br ure C IL A GFP
ure 1C). IL-17A-GFP reporter expression was confined to various lymphoid (Figure 1D) but not epithelial, stromal, or myeloid GastrinI (Figure S1E). IL-1Ra treatment significantly reduced IL-17A-GFP reporter expression by the lymphoid cells (Figures 1D and 1E). Therefore IL-1 signaling is required for IL-17A and TEI cyto-kine expression and even short-term inhibition of IL-1 resulted in reduction of intratumoral IL-17A and TEI in CRC.
Genetic Inactivation of IL-1 Signaling Reduces IL-17A TEI But Has Only a Limited Effect on CRC Tumorigenesis Next, we ablated IL-1R1 expression by crossing CPC-APC mice to Il1r1 / background. Il1r1 / CPC-APC mice and their co-housed littermate Il1r1+/ CPC-APC controls were allowed to spontaneously develop CRC. Genetic ‘‘whole-body’’ ablation of IL-1R1 resulted in significant drop of Il17a, Rorc, and Il22
mRNAs (Figure 1F). Flow cytometry analysis of intratumoral LPL and IEL fractions from Il1r1 / CPC-APC-Il17aGFP+/ re-
porter mice revealed that IL-17A expression was reduced in the absence of IL-1 signaling (Figure 1G).
We and others previously reported that inactivation of IL-17A, RORgt, or IL-22 results in a prominent reduction in CRC (Blatner et al., 2012; Grivennikov et al., 2012; Huber et al., 2012; Kirch-berger et al., 2013; Wu et al., 2009). Thus, we expected that the significant decrease in IL-17A and IL-22 cytokine expression in Il1r1 / CPC-APC mice would culminate in reduced CRC. However, tumor multiplicity was only modestly reduced and tu-mor size was not affected by IL-1R1 deficiency, leaving tumor load essentially unchanged (Figures 1H and 1I).
As hematopoietic cells are the main source of IL-17A and IL-22 (Figures 1D and S1E), hypothetically the ability of IL-1 to regulate TEI should be more evident within the hematopoietic compartment. Therefore, we lethally irradiated 6-week-old CPC-APC mice and reconstituted them with bone marrow (BM) from Il1r1 / and Il1r1+/+ control mice. Similarly, to what we observed in the whole-body Il1r1 deficiency, lack of IL-1R1 on hematopoietic cells led to a significant decrease in Il17a, Rorc, and Il22 mRNA expression and protein release (Figures S2A and S2B and data not shown) in CRC tumors. Intracellular cytokine staining confirmed the reduction in pro-tumorigenic IL-17A (Figures S2C and S2D); however, hemato-poietic IL-1R1 deficiency did not affect CRC tumor develop-ment (Figures 1J and S2E).
To confirm these unexpected results, we used an inducible CRC model, CDX2-ERCre (CDX2ERT)-Apcf/f, where bi-allelic deletion of the Apc gene and rapid development of CRC tumors are driven by injection of tamoxifen (Feng et al., 2013; Wang et al., 2014). Recipient CDX2ERT-Apcf/f mice were lethally irradi-ated, injected with Il1r1 / or Il1r1+/ BM, allowed to recover for 2 months during which their hematopoietic system was replaced (over 90% chimerism), and then injected with tamoxifen. No reduction in CRC was found in mice with hematopoietic
(F–I) Analysis of tumor development in global Il1r1 (Il1r) / and Il1r+/ 6-month-old tumor bearing CPC-APC mice. (F) qRT-PCR analysis of TEI cytokines and markers in tumors, p*<0.05; N R 10. (G) LPL and IEL fractions of tumors from CPC-APC-Il17aGFP mice with Il1r / and Il1r+/ alleles were analyzed by FACS. Representative of three independent experiments, total N R 10. (H) Tumor multiplicity, load and size in Il1r+/ and Il1r / CPC-APC mice, N R 17. (I) H&E stained paraffin sections of colons from CPC-APC- Il1r / and Il1r+/ mice, tumors are pointed, representative of N R 17.
(J) Analysis of CRC in 6- to 8-week-old CPC-APC mice reconstituted with Il1r1 / or Il1r1 / bone marrow (BM) and allowed to develop CRC for 4 months. H&E stained paraffin sections of colons from Il1r1 / or Il1r1 / BM chimera CPC-APC mice, representative of N R 10. Data are mean ± SEM. Representative of 3 independent experiments. See also Figures S1 and S2.
deficiency of IL-1 signaling (Figure S2F) whereas TEI factors Il17a, Rorc and Il22 expression were reduced (Figure S2G), as was the production of the IL-17A protein (Figure S2H).
Together, the results from two models of CRC revealed that IL-1 signaling in hematopoietic cells controlled key components of TEI, but global or hematopoietic cell specific ‘‘blanket’’ inacti-vation of IL-1R did not significantly alter CRC tumorigenesis.
IL-1R1 Expressed by Epithelial Cells Is Required for CRC Tumorigenesis, NF-kB Activation and Cell Proliferation Independent of TEI
We next hypothesized that IL-1R signaling in particular cells may account for the pro-tumorigenic inflammatory responses, but IL-1 activity in other, distinct cell types may have contrast-ing effects on CRC, ultimately neutralizing each other. We abla-