The genotyping of selected SNPs (Table 1) was performed by Sanger sequencing on 155 MDs diagnosed with ICRP, 90 age-controlled MDs without ICRP, 36 juvenile MDs aged < 1 year, and 40 dogs of other breeds. The details of the animals recruited and the samples used in this study are summarized in Supplementary Table 1. A DNA Blood & Tissue Kit (QIAGEN, Hilden, Germany) was used to derive genomic DNA (gDNA) from EDTA-archived blood, fresh colon tissue samples, or formalin-fixed paraffin-embedded colon tissue samples of the recruited dogs. Blood was collected by a veterinarian or a veterinary student supervised by a licensed veterinarian through a jugular venipuncture as part of routine blood workup for screening or diagnosis purposes. A signed consent form was obtained from the owners of all dogs for the tissue sample collection and use in this study.

Endoscopic examinations were performed under general anesthesia for all dogs to retrieve the colon tissues. Each dog was administrated with midazolam (0.1 mg/kg) and butorphanol tartrate (0.2 mg/kg) intravenously as pre-medication and then with propofol (4 to 6 mg/kg) using the same route. Anesthesia was then maintained through the inhalation of isoflurane with oxygen, where additional butorphanol was administrated when necessary. Pulse oximetry readings, electrocardiograph, capnograph, arterial blood pressure, and rectal temperature were monitored throughout the anesthesia to ensure a smooth endoscopic procedure. Endoscopic procedures were completed within 2 h in all dogs, and all dogs recovered uneventfully. In MDs with ICRP, samples were collected as part of the diagnostic procedure, where polyp lesions deemed inflammatory on gross appearance were collected endoscopically with forceps or through electrosurgical snare polypectomy. Normal colorectal mucosae were collected endoscopically from the colorectal region adjacent to the diseased area based on gross appearance. At least six tissue samples were collected from both the polyp lesion site and normal colorectal mucosal region in MDs with ICRP. All biopsy specimens were assessed by a board-certified veterinary pathologist (YK) according to histopathological standards established by the World Small Animal Veterinary Association Gastrointestinal Standardization Group (40). The severity of ICRP inflammation in each case was staged according to a previous report (3). The information on the medication and histopathology for each group is shown in Supplementary Table 2. Each tissue sample was stored at −80°C for protein analysis or RNALater RNA Stabilization Solution (Ambion Inc., Austin, TX, USA) for 24 h at 4°C to allow penetration and stabilization and then at −80°C for prolonged storage. Five specimens were collected for the qPCR analysis to perform a definitive diagnosis of ICRP.

Polymerase chain reactions (PCRs) and Sanger sequencing were utilized to validate the genotype variants selected from WES data (12). The WES data have been deposited with links to BioProject accession number PRJDB16014 in the DDBJ BioProject database. Primer pairs used for the variant validation were designed using Primer3Plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi; Table 1). Amplicons post-PCR were purified using a commercial clean-up reagent, ExoSAP-IT Express (Thermo Fisher Scientific, Waltham, MA, USA), and sequencing was performed using the Sanger method utilizing BigDye Terminator v3.1 (Thermo Fisher Scientific) and ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). FinchTV (https://digitalworldbiology.com/FinchTV) was used to interrogate the reads generated, and the allele frequency was compared between groups using Fisher's exact test.

The H4 human cancer cell line was purchased from ATCC (Sumitomo Dainippon Pharma, Osaka, Japan). All cell lines were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA) enriched with 10% fetal bovine serum (Thermo Fisher Scientific) and treated with 1% penicillin and streptomycin at 37°C under 5% CO₂. For the cytokine stimulation, cells were seeded into 96-well plates (1 × 10⁴ cells/well) and stimulated with human IL-6 (30 ng/ml; Toray Industries, Tokyo, Japan) plus human soluble IL-6 receptor (30 ng/ml; R&D Systems, Minneapolis, MN) and/or TNF- α (10 ng/ml; PeproTech, Tokyo, Japan) for 3 h after 2 h of serum starvation in Opti-MEM (Thermo Fisher Scientific, Waltham, MA). For the TG stimulation, the cytokine stimulation was modified to include cells stimulated with a 5x dilution of the cytokine with the serial addition of recombinant human TG (1 μg/ml, 5 μg/ml, and 10 μg/mL) as per the manufacturer's recommendation (Cloud-Clone Corp., USA). After stimulation, the cells were lysed, and the total RNA was retrieved for real-time PCR.

