The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-myc in Apc Min/+ mice

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1 Ono et al. Journal of Biomedical Science (7) :79 DOI./s z RESEARCH The TLR/TICAM- signal constitutively controls spontaneous polyposis through suppression of in mice Junya Ono,, Hiroaki Shime,, Hiromi Takaki,, Ken Takashima,, Kenji Funami,, Sumito Yoshida,, Yohei Takeda, Misako Matsumoto, Masanori Kasahara, and Tsukasa Seya Open Access Abstract Background: Intestinal tumorigenesis is promoted by myeloid differentiation primary response gene (MyD) activation in response to the components of microbiota in mice. Microbiota also contains double-stranded RNA (dsrna), a ligand for TLR, which activates the toll-like receptor adaptor molecule (TICAM-, also known as TRIF) pathway. Methods: We established Ticam / mice and their survival was compared to survival of Myd / and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis. Results: We demonstrate that TICAM- is essential for suppression of polyp formation in mice. TICAM- knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD in the background. Polyps were more frequently formed in the distal intestine of Ticam / mice than in mice. Infiltration of immunecellssuchascdb + and CDα + cells into the polyps was detected histologically. CDb and CDα mrnas were increased in polyps of Ticam / mice compared to mice. Gene expression of inducible nitric oxide synthase (inos), interferon (IFN)-γ, CXCL9 and IL-p was increased in polyps of Ticam / mice. mrna and protein expression of, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of Apc Min/ + Ticam / mice. A Lactobacillus strain producing dsrna was detected in feces of mice. Conclusion: These results imply that the TLR/TICAM- pathway inhibits polyposis through suppression of expression andsupportslongsurvivalin mice. Keywords: TLR, TICAM- (TRIF),, Intestinal polyposis Background Tumor progression is closely linked to inflammation []. The intestine contains microorganisms that influence the incidence of inflammation-associated cancer via toll-like receptors (TLRs) expressed in gut mucosal cells. TLR/ and Nod-like receptor (NLR) are expressed in mucosa and detect intestinal bacterial patterns []. Nod signal may ameliorate inflammation-induced polyposis through nuclear factor (NF)-κB and activator protein (AP). Nod is reportedly important for maintaining the Correspondence: seya-tu@pop.med.hokudai.ac.jp Equal contributors Department of Vaccine Immunology, Hokkaido University Graduate School of Medicine, Kita, Nishi 7, Kita-ku, Sapporo -, Japan Full list of author information is available at the end of the article integrity of the intestinal epithelium to protect it against injury, inflammation and subsequent carcinogenesis []. TLRs except for TLR activate the adaptor MyD, which serves as a key factor for promotion of carcinogenesis and development in colon cancer [, ]. TLR/ respond to bacteria and modulate NF-κB activation during the inflammatory response in the TLR/MyD pathways []. Other receptors which activate MyD also participate in inflammation and tumorigenesis in intestinal epithelial cells [, 7], suggesting a crucial role for MyD in homeostasis of intestine. Epithelial cells and microflora work together cooperatively to maintain mucosal homeostasis via MyD signaling. Several reports have suggested that lactobacillus produces partial or structural double-stranded (ds) RNA, The Author(s). 7 Open Access This article is distributed under the terms of the Creative Commons Attribution. International License ( which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

