Female (16-22 g) and male (22-30 g) C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) aged 7-8 weeks on arrival, were group housed (3-4 mice per cage), under a 12 h light–dark cycle (lights off 19:00) in plastic sawdust floor cages (22°C, 40% humidity, standard laboratory chow and water ad libitum) in a specific pathogen-free facility. After one week of acclimatization, cages of mice were randomly assigned to treatment groups. All experimental procedures were approved by the Health Sciences Animal Care Committee of the University of Calgary and were carried out in accordance with the guidelines of the Canadian Council on Animal Care (Protocol #’s AC17-0093, AC15-0129).
Mice were given DSS (40–50 kDa, Affymetrix, Cleveland, OH, USA) ad libitum in their drinking water (2.5%-3.5% wt/vol); on day 5 this was replaced with tap water until day 7. Control mice received tap water alone for 7 days. Body weight was measured three times per week. Day 7 has previously been determined to be the peak of colonic inflammation in this model .
In all cases, mice were euthanized by cervical dislocation under isoflurane or ketamine-xylazine anesthesia on day 7. Body weight score was calculated as the % weight loss on day 7 from the initial body weight on day 0 (0 = 0 %, 1 = <0-≤5 %, 2 = >5-≤ 10 %, 3 = >10-≤ 15 %, 4 = >15 %). The colon was dissected and examined by a blinded observer for macroscopic evidence of colitis. Colon length score was calculated as a % of control colon length, with the average control length in females being 6.0 cm and in males 7.4 cm: (0 = 85-100%, 1 = 75-84 %, 2 = 65-74 % and 3 < 65 %). The presence (score = 1) or absence (score = 0) of adhesion, erythema, gross fecal blood and diarrhea was recorded. A total damage score was calculated for each animal comprising, body weight score, adhesion, colon length score, erythema score, fecal blood score, diarrhea score, length of inflamed colon as % of total length, ulcer length and bowel thickness (mm). The macroscopic damage score is presented as mean ± SEM for ease of comparison with the literature, while acknowledging that these are non-parametric values.
To determine the proportions of leukocyte population in colitis, we performed a flow cytometric analysis. After cervical dislocation, blood was immediately withdrawn by cardiac puncture. A whole blood staining method was used to investigate the phenotypic profile of peripheral blood leucocytes. In order to block non-specific binding to Fc III/II receptors, 100 µL of anticoagulated whole blood were added to a 5 mL polystyrene tube and incubated with anti-CD16/CD32. Following incubation at room temperature for 15 min, a predetermined optimum concentration of desired fluorochrome-conjugated primary antibodies was added and incubated for 30 min. Red blood cells were lysed by adding 2 mL of Ammonium-Chloride-Potassium lysis buffer. Following incubation at room temperature for a further 10 min, cells were washed twice in staining buffer by centrifugation at 500xg for 10 min. Samples were acquired either using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) or Attune™ Acoustic Focusing flow cytometer (Applied Biosytems, Mainway, Burlington, ON, Canada). Data were analyzed using FlowJo® software (Treestar, Ashland, OR, USA). Flow cytometry dot-plots showing the gating strategy used in the identification of α4β7 expressing monocytes and neutrophils in mouse blood are shown in Supplementary Figure 1. The following antibodies were obtained from sources indicated: anti-mouse CD16/CD32 (93), anti-mouse Ly6C (HK1.4),) (ThermoFisher Scientific, Waltham, MA, USA). Anti-mouse CD11b (M1/70), anti-mouse Ly6G (1A8), anti-mouse CD3ε (145-2C11), anti-mouse Integrin α4β7 (DATK32) (BioLegend, San Diego, CA, USA). Data are shown as mean ± standard error of the mean (SEM) of 5 mice per group. For comparisons between two groups, an unpaired Student's t-test was performed (GraphPad Prism version 9, GraphPad, San Diego, CA, USA). A P value of ≤0.05 was considered significant.
To examine leukocyte – endothelial interactions in colitis, we performed intravital microscopy using previously published approaches . On day 7 of colitis, mice were anesthetized using a ketamine and xylazine mixture (i.p.; 200mg/kg and 10mg/kg, respectively). The tail vein was cannulated for administration of dyes and conjugated antibodies for imaging. The skin was blunt dissected from the skull and the parietal bone thinned to approximately 30 μm using a high-speed dental drill, resulting in an intact cranial window over the parietal cortex of approximately 5 mm x 5 mm . The window was covered with a drop of saline and the mouse placed on the microscope stage.
Dyes or conjugated antibodies were administered i.v. immediately before imaging: Rhodamine-6G (0.225 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) was used to visualize all leukocytes , phycoerythrin (PE)- or allophycocyanin (APC) -labelled CD-31 (390; 2 μg/mouse; eBioscience; catalog #17-0311-82 (APC)- #12-0311-81, (PE)) was used to label cerebral endothelial cells, PE-labelled Ly6G (1A8, 2 μg/mouse; eBioscience; catalog #12-9668-80) was used to label neutrophils, APC-labelled Ly6C (HK1.4; 2 μg/mouse; eBioscience; catalog #17-5932-80) was used to label monocytes, and APC-labelled MAdCAM-1 (MECA-367; 2 μg/mouse; Biolegend, San Diego, CA, USA; catalog #120711) was used to label MAdCAM-1.
