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Figure 2 IL-17-Dependent NF-κb Signaling in CECs Triggers Higher Expression of Cxcl1, Cxcl2, and Cxcl5 in the Distal Compared to Proximal Segments of ETBF-Colonized Colon Show full caption (A) Il17a, Il17ra, Il17rc, Cxcl1, Cxcl2, and Cxcl5 mRNA expression in whole tissue sections from proximal (P; C6) to distal (D; C1) colon of WT (sham, blue circles; ETBF, red squares), Il17a−/− (Il17a−/−, ETBF, red triangles), and Il17ra−/− (Il17ra−/−, ETBF, red stars) C57BL/6 (WT) mice. A representative image of colon segments C6 to C1 is shown. (B–D) Ct are normalized with gapdh Ct (ΔCt) and mRNA expression is calculated as 1,000 × 2-ΔCt. Colon segments C6 to C1 are grouped into P (C6, C5), M (mid, C4, C3), and D (C2, C1). (B) CECs were isolated from proximal (P), mid (M), and distal (D) portions of colons obtained from either sham or ETBF-colonized C57BL/6 mice at day 7 post-infection. At top, nuclear fractions derived from the indicated CECs were immunoblotted (IB) for p65 and pStat3. Caspase-3 (Casp3) and PARP1 served as loading controls for cytosolic and nuclear markers, respectively. At bottom, whole-cell lysates (WCL) derived from the indicated CECs were IB for IL-17R, with β-actin as a loading control. Data are representative of two independent experiments. (C) CECs were isolated from the mid (M) and distal (D) portions of colons obtained from either sham or ETBF-colonized IL-17ra+/+ (WT) or IL-17ra−/− C57BL/6 mice at day 7 post-infection. (D) HT29/c1 cells were stimulated with the indicated concentration of TNFα, purified BFT, or IL-17 for 6 hr. Nuclear fractions were derived and IB for p65. Casp3 and PARP1 served as loading controls and cytosolic and nuclear markers, respectively.

The mechanism(s) driving the marked distal localization for colon tumorigenesis is unknown. Thus, to understand the CEC-associated molecular mechanisms involved in IL-17-dependent ETBF distal colon tumorigenesis, we first compared the expression of inflammation-related (Ir) genes in CECs isolated from ETBF-colonized WT versus Il17C57BL/6 mice. Colons were divided into six juxtaposed sections from distal (C1) to proximal end (C6) ( Figure 2 A ). CD45EpCAMCECs were cell sorted from the distal C1+C2 colon sections (DC) of 7-day-colonized WT mice instead of Apcmice in order to exclude interference by early ETBF tumorigenesis and Apc-mutation-augmented Wnt signaling on NF-κB activation. CECs expressed Defb2 (encoding Defensin β2), Cxcl12 (encoding SDF-1), Cxcl1 (encoding KC), Ptgs2 (encoding COX2), and Reg3b at a higher level when isolated from the distal colon of WT mice compared to Il17amice 7 days after ETBF colonization ( Figure S1 ). Our observations that ETBF-induced CEC expression of the IL-17 target genes encoding KC (Cxcl1) and SDF-1 (Cxcl12), critical chemokines for leukocyte trafficking, led us next to test the hypothesis that ETBF colonization induced a distal colon gradient of IL-17 potentially underlying the regional ETBF inflammatory and tumor effects. We used Taqman-based qRT-PCR to measure the expression of Il17a and IL-17-target genes Cxcl1, Cxcl2, and Cxcl5 in each of six colon sections after 7 days of ETBF colonization ( Figure 2 ). Strikingly, while gradients of Il17a, Il17ra, and/or Il17rc mRNA were not detected along the colon axis, there was a pronounced gradient of Cxcl1, Cxcl2, and Cxcl5 mRNA expression that increased from the proximal to distal colon ( Figure 2 A). Chemokine gene expression was abrogated in Il17aand Il17ramice, demonstrating that IL-17 triggered the CXC chemokine production ( Figure 2 A). We confirmed in 7-day-colonized C57BL/6 mice that the chemokine gradient was induced in the CEC fraction by assessing the