2 Ono et al. Journal of Biomedical Science (7) :79 Page of 9 which can activate TLR in the intestine []. TLR is expressed in mucosal epithelial cells and myeloid cells, which may sample the bacterial by-products of dsrnas via phagocytosis [9]. TLR couples with the adaptor TICAM- (TRIF) to activate transcription factors, IRF and AP. If this is the case, both MyD and TICAM- pathways participate in polyposis under the presence of complex innate stimulation. To test the relationship between TLR/TICAM- and intestinal polyposis, we employed the mouse model []. We found TICAM- is important for suppression of tumorigenesis and homeostasis of innate sensing of bacteria. We herein addressed the mechanism by which TLR/TICAM- participates in polyp formation in mice. Methods Mice mice on a C7BL/ background were purchased from the Jackson Laboratory. Myd / C7BL/ mice were provided from Dr. S Akira (Osaka University). Ticam / C7BL/ mice were established in our laboratory. Apc- Min/+ mice were crossed to Myd / or Ticam / mice to generate Apc Min+/ Myd +/, Apc Min+/ Myd /, Apc Min +/ Ticam +/ and Ticam / littermates. Mice were bred and maintained under specific pathogen free conditions. No abnormal behavior was observed in Apc Min/ + Ticam / during the period we maintained. In several individuals, growth retardation was observed for unknown reason, but we used individuals with normal body weight. Female and male mice were used for the present experiments. All animal experiments were approved by the University s Committee on Use and Care of Animals. Harvesting of polyps Mice were sacrificed by cervical dislocation. The small and large intestines were harvested and washed with cold PBS. The intestines were longitudinally slit open to grossly count tumors with the aid of a magnifier and stereomicroscope. Polyps mm were collected by forceps and used for the following experiments. RT-qPCR and PCR For quantitative PCR, total RNA was extracted with TRIzol, and. μg of RNA was treated with DNase I, and then reverse-transcribed using the High Capacity cdna Transcription Kit (ABI) with random primers according to the manufacturer s instructions. qpcr was performed using the Step One Real-Time PCR system (ABI). The RNA expression levels were normalized to Gapdh. To detect genomic DNA of Lactobacillus Johnsonii, DNA was extracted with QIAamp Stool Mini kit (Qiagen) form feces according to the manufacturer s instructions. Purified genomic DNA was subjected to PCR using specific primers for Lactobacillus Johnsonii. PCR product was confirmed as amplified Lactobacillus Johnsonii genome by sequencing. The primers for detection of this bacillus were described in an early report []. Primer sequences used in this study are listed in Additional file : Table S. Immunofluorescence The small intestines were fixed with % paraformaldehyde (PFA)/PBS for h at C. Fixed tissues were impregnated with % sucrose/pbs for h following % sucrose/pbs for overnight at C with rotation. Tissues were then embedded in O.T.C. compound (Sakura Finetek Japan) and the frozen tissue blocks were sectioned by using cryotome (LEICA CM). Sections were fixed with acetone on ice for min. After three washes with PBS, the sections were blocked with mouse serum IgG in % BSA/PBS for h at R.T. Sections were stained with FITC-labeled anti-cdc, anti-cdb, anti-cd or anti-cd, and mounted with Prolong Gold (Thermo Fisher Scientific). Samples were monitored at or magnification using an LSM META microscopy (Zeiss). Flow cytometry Single cell suspensions isolated from small intestine were stained with fluorescent dye-labeled Abs after blocking with an anti-cd/ Ab []. The following Abs were used: FITC- or APC/Cy7-CD (-F), PE-anti-CDb (M/7), APC-anti-CDc (N), APC-anti-CDe (- C), FITC-anti-CD (GK.), PE-anti-CDa (-.7) (Biolegend). Dead cells were stained with 7AAD (Sigma). Samples were analyzed by a FACS Calibur or FACS Aria II (BD Bioscience); data analysis was performed using Flow Jo (Tree star). SDS-PAGE/western blotting Proteins were extracted from polyps by SDS-containing sample buffer ( mm Tris-HCl, ph., % SDS, % glycerol, and BPB). The samples were resolved on SDS- PAGE (7. or % gel), and blotted onto PVDF membranes (Millipore). Proteins were detected by rabbit antibodies against and GAPDH (Cell signaling technology). The blot was labeled with Horseradish peroxidase-conjugated goat Ab against rabbit Ig s (Biosource). The color was developed by ECL Prime Western Blotting Detection Reagent (GE Healthcare). Statistical analysis P-values were calculated with Student t-test. Error bar represent the standard deviation (SD) between samples. Results Ticam deficiency results in short survival of Apc Min+ mice TLR transmits signal through two major adaptors, MyD and TICAM- []. We generated

3 Ono et al. Journal of Biomedical Science (7) :79 Page of 9 a Percent survival Myd -/- Days b Survival days Myd -/- Fig. TICAM deficiency results in short survival of mice. Kaplan-Meier survival curves of (n =, circle), Myd / (n = 7, square), Ticam / (n =, diamond) mice. Panel a: survival curves of mice; Panel b: survival days of individulal mice mice on MyD- or Ticam-deficient background by crossing mice to Myd / or Ticam / mice, respectively. The average survival of mice was. weeks. Myd / mice survived longer than mice, consistent with previous reports [7]. In contrast, survival times for Ticam / mice were significantly shorter at ~ weeks (Fig. a, b). Myd +/ and Ticam +/ mice showed similar life-spans, the average survival was 9 weeks indistinguishable from that of mice (Additional file : Figure S), suggesting the Ticam gene disruption was the event that affected life span. Only homologous deficiency of MyD or TICAM- affected the survival time. Multiple polyposis occurs in the intestine of mice, which represents intestinal tumorigenesis by adenomatous polyposis coli (APC) under the regulation of TLR signal [7]. Myd / mice reportedly have fewer polyps in the intestine than mice [7]. We then examined the polyp formation in Ticam / mice at ~9 weeks of age (Fig. ). mice showed a high frequency of polyp formation as reported previously [, 7]. Ticam / mice had more polyps than mice in the distal small intestine (Fig. ). Thus, the incidence of tumor formation is high in the middle and distal intestine of Ticam / mice compared to mice. Only a few polyps were observed in the proximal intestine and colon of mice, and no or minimal increases in polyp numbers was observed in Ticam / mice (Fig. ). Immune cell infiltration in polyps TLRs on epithelial cells recognize microbial products of commensal bacteria and induce inflammatory responses, including oncogene expression []. TLRs except TLR provoke MyD signaling and accelerate the proliferation of intestinal epithelial cells and prohibit apoptosis []. We focused on immune cells infiltrating into mucosal polyps. CD and CD mrnas were minimally detected in unaffected mucosa in and were slightly increased in Ticam / mice at ~ weeks age (Fig. a). CDb and CDc mrnas levels were more increased in polyps of Ticam / mice than mice (Fig. a). mrna for CD and CDα, markers of Number of polyps Proximal small intestine (N=) N.S (N=) Number of polyps Middle and distal small intestine (N=7) P <. (N=) Number of polyps Large intestine (N=) N.S (N=) Fig. The number of polyps is significantly increased in Ticam / mice. The small and large intestines were collected from to 9-weeks-old mice. The small intestine was divided to three equal parts, proximal small intestine, middle and distal small intestine, and large intestine. Polyps ( mm) were counted in the indicated mice. The graphs show the number of polyps in proximal small intestine, distal small intestine, and large intestine. Error bars show SD. p <.instudent s t-test and n.s.; not significant

4 Ono et al. Journal of Biomedical Science (7) :79 Page of 9 a Cdb Cdc b Cd CD Cda CD CDb CDb µm Fig. (See legend on next page.) µm

