Shea J. J., Generation of pathogenic TH17 cells in the absence of TGF-β signalling. Nature 467, 967–971 (2010). - PMC - PubMed
    1. Lee J.-Y., Hall J. A., Kroehling L., Wu L., Najar T., Nguyen H. H., Lin W.-Y., Yeung S. T., Silva H. M., Li D., Hine A., Loke P., Hudesman D., Martin J. C., Kenigsberg E., Merad M., Khanna K. M., Littman D. R., Serum amyloid a proteins induce pathogenic Th17 cells and promote inflammatory disease. Cell 180, 79–91.e16 (2020). - PMC - PubMed

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