gene expression selectively in CECs isolated from the successive colon sections from the proximal to distal colon ( Figure S2 A). We further evaluated whether ETBF preferentially colonizes the mucosa of the distal colon of Apcmice to drive the preferential colon tumorigenesis in the distal region. Paradoxically, we found that ETBF mucosal adherence along the colon axis was significantly less in the distal than the proximal colon of Apcmice (p = 0.008) ( Figure S2 B). In contrast, ETBF mucosal adherence was similar in the proximal and distal colon of Il17aand Il17raApcmice ( Figure S2 B). These results demonstrated that increased distal colon Apcmouse ETBF colonization does not drive ETBF colon tumor localization; conversely, reduced ETBF colonization does not underlie the decreased tumorigenesis in Apcmice deficient in IL-17 CEC signaling. Nevertheless, BFT secretion by ETBF is required to generate the gradient of Cxcl1, Cxcl2, and Cxcl5 gene expression, as the gradients of chemokine gene expression are lost in mice colonized with the ETBF(Δbft) strain ( Figure S2 ). Similar to the Il17ra and Il17rc mRNA levels, the IL-17R protein level in CEC was largely comparable along the colon axis upon ETBF colonization ( Figure 2 B), indicating that the elevated C-X-C chemokine gene expression in the distal colon of ETBF-colonized mice most likely does not result from enhanced IL-17R expression. We did not find significant ETBF-mediated changes in expression of either Tnfaip3, which encodes the deubiquitinase A20, a negative regulator of IL-17R (), or Elavl1, which encodes the protein Hur () that is involved in Act1-mediated stabilization of Cxcl mRNA upon IL-17 signaling ( Figure S2 ). These results suggested that increased expression of the inflammatory cytokines along the colon axis is not due to regulatory feedback on epithelial IL-17R signaling or mRNA stabilization. Since C-X-C chemokines are known to be tightly regulated by NF-κB and Stat3 transcription factors, we tested the activation of NF-κB and Stat3 along the colon axis by immunoblotting for the nuclear translocation of p65 and pStat3, respectively, in the CECs derived from ETBF-colonized and sham colons. The nuclear pStat3 levels were comparable along the colon ( Figure 2 B), consistent with our previously reported IHC staining results (). In contrast, the nuclear translocation of p65 was substantially enhanced in the CECs isolated from middle (M; C3+C4) and distal (D; C1+C2) colon sections versus the proximal (P; C5+C6) section of ETBF-colonized colons compared to sham colons ( Figure 2 B), suggesting that ETBF colonization triggers more robust NF-κB signaling in the distal colon. Moreover, p65 nuclear accumulation in the ETBF-colonized M and D colon sections was diminished when Il17ramice were used ( Figure 2 C), demonstrating that the ETBF-induced NF-κB activation is IL-17R dependent. In support of this notion, we observed that BFT and IL-17 each directly induced p65 nuclear translocation in the BFT-sensitive human colon carcinoma cell line HT29/C1 ( Figure 2 D). In contrast, neither BFT nor IL-17 directly induces Stat3 activation in HT29/C1 cells (). Finally, we confirmed the functional relevance of BFT and IL-17-initiated NF-κB activation by determining that both significantly increased IL-8 (human homolog to murine CXCL1/KC) secretion by HT29/C1 cells after a 4-hour treatment. ( Figure S3 ). Together, our findings suggest that ETBF colonization, via BFT, initiates selective distal colon NF-κB activation dependent on IL-17 and the CEC IL-17R that results in a gradient of C-X-C chemokine production. This BFT/IL-17:IL-17R/NF-κB distal colon cascade correlates with the distal localization of ETBF-induced colon tumors ().