The Institutional Animal Care and Use Committee (IACUC) at the University of California, Davis, approved all procedures involving live animals and methods of euthanasia; experiments were performed in following AVMA guidelines and with adherence to IACUC-approved protocols. Mice were euthanized under deep anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg).

The Itln1 gene variant mouse strain was created in collaboration with the Mouse Biology Program at the University of California, Davis. Detailed methods used to create the tm1a allele have been reported previously (26), and the tm1a trapping-allele vector was generated by the trans-NIH Knock-Out Mouse Project (KOMP). Both the trapping allele vector and the C57BL/6N derived JM8.F6 embryonic stem (ES) cell line used for electroporation were obtained from the KOMP Repository (www.komp.org). The tm1a allele was designed to disrupt a critical region of the Itln1 gene ( Figure 1E ). Within the tm1a (“trap”) allele, a human β-actin gene promoter is located upstream of a neomycin-resistance gene to enable clonal selection in the pool of ES cells. A second key design feature of the trapping allele is an internal ribosome entry site (IRES) upstream of LacZ reporter gene, which (i) interferes with expression of the native gene (insertion of the trapping cassette causes disruption of Itln1 gene expression) and (ii) enables selective expression the LacZ reporter with specificity of the endogenous Itln1 gene. For germline transmission, high percentage chimeric offspring (27, 28) were crossed with wild-type C57BL/6N mice to generate heterozygous Itln1 ^trap/+ colony founders on a pure C57BL/6N background.

Tissue samples were digested in 25 mM NaOH/0.2 mM EDTA at 65°C overnight in a water bath, and then neutralized with 40 mM Tris HCl (pH 5.0). Isolated genomic DNA was amplified to detect the wild-type (WT, Itln1⁺ ) or the targeted Itln1^trap allele using the following primers: Itln1^trap (5’-GAGATGGCGCAACGCAATTAATG-3’, 5’-GAAAGCTAAAGCTAAACCCTGGGTGG-3’) and Itln1⁺ (5’- AAGTCCTCTGATAGAGCAGTGCTTGC-3’, 5’- CAAGACCTGAAAGGCAGAAACAACC-3’). PCR: Briefly, sample DNA and primers were mixed with Taq DNA Polymerase, reaction buffer, and dNTP(s) according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA). PCR reactions were as follows: 94°C for 5 min, 40 cycles [94°C 30 sec, 55°C 30 sec, and 72°C, 40 sec], and 72°C for 5 min. The PCR products were assessed by agarose gel electrophoresis, where the Itln1⁺ allele produces a 340 BP product, and the Itln1^trap allele produces a 439 BP product.

The general procedures for RNA isolation and synthesis of cDNA were previously described by our group (29). Briefly, tissue samples were dissected immediately upon euthanasia and placed in RNAlater (Ambion; Millipore Sigma, Burlington MA) incubated overnight at 4°C, and stored long-term at -20°C. Tissues were homogenized in guanidine thiocyanate buffer (29, 30). Total RNA was isolated using cesium chloride gradient ultracentrifugation (16, 29, 31), and then quantified using ultraviolet absorption spectrometry at 260 nm. For cDNA synthesis, 1 to 5 µg of total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) using an oligo- (dT)12-18 primer (16, 29, 31). The single-stranded cDNA product was purified using Qiagen PCR purification kit (Qiagen, Valencia, CA), and diluted to 10 ng/µl based on the input concentration of total RNA. Primers specific for target genes (Actb [5’-GCTGAGAGGGAAATCGTGCGTG-3’, 5’-CCAGGGAGGAAGAGGATGCGG-3’], Itln1 [5’-ACCGCACCTTCACTGGCTTC-3’, 5’- CCAACACTTTCCTTCTCCGTATTTC-3’], Lyz1 [5’-GCCAAGGTCTACAATCGTTGTGAGTTG-3’, 5-CAGTCAGCCAGCTTGACACCACG-3’], and Reg3g [5’-CCTCAGGACATCTTGTGTC-3’, 5’- TCCACCTCTGTTGGGTTCA-3’) were designed using MacVector Software (MacVector, Cary, NC) (23, 31). Real-time quantitative PCR was performed as described previously (16, 29, 31) using a Roche Diagnostics Lightcycler 2.0 (Roche, Indianapolis, IN). The melting temperatures for PCR products of each reaction were determined to assure that they were indistinguishable from the cloned assay internal standard. Previous control experiments by our group demonstrated that when RNA from a single specimen was used to independently synthesize, isolate, and purify the cDNA, the reaction-to-reaction variability was ≤15% (29). Other reproducibility assessments of this approach were previously reported (29).