For the depletion of specific cells, mice with colitis were i.p. administered antibodies or isotype control, 18-22h prior to the imaging experiment. Circulating neutrophils were depleted using anti-Ly6G (1A8; 200 μg/mouse; Bio X Cell, Lebanon, NH, USA; catalog #BE0075-1), while the controls received rat IgG2a antibody (200 μg/mouse; Bio X Cell; catalog ##BE0089). The efficiency of the mAb anti-Ly6G to specifically deplete classical monocytes in C57BL/6 mice was confirmed using flow cytometric analysis as shown in Supplementary Figure 2. Anti-α4 integrin (PS/2; 200 μg/mouse; Bio X Cell; catalog #BE0071) and anti-α4β7 integrin (DATK32; 200 μg/mouse; Bio X Cell; catalog #BE0034) were administered to investigate the role of integrins in leukocyte recruitment. The controls were administered rat IgG2b (200 μg; Bio X Cell; catalog #BE0090) and rat IgG2a, respectively. Other mice were administered anti-MAdCAM-1 (MECA-367; 200 μg/mouse; Bio X Cell; catalog #BE0035) or rat IgG2a. Circulating monocytes were depleted using anti-Ly6c (HK1.4; 100 μg/mouse; eBioscience; catalog #16-5932-85), with controls receiving rat IgG2c (100 μg; Biolegend; catalog #400710). The efficiency of the mAb anti-Ly6C to specifically deplete classical monocytes in C57BL/6 mice was confirmed using flow cytometric analysis as shown in Supplementary Figure 3. We have previously used this anti-alpha4 integrin neutralizing antibody strategy to prevent monocyte adhesion to cerebral endothelial cells .
Leukocyte-endothelial interactions were imaged in the parietal cortex using a Quorum WaveFX spinning disk confocal microscope (Quorum Technologies, Puslinch, ON, Canada) driven by Volocity 6.1 acquisition software (PerkinElmer, Waltham, MA, USA). Labeled cells were imaged using 561, or 635 nm laser excitation and visualized with the appropriate long pass filters using a 20X/0.95 NA water objective. A 512 X 512 pixel back-thinned EMCCD camera (Model C9100-13; Hamamatsu Corp., Hamamatsu City, Japan) was used for fluorescence detection. 3-5 venules (17-40 μm diameter) were recorded for 1 min and data averaged per mouse. Rolling leukocytes were classed as those that moved at a velocity less than that of an erythrocyte down a 100 μm segment of vessel. Adherent cells were classed as those that were stationary for 30 sec or longer within the 100 μm segment of vessel . Data are shown as mean ± standard error of the mean (SEM). For comparisons between two groups, an unpaired Student's t-test was performed (GraphPad Prism). A P value of ≤0.05 was considered significant. A total of 90 animals were successfully used in 32 cohorts. Four outliers were identified using the Grubbs’ test and were removed.
To delineate the importance of leukocyte - cerebral endothelial cell interactions in initiating neuroimmune activation in the brain, we measured prefrontal cortical cytokine levels 7 days after DSS treatment. On day 4 and 6 of DSS treatment, the control group (n=6) was administered sterile PBS 10mL/kg, i.p. while the DSS-treated mice were given either control IgG2a antibody (200 μg/mouse, IP; Bio X Cell; catalog #BE0089, n=6), or anti-α4β7 integrin antibody (200 μg/mouse, IP; Bio X Cell; catalog #BE0034, n = 6) to investigate the role of integrins in cytokine changes in the brain. On day 5, DSS administration was stopped, and all mice were given tap water. In the morning of day 7, mice were anesthetized with isoflurane and transcardially perfused with cold PBS buffer for 5 min while under anesthesia. The prefrontal cortex was microdissected and immediately snap-frozen in liquid nitrogen and stored at -80°C until processing. The isolated cortical tissue was homogenized using a Micro-Tube homogenizer in tissue protein extraction buffer (RIPA buffer containing protease inhibitor cocktail). The homogenate was centrifuged at 15000 × g for 15 min at 4°C, and supernatants were collected and stored at −20˚C until analysis. Mouse cytokines (IFNy, IL-1β, GM-CSF, IL-2, IL-4, IL-6, IL-10, IL-12(p70), CCL2, TNFα) were simultaneously measured in tissue homogenate samples using a mouse MILLIPLEX kit (Millipore, Burlington, MA, USA) according to the manufacturer's protocol. The multiplexing analysis was performed using the Luminex 100 system (Luminex®, Austin, TX, USA) by Eve Technologies Corporation (Calgary, AB, Canada). Total protein concentration in tissue homogenates was quantified using a BCA Protein Assay kit (ThermoFisher) according to the manufacturer's instructions. Results were expressed as pg of analyte/mg of protein. All data are shown as mean ± standard error of the mean (SEM) of 6 mice per group. For comparisons between groups, an analysis of variance (ANOVA) followed by the Student-Newman-Keuls post hoc test was performed (GraphPad Prism). A P value of ≤0.05 was considered significant. The Grubbs’ test was used to identify and exclude potential statistical outlier data points.
Intracerebroventricular (ICV) cannulation and infusion.
To assess whether blocking elevated IL-1β levels in the brain would alter behavior we administered IL-1ra intracerebroventricularly. Mice were anesthetized with isoflurane and a guide cannula (23G, 8 mm) was implanted under stereotaxic guidance above the right lateral ventricle (from Bregma: +0.5 mm, lateral: +1.0 mm, depth: +1.4 mm). Mice were given analgesic treatment (buprenorphine, provided by the Health Science Animal Resource Centre, Calgary, AB, Canada, 0.05 mg/kg subcutaneously before and after the surgery) and allowed to recover for 5 days, after which DSS was administered in drinking water for another 5 days as described above. Body weight was recorded pre-and post-surgery, as well as during and after DSS administration/ICV infusions.
At the onset of DSS administration, both control and DSS-treated mice were infused ICV (0.5μL/30 sec) with IL-1ra (2 μg/2μL, R&D Systems, Minneapolis, MN, USA, Catalog #280-R) or vehicle (sterile PBS with