5 Ono et al. Journal of Biomedical Science (7) :79 Page of 9 (See figure on previous page.) Fig. Immune cells infiltrate into polyps in Ticam / mice. a Gene expression in polyps prepared from to -weeks-old mice were quantified by RT-qPCR. More than mice in each group were used. ; p <.instudent st-test.b Immune staining of the small intestine prepared from to -weeks-old mice using anti-cdb and anti-cdα antibodies. Data shows representative results of two independent experiments myeloid cells, were also increased in Ticam / polyps (Fig. a). We detected more accumulation of CDαpositive cells in polyps of Ticam / mice compared to mice by immunohistological staining (Fig. b). Similar tendencies were obtained with anti-cdb antibody. The FACS profiles of the CD-, CD-, CDb- and CDc-positive cells in the intestine of Ticam / mice vs. mice are shown in Additional file : Figure S. The mrnas of these immune cells were only marginally increased in the normal (non-polyp) region of the small intestine in and Ticam / polyps (Additional file : Figure S). Inflammatory parameters were also increased in the polyps of Ticam / mice (Fig. ). mrna expression of inos (Nos) was highly induced in Apc Min/ + Ticam / polyps compared to polyps (Fig. ). In addition, mrna expression of CXCL9 (Cxcl9), IFN-γ (Ifng) and IL-p (Ilp) was slightly but significantly increased. In the normal (non-polyp) region, this tendency was not prominent (data not shown). Thus, tumor-related inflammation was induced to a greater extent in the intestine of Ticam / mice than in mice. High expression of in Ticam / polyps Previous reports suggest that mrna is not increased in epithelial cells in response to MyD activation [7, ]. Instead, β-catenin signaling is amplified by constitutive inactivation of APC and transcriptionally up-regulates the mrna [7]. We next checked the levels of the mrna in and Apc Min/ + Ticam / polyps. The levels of mrna were high in Ticam / polyps compared to polyps (Fig. a). The protein was abundant in Apc Min/ + Ticam / polyps in comparison with polyps (Fig. b). The results were confirmed with confocal analysis (Fig. c). Although the staining density does not reflect the protein levels, the protein level appears high in Ticam / polyps in Fig. c (and data not shown). The mrna levels of PD-L (Pdl) and COX Nos Ifng Cxcl9 Ilp Fig. Inflammation is promoted in polyps of Ticam / mice. Gene expression in polyps prepared from to -weeks-old mice were quantified by RT-qPCR. More than mice in each group were used. ; p <., ; p <. in Student s t-test

6 Ono et al. Journal of Biomedical Science (7) :79 Page of 9 a b 7 c EpCAM Merged GAPDH µm Fig. High expression of in polyps of Ticam / mice. a Gene expression in polyps prepared from to -weeks-old mice were quantified by RT-qPCR. More than mice in each group were used. ; p <. in Student s t-test. b Protein was extracted from pooled three polyps in distal small intestine and separated by SDS-PAGE. Expression levels of protein were detected by Western blotting. GAPDH is used as an internal control. Each lane shows results from pooled samples prepared from individual mice. Lane -: polyps from mice, Lane -: polyps from Ticam- / mice. c Immune staining of polyps in small intestine prepared from to -weeks-old Ticam / mice using anti- and anti-epcam antibodies. Data show representative results of two independent experiments (Ptgs) appearedhigherin high polyps than low polyps. Conversely, expression of CDα (Cda) and Perforin- (Prf) in high polyps was lower than c- Myc low polyps. high polyps are likely to form a microenvironement favorable for tumor growth (Fig. ). The TLR level was barely affected by environment in c- Myc low and high polyps (not shown): the genes affected by environment in low vs. high polyps are shown in Additional file : Table S. Intestine of mouse contains bacteria with TLR- stimulating capacity Since TICAM- is the adaptor of TLR and TLR [, ], may be suppressed by TLR/-TICAM- signaling. 7 low low Pdl Cda high high low low Ptgs Fig. Differential gene expression in low and high polyps. Polyps prepared from to -weeks-old Ticam / mice were divided into two groups according to the high (.) and low (<.) expression levels of and gene expression was compared by RT-qPCR. ; p <., ; p <. in Student s t-test Prf high high

7 Ono et al. Journal of Biomedical Science (7) :79 Page 7 of 9 A previous report suggested that TLR is activated in response to dsrna moieties yielded by Lactobacillus in mouse intestine []. PCR analysis using the specific primer sets detected the genome DNA of Lactobacillus johnsonii in the feces of mice, implying that the TLR signaling is constitutively activated in the intestine (Fig. 7). Discussion Carcinogenesis is established through multi-step gene mutations in intestinal epithelial cells. Loss-of-function of APC occurs in most patients of familial-associated polyposis [] and causes malignant polyposis. mice have a mutation in the APC gene and accelerate polyposis in the intestine but not colon []. While mice die ~ weeks from complication of tumorigenesis, their survival is prolonged by MyD disruption [7]. Thus, MyD signal of TLRs enhances protumor activity to shorten the survival. TICAM- transmits the other signal to activate a transcription factor IRF. Here we showed that knockout of TICAM- results in short survival in mice, which suggests that the TLR/TICAM- signal reduces polyposis promoted by the TLR//MyD signaling pathway. Intestinal epithelial cells express TLRs which utilize two adaptors, MyD and/or TICAM-, as well as immune cells and tumor cells in mice [, ]. MyD evokes inflammatory signal that causes nuclear translocation of NF-κB and regulates apoptosis in tumor cells. Liberation of inflammatory cytokines sustains tumor-supporting microenvironment. In the mouse model of intestinal tumorigenesis, activation of the MyD pathway is related to stabilization of protein but not to up-regulation of its mrna in epithelial cells, resulting in a decrease in tumor growth in Myd / mice [7, ]. On the other hand, the role of TICAM- in WT No template (bp) Fig. 7 Detection of genomic DNA of Lactobacillus Johnsonii in feces. Genomic DNA of Lactobacillus Johnsonii in feces was detected by PCR using specific primers for Lactobacillus Johnsonii genome. Data shows representative results of two independent experiments the regulation of expression and tumorigenesis has been controversial [9, ]. TICAM- has been identified in myeloid cells including dendritic cells and several subsets of macrophages [ ]. The TLR/TICAM- pathway takes part in cross-priming and IL- production that in turn causes DC priming and cytotoxic T cell (CTL) induction [,, ]. Moreover, some tumor cell lines express TLR []. We show that expression is suppressed via TICAM-: TICAM- signals constitutively suppress expression and TICAM- loss results in up-regulation. Over-expression of abrogates its regulatory function in the cell cycle and induces tumorigenesis [7]. TLR signaling suppresses tumor cell growth through down-regulation of [9]. The c- Myc level, however, barely affect the TLR expression. Thus, it would be reasonable to hypothesize that the TICAM- signaling pathway suppress transcription and reduces intestinal polyp formation in mice. We have examined Ticam-associated gene clusters by comprehensive method [7]. So, we selected inflammatory-induced genes form the Ticam-associated genes (Additional file : Table S). PolyI:C-activated TLR-TICAM- signaling also suppresses tumor growth via immune activation []. Hence, TLR ligand may bimodally act on TLR expressed in tumor cells and immune cells, leading to tumor regression. Our results imply that constitutive activation of the TLR/TICAM- signaling pathway occurs in intestinal mucosa of mice. TLR stimulation also occurs with Lactobacillus dsrna in the intestine []. In this scenario, bacterial-derived dsrna behaves like a tumor suppressor via regulation through the TLR signaling pathway. Thus, Lactobacillus may support good flora conditions to constitutively activate TLR in the intestinal epithelial cells or immune cells. TLR/TICAM- signaling is likely to suppress mrna expression through direct stimulation of TLR on epithelial cells or by indirect stimulation via TLR-expressing immune cells. Further study is required to elucidate the mechanism of TICAM--mediated suppression of expression through intestinal microflora. Many reports suggest that MyD induces protumor signal in tumor or transformed cells, but in dendritic cells MyD induces priming of T cells to regress tumor cells (). Myeloid-derived suppressor cells and tumorassociated macrophages express TLR that activates MyD signaling to promote invasion and metastasis [9]. However, TLR/TICAM- signals convert these myeloid cells to tumoricidal effectors in tumor microenvironment [, ]. Even in epithelial and tumor cells, stimulation of TLR does not promote cell growth or inflammation, which may be attributable to suppression of. TLR adjuvant is now considered more successful in tumor immunotherapy compared to other TLR adjuvants. This study demonstrates an additional

8 Ono et al. Journal of Biomedical Science (7) :79 Page of 9 advantage of TLR adjuvant for direct therapeutic application to tumor cells: TLR-targeted therapy may be of benefit to cancer patients by acting on both immune cells and tumor microenvironment. Conclusion The TLR/TICAM- signaling suppresses mrna expression through direct stimulation of TLR in intestinal cells to suppress mucosal polyposis in mice. Survival time is shortened by knockout of Ticam- in mice. Additional files Additional file : Table S. Primer sequences used for real-time RT-PCR. (DOCX 9 kb) Additional file : Figure S. Survival curve and days of ApcMin/+ mice. Survival curves (upper panel) and survival days (lower panel) were monitored in ApcMin/+, ApcMin/+Myd /+ and ApcMin/+Ticam /+ mice. N >in each group. (PDF kb) Additional file : Figure S. FACS analysis of immune cells in the small intestine. We checked the degrees of infiltration of immune cells into small intestine in ApcMin/+Ticam / mice by FACS analysis. The whole small intestine was harvested from WT, ApcMin/+ and ApcMin/+ Ticam / mice. The proportions of small intestine-infiltrating CDb+, CDc+, CD+ T and CD+ T cells were evaluated on FlowJo ver.9.9. (Tree Star). (PDF kb) Additional file : Figure S. Immune cell markers in the non-polyp region of the distal intestine. Gene expression in the non-polyp region of the distal intestine prepared from to -weeks-old ApcMin/+ mice (n = ) or ApcMin/+Ticam / mice (n = ) was quantified by RT-qPCR. n.s.; not significant in Student s t-test. (PDF kb) Additional file : Table S. levels of inflammatoryassociated genes in low and high polyps. (DOCX 9 kb) Abbreviations AP: Activation protein ; APC: Adenomatous polyposis coli; CTL: Cytotoxic T lymphocytes; IFN: Interferon; IL: Interleikin; IRF: Interferon regulatory factor; MyD: Myeloid differentiation primary response gene ; Nod: Nucleotidebinding oligomerization domain-containing protein ; TICAM-: TIR domaincontaining adapter molecule ; TLR: Toll-like receptor Acknowledgements We are grateful to Drs. Kiyoshi Takeda and Eiji Umemoto (Osaka University, Osaka) for their kind support of this study. We thank Ms. N. Ishii-Mugikura, and A. Morii-Sakai for their technical support. Funding This work was supported in part by the Grants-in-Aid from the Ministry of Education, Science, and Culture (MEXT), the Carcinogenic Spiral a MEXT Grant-in-Project (T. Seya), the Ministry of Health, Labor, and Welfare of Japan (T. Seya, M. Matsumoto), Takeda Science Foundation (H. Shime), the Uehara Memorial Foundation, Smoking Research Foundation, and the Iskura Research Foundation (T. Seya). Availability of data and materials Not applicable. Authors contributions Design of the research, HS, MM, TS; Performing experiments, JO, HT, KT, KF, YT, SY; Data interpretation, HS, MM, TS; Writing manuscript, HS, MK, TS. All authors read and approved the final manuscript. Ethics approval All animal research protocols for this work were reviewed and approved by the Animal Safety Center, Hokkaido University, Japan. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details Department of Vaccine Immunology, Hokkaido University Graduate School of Medicine, Kita, Nishi 7, Kita-ku, Sapporo -, Japan. Department of Microbiology Immunology, Hokkaido University Graduate School of Medicine, Sapporo, Japan. 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