To characterize developmental expression patterns of Itln1 in small intestine and colon, tissues were isolated from C57BL/6N mice at postnatal day (PD) 5, 10, 20, and 50 as previously described (31). Briefly, the small intestine was sectioned into proximal and distal portions. In adult mice (PD50, n = 4), 10 cm of proximal and distal small intestine was collected; full length of colonic tissue was also collected. In a separate experiment to investigate the topographical expression patterns of Itln1 in adult mice, the small intestine (n = 4) was divided into 3 cm sections (n = 10 sections) proximally to distally (31). Extra-intestinal tissues of adult mice (n = 4) were similarly dissected for RNA extraction.

General methods for western blot analysis have been reported (16, 20). Briefly, mouse intestinal tissue was homogenized in assay buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) containing a cocktail (1:100) of protease inhibitors (Protease Inhibitor Cocktail III; Calbiochem/EMD Millipore, Burlington, MA). Protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce, ThermoFisher Scientific, Waltham, MA). Following heating at 95°C (either with or without reducing agent – 2-mercaptoethanol) for 30 min, protein (25 μg/lane) was loaded to a SDS-polyacrylamide gel (10% acrylamide) and electrophoresed for 40-50 min at 200 V, prior to wet transfer (Towbin Buffer: 25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3) to a nitrocellulose membrane (0.2 μM pore, Bio-Rad Laboratories, Hercules, CA) at 350 mA for 60-80 min. Estimated molecular size of Itln1 monomer was deduced using the MagicMark™ XP Western Protein Standard (ThermoFisher Scientific). The membrane was blocked in PBS-T (PBS with 0.1% Tween 20) containing 5% w/v skim milk for 30 min, and incubated with primary antibody at 4°C. Following overnight incubation, the membrane was washed with PBS-T for 1 hr, probed with donkey anti-rabbit IgG horseradish peroxidase (HRP) linked secondary antibody (GE Healthcare Amersham, Pittsburgh, PA) for 3 hr at room temperature, rinsed in PBS-T, and visualized using Femto and ECL West chemiluminescent substrates (ThermoFisher Scientific). Chemiluminescent signal was detected using a Biospectrum AC Imaging System (UVP, Upland, CA).

Mouse small intestinal and colonic tissues were fixed in 4% w/v paraformaldehyde (Sigma-Aldrich, St. Louis, MO), sequentially dehydrated in increasing concentration of ETOH and xylene, embedded in paraffin, and sectioned (4-5 μM) by microtome. Tissues were placed on X-tra™ positive-charged slides (Leica Biosystems, Buffalo Grove, IL). Slides were cleared of paraffin using xylene, washed with ETOH, and cleared of peroxidase activity by incubation in methanol containing 0.3% H₂O₂. Rehydrated samples were blocked with 5% v/v serum (donkey) in PBS prior to overnight incubation at 4°C with an in-house rabbit anti-ITLN antibody that recognizes a highly conserved region of mammalian intelectins, as previously reported (16, 20, 32, 33). Following incubation, slides were washed, and probed with a goat anti-rabbit secondary antibody and streptavidin-biotin ABC/horseradish peroxidase detection kit as outlined by the manufacturer (Vector Laboratories, Burlingame, CA). Slides were activated with 3,3- diaminobenzidine substrate (Vector Laboratories), and counterstained with light green (0.4% w/v in 0.2% glacial acetic acid) for 5 min prior to dehydration with denatured ETOH and xylene. Mounted slides were visualized using an Olympus BX51 microscope (Center Valley, PA). For histology, mounted tissue was stained with either hematoxylin and eosin, Masson’s trichrome or Alcian blue using standard protocols.

Small intestinal and colonic tissues of 10-week-old Itln1^trap/trap homozygous mice were harvested, perfused with cold PBS, and fixed for 1 – 2 hr in fixation buffer (2% paraformaldehyde, 0.2% glutaraldehyde) at room temperature in a 6-well cell culture dish. LacZ staining was achieved using published protocols (34–36) and instructions outlined by the β-galactosidase Reporter Gene Staining Kit (Millipore-Sigma). Briefly, fixed tissue segments were washed with PBS and then incubated in the X-gal staining solution (5mM Potassium ferricyanide, 5mM Potassium ferrocyanide, 1mg/ml X-gal solution [X-gal dissolved in dimethylformamide]) for 14 – 24 hr at 37°C sealed and covered in foil. Following incubation (intestines appeared blue), the X-gal staining solution was removed, and tissues were washed three times in PBS (30 min each) under gentle rocking. Thereafter, tissues were further fixed with 4% paraformaldehyde overnight at 4°C, washed with PBS, and then transferred to tissue cassettes for paraffin embedding. Following tissue sectioning, specimens were cleared, rehydrated, and counterstained with eosin Y solution prior to visualization by light microscopy.

Study animals were provided ad libitum access to water and food (Purina Lab Diet 5001, Purina, St. Louis, MO). Heterozygous (Itln1 ^trap/+) breeding pairs on a pure C57BL/6N background were crossed to generate experimental wild-type (Itln1 ^+/+) and homozygous (Itln1 ^trap/trap) offspring. For the acute colitis model, singly housed male Itln1 ^trap/trap and littermate Itln1 ^+/+ mice were administered 2% (w/v) dextran sulfate sodium (DSS: 36-50,000 Da; MP Biomedicals, Santa Ana, CA) as drinking water for 7 days, followed by pure H₂O for an additional 6 days prior to necropsy. For the chronic colitis – carcinogenesis model, 12-week-old male Itln1 ^trap/trap and Itln1 ^+/+ mice were injected intraperitoneally with 10 mg/kg body weight azoxymethane (Millipore-Sigma) one-week prior to the initial administration of 1.5% w/v DSS. The protocol for repeated DSS exposure was previously described with minor modifications (37, 38). Briefly, following the first DSS treatment for 7 d, animals were provided H₂O for 14 d prior to the subsequent 7 d DSS treatment. In total, animals were challenged three times with DSS (7 d each) and provided H₂O for an additional 14 d following the last challenge prior to necropsy. Animals were monitored for induction, progression, and resolution of disease across the study period. Body weight and fecal markers of disease activity were assessed daily. Feces were collected and scored for presence of occult blood (Hemoccult test kit, Beckman Coulter, Brea, CA) and consistency (texture and water content) based on an established scoring system (39). The scoring measures were as follows: stool texture, 0 (normal), 1 (increased moisture content), 2 (loose stool – soft but still discontinuous/formed), 3 (very soft/loose and/or continuous stool), and 4 (diarrhea, feces present on mouse anus/hindquarters); fecal blood: 0 (no blood), 1 (hemoccult positive but no visible blood), 2 (strong hemoccult positive and visible but discontinuous blood), 3 (visible, continuous blood), and 4 (gross bleeding and/or blood at anus). Intermediate scores of 0.5 were included where necessary. At necropsy, small intestinal length, colon length, and tumor count per colon were assessed.

8-week-old male wild-type (Itln1 ^+/+) and homozygous (Itln1 ^trap/trap) mice were randomized to receive one of three Research Diets (New Brunswick, NJ): control (CD; D12450K [10% kcal fat, 0% sucrose]), high fat-sugar (HFS; D12451 [45% kcal fat, 17% sucrose]), or very high fat (VHF; D12492 [60% kcals fat, 7% sucrose) for the duration of the experiment. Body weight and food intake were recorded daily. For the glucose tolerance test (GTT), male wild-type (Itln1 ^+/+) and homozygous (Itln1 ^trap/trap) mice fed CD (D12450K) for approximately 14 wk were fasted 6 hr (morning) prior to intraperitoneal injection with 2 g/kg body weight D-glucose (VetOne, Boise, ID) as described (40). Glucose concentrations were measured by glucometer (OneTouch Ultra2, LifeScan Inc., Milpitas CA) at t = 0-, 15-, 30-, 60-, and 120-min post glucose injection. For the insulin tolerance test (ITT), mice were fasted for 5 h prior to intraperitoneal injection with human recombinant insulin (0.75 U/kg body weight, Novolin, Princeton, NJ) (40). Blood collection procedures paralleled those of the GTT.

At the experimental endpoint of the diet-induced obesity study, plasma was obtained from blood collected via cardiac venipuncture using EDTA-treated capillary tubes (Sarstedt, Newton, NC). Plasma analytes were assessed by Bio-Plex Multiplex Immunoassay (Bio-Rad, Hercules, CA). Sample preparation and assay procedure was performed as outlined by the manufactures’ guidelines.

Statistical analysis was performed using Prism software, version 9.3.1 (GraphPad Software, Inc). Specific analysis details are indicated in figure legends.

Using a quantitative real-time PCR assay, with an external plasmid standard of known concentration, the expression of intelectin-1 (Itln1) mRNA transcript in C57BL/6N mouse tissues was determined (29, 31). To characterize the expression patterns of Itln1 in small intestine, we isolated samples from pre-weanling and adult mice. In both proximal and distal small intestine, Itln1 mRNA expression across the pre-weaning period increased ~10-fold, and a further ~50-fold by postnatal day 50. Conversely, Itln1 mRNA in the colon was present at manifold lower levels (~1,000 – 10,000-fold) and expression was independent of developmental period ( Figure 1A ). The patterns of Itln1 mRNA expression in mouse intestine reported here mirrored other abundant Paneth cell products, which we previously described (31) such as Lyz1 and a subset of α-defensins (e.g., Defa3, Defa23, and Defa26). To delineate the proximal to distal expression of Itln1 in the small intestine, we divided the small intestine into ten adjacent 3-cm sections and absolute quantity of Itln1 mRNA was determined. Expression of Itln1 was found to be similar (within threefold) across the length of the small intestine ( Figure 1B ). Additionally, at necropsy we dissected the colon of adult mice into two equal length portions (i.e., proximal and distal halves) to better characterize Itln1 expression, as molecular regionalization has also been reported in murine colon ( Figure 1B ) (41). Constitutive Itln1 mRNA expression was confirmed to be low in both regions. Using a threshold of detection for RNA transcripts of 10² copies per 10 ng RNA, no expression of Itln1 mRNA was detected in the extra-intestinal tissues tested, except testes ( Figure 1C ). Of note, two tissues with high ITLN1 expression in humans, adipose and lung, were below the expression threshold for Itln1 in mice ( Figure 1C ). These data are consistent with expression patterns of Itln1 previously reported (23). Together, these results demonstrate that in C57BL/6 mice, Itln1 mRNA expression is largely restricted to the small intestine.

Immunoblot analysis of small intestinal tissue extracts detected a band of ~35 kDa by SDS-PAGE under reducing conditions ( Figure 1D ), consistent with the expected size of a monomeric isoform based on the deduced amino acid sequence. Unlike human ITLN1, which forms disulfide-linked trimers, mouse Itln1 is predicted to generate only monomers, as it lacks the cysteine residues ascribed to trimer formation (24). Nevertheless, under non-reducing but denatured conditions, a single band was detected at ~220 kDa ( Figure 1D ). More work is required to further characterize this potentially higher-order oligomer that appears to migrate as a hexamer.

To investigate the biological activities of Itln1 in vivo we developed a gene variant mouse model on a pure C57BL/6 genetic background (see methods). A trapping allele (tm1a (26)) was designed to insert into and disrupt the Itln1 gene ( Figure 1E ). The tm1a vector includes a neomycin resistance gene for clone selection, as well as FRT and LoxP sites (for future site-specific recombinase-mediated excision of the trapping cassette) and Itln1 exons 4-7. The trapping allele has an internal ribosome entry site (IRES) upstream of the reporter LacZ ( Figure 1E ). Following germline transmission of the transgenic trapping allele (designated Itln1 ^trap), chimeric mice were bred to C57BL/6N mice, creating heterozygous mice - henceforth referred to as Itln1 ^trap/+. When crossed, Itln1 ^trap/+ mice generated offspring expected for Mendelian-inheritance patterns (data not shown; DNS). The gene expression of Itln1 in mice homozygous for the trapping cassette (Itln1 ^trap/trap) had Itln1 transcript levels reduced by ~10,000-fold in the small intestine compared to wild type littermate control mice (DNS).

Previous in situ hybridization analysis established that Itln1 mRNA localizes specifically to Paneth cells in the small intestine of C57BL/6 mice (8). To confirm this cellular localization of Itln1 protein, we performed immunohistochemistry, and observed Itln1 staining in mouse intestinal crypts – indicative of Paneth cells ( Figure 1H ). By immunohistochemistry, Itln1 was undetectable in Paneth cells of Itln1 ^trap/trap mice, and no Itln1 staining was observed in colons of either Itln1 ^+/+ or Itln1 ^trap/trap animals in support of western blot and mRNA analysis ( Figure 1G ). The knockout Itln1 ^trap/trap mice showed no signs of systemic or small intestine-specific pathology when unchallenged ( Figure 1I ), and the mice gained weight like Itln1 ^+/+ littermates (DNS and as observed in Figure 3A ). The IRES of the trapping cassette enables expression of a LacZ reporter to be driven by the upstream endogenous Itln1 gene promoter. Accordingly, expression of LacZ in Itln1 ^trap/trap mice is evident in at the base of the small intestinal crypts ( Figure 1F ), consistent with established Paneth cell expression for Itln1 mRNA by in situ hybridization (8).

Human ITLN1 is implicated in the etiology of inflammatory bowel disease (10–15). We assessed susceptibility to chemically induced colitis in Itln1 ^trap/trap mice using a dextran sulfate sodium (DSS) model. Itln1 ^trap/trap mice and Itln1 ^+/+ littermate controls were challenged with 2% w/v DSS in drinking water for 7 days. The DSS was then removed, and the mice were allowed to drink water until the termination of the experiment 6 days later ( Figure 2A ). Both knockout (Itln1 ^trap/trap) and wild type (Itln1 ^+/+) mice exhibited high sensitivity to 2% w/v DSS, including weight loss beginning at 7 d treatment ( Figure 2B ). Following DSS removal, animals continued to lose weight until day 10 at which point wild type mice began to regain weight. Compared to wild type control mice, Itln1 ^trap/trap mice exhibited more pronounced weight loss, as well as impaired weight regain upon DSS removal and shortening of colonic length ( Figure 2B ). Mortality, which did not differ between genotypes, was approximately 15 – 25% ( Figure 2B ). Together, these results suggested that Itln1 ^trap/trap mice are more susceptible to DSS.

Nevertheless, due to both the acute treatment period and relatively high mortality rate of mice receiving 2% DSS, we chose to pursue another established model of chronic colitis, which enabled a detailed view of disease activity across a protracted period ( Figure 2C ). Singly housed Itln1 ^trap/trap mice and Itln1 ^+/+ littermate controls were pretreated with azoxymethane, and then challenged with three rounds of 1.5% w/v DSS (7 days). Between each DSS challenge, animals were provided DSS-free water for 14 d ( Figure 2C ). Body weight, disease activity (fecal consistency and hemoccult), and food/water intake were measured daily across the experimental period (63 days). Both Itln1 ^+/+ and Itln1 ^trap/trap mice lost body weight and exhibited increase disease activity across the study period, where the second DSS treatment period resulted in a clear exacerbation ( Figure 2D ). At the lower DSS dose (1.5% w/v), no differences in body weight, the partitioned fecal texture and hemoccult scores, or food/water intake (DNS) were observed; however, there was a subtle but significant genotype over time effect for summation (combined fecal scores) of disease activity, suggesting Itln1 ^trap/trap mice were slightly more sensitive to challenge ( Figure 2D ). At necropsy, neither small intestine nor colon lengths differed ( Figure 2E ). The colonic tumor burden induced by carcinogen azoxymethane appeared to be equivalent between the two groups ( Figure 2E ). Distal small intestine (last 5 cm) mRNA expression of Paneth cell effectors, Lyz1 and Reg3g were similar between Itln1 ^+/+ and Itln1 ^trap/trap mice ( Figure 2F ). Together, these results suggest that Itln1, a Paneth cell product in C57BL/6N mice, likely plays a minor role in the pathophysiology of chemically induced colitis.

Human ITLN1, expressed in visceral adipose, has been identified as a biomarker for obesity and related metabolic dysregulation (17–20, 42–45). To investigate the potential role of mouse Itln1 during diet-induced obesity, we assigned Itln1 ^trap/trap mice and Itln1 ^+/+ littermates to receive either control or one of two western-style diets, with varied amounts of sugar (as sucrose) and saturated fat (as lard). Body weight and food intake was recorded daily for the duration of the experimental period ( Figures 3A, B ). Itln1 ^trap/trap and Itln1 ^+/+ mice gained similar weight on each diet and consumed an equivalent amount of chow ( Figures 3A, B ). At necropsy, adipose depot mass was indistinguishable between genotype, reflective of body weight findings ( Figure 3C ). Furthermore, plasma concentrations of markers for obesity (leptin), inflammation (resistin), and insulin signaling (insulin and plasminogen activator inhibitor 1) did not differ between genotypes ( Figure 3D ). Lastly, in a cohort of control diet-fed animals, we investigated glucose and insulin tolerance to determine if under normal physiological conditions Itln1 plays a role in metabolic response. Itln1 ^trap/trap and Itln1 ^+/+ mice demonstrated equivalent responses to glucose and insulin challenge ( Figure 3E ). Together, these findings demonstrate that ablation of Itln1 in C57BL/6 mice does not result in increased susceptibility to diet-induced obesity.