The Bio-Rad CFX96 real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) and THUNDERBIRD SYBR quantitative PCR (qPCR) Mix (TOYOBO Co. Ltd., Osaka, Japan) were used to quantify the levels of target mRNA and internal control mRNA (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). Total RNA was prepared from cells using a SuperPrep Cell Lysis Kit for qPCR (TOYOBO). The conditions for real-time qPCR were 40 cycles at 94°C for 15 s followed by 40 cycles at 60°C for 60 s. Relative IL6 mRNA expressions were normalized to the level of GAPDH mRNA expression. Primers and their sequences for the qPCR are described in Table 2. For the in vitro experiments, all the experiments were performed in triplicate but also performed twice to ensure replication.

Human small-interfering RNAs (siRNA) were transfected into H4 cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific). The sequences for the sense oligonucleotides of the knockdown constructs were human si-TG (1: CCUUAUGAGUUCUCACGGAtt and 2: GCUGCUACAUGGUAUUACUtt; Ambion Silencer Select siRNA, Thermo Fisher Scientific), human si-p65 (Ambion Silencer Select RELA siRNA, Thermo Fisher Scientific), and human siRNA negative control (Ambion Negative Control #1 siRNA, Thermo Fisher Scientific).

Serum samples from 22 MD-ICRP, 22 MD-Control, and 36 juvenile MDs were enrolled in this study. For the 44 adult MDs, blood samples were collected from April 2017 to April 2021 by a licensed veterinarian or a supervised veterinary student by venipuncture during the dogs' first visits to the Hokkaido University Veterinary Teaching Hospital (HUVTH) for routine diagnostics with written informed consent obtained from the owners. For the juvenile MDs, blood samples were collected by a licensed veterinarian (YBT) using a venipuncture. The juvenile MDs were owned by a small and medium enterprise pet store in Hokkaido, Japan; written informed consent was obtained from the enterprise's owner and veterinarian on duty, and sample collection was performed from November 2021 to February 2022. Serum samples separated after centrifugation were stored at −80°C until the TG analysis. Serum TG concentrations were measured as per protocol provided by the manufacturer using the Canine Thyroglobulin ELISA Kit (MyBioSource, San Diego, CA, USA, Catalog No: MBS2608140). The intra-assay coefficient of variability between sample replicates was < 20%. The information on the regional/systemic inflammation and medication (e.g., corticosteroids) is given in Supplementary Table 3.

For qPCR using canine colonic mucosa samples, seven MD-ICRP (three with T/T allele and four with C/C allele), which visited HUVTH between April 2017 and April 2021, were included; total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA); and genomic DNA was removed using an RNase-free DNase Set (Qiagen) following the manufacturer's instructions. cDNA synthesis was then performed using oligo (dT) and M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA) from 1 μg total RNA as per the manufacturer's recommendation. Real-time TaqMan qPCR was performed using a commercially available set of a pre-designed probe and primers (Applied Biosystems) for TG (product no: Cf02701382_m1) with an endogenous control for colonic samples using SDHA (product no: Cf02664981_m1) confirmed by a previous study (41). Real-time qPCR was performed using TaqMan PCR probe master mix (KAPA Biosystems) and at the following cycle conditions: 40 cycles at 94°C for 3 s and 40 cycles at 60°C for 30 s. The relative TG mRNA expression levels were normalized to the level of SDHA mRNA expression. THUNDERBIRD SYBR qPCR Mix (TOYOBO Co. Ltd., Osaka, Japan) was used to quantify the levels of the target mRNA (IL6, CCL2) and internal control mRNA (succinate dehydrogenase complex, subunit A; SDHA). The condition for real-time qPCR was 40 cycles at 94°C for 15 s followed by 40 cycles at 60°C for 60 s. Relative IL6 and CCL2 mRNA expressions were normalized to the level of SDHA mRNA expression. The primers and their sequences for the qPCR are described in Table 3 and reported previously (42, 43). For the qPCR using these samples, all the experiments were performed in duplicates and performed twice to ensure replication.

Two-tailed Student's t-test and Fisher's exact test were used to analyze differences between the two groups. Values of P < 0.05 were considered significant.

The frequency of TG c.4567C>T (p.1523R>W) allele in MDs (n = 281) was 69.04% (48 homozygous and 246 heterozygous), which was significantly higher than in dogs of other breeds (22.5%; one homozygous and eight heterozygous, n = 40) (P < 0.0001, Figure 1A). Sequence chromatograms of the target variant are shown in Supplementary Figure 1A. Furthermore, the frequency of the T risk allele (84.52%) was significantly higher in MD-ICRP (median age = 10 years old; 34 homozygous, 97 heterozygous, n = 155) than in MD-controls (47.78%; median age = 12 years old; 9 homozygous, 34 heterozygous, n = 90) (P < 0.0001, Figure 1B). Thus, the TG c.4567C>T (p.1523R>W) allele was increased in MDs with ICRP. Finally, we found that one amino acid of the TG gene was conserved in dogs and other mammals (Supplementary Figures 1B, C).

Because we found that TG is one positive regulator candidate of the IL-6 amplifier (39) and showed that TG c.4567C>T (p.1523R>W) allele is increased in MDs with ICRP, we hypothesized that TG is functionally involved in ICRP development via activation of the IL-6 amplifier. Consistently, we found that TG expression was increased during the activation of the IL-6 amplifier (Figures 2A, B), suggesting that TG is a target gene of the IL-6 amplifier. We then treated H4 cells, a human neuroglioma cell line, with siRNA of TG to investigate cytokine-mediated IL-6 amplifier (39) and found that IL-6 expression and TG expression were reduced after cytokine stimulation (Figures 3A, B). Furthermore, recombinant human TG enhanced IL-6 amplifier activation in H4 cells (Figure 3C). These results suggested that TG is both a positive regulator and a target of the IL-6 amplifier.

We hypothesized that the TG SNP functions to increase TG expression either systematically or locally. To test this hypothesis, we investigated the serum concentrations of TG but found they were unchanged in MDs with or without ICRP and with or without the risk T/T SNP (Figures 4A–C). In addition, we found no difference in serum TG concentrations between dogs receiving systemic anti-inflammatory medication or across genotypes (Supplementary Figures 2A–C). We then investigated the expression levels of TG in the non-inflammatory colonic mucosa of MD-ICRP. We found that TG expression in samples with the risk T/T SNP was significantly higher than those with the non-risk C/C SNP (Figure 5A), suggesting that the TG SNP is an expression quantitative trait locus (eQTL) in the colon of MDs. We next investigated whether TG is induced by the IL-6 amplifier in the colon. Because the IL-6 amplifier reflects the activation of NF-κB in non-immune cells, we analyzed the expression of two NF-κB targets, IL6 and CCL2, in the same colon samples. We found that IL6 and CCL2 were also enhanced in colon samples with the risk T/T SNP (Figures 5B, C). These results indicate that the risk T/T SNP of TG is an eQTL and enhances TG expression. They also indicate that TG is involved in the activation of the IL-6 amplifier in the colon of MD-ICRP. Therefore, the risk T/T SNP of TG may be critical in the pathogenesis of ICRP through the activation of the IL-6 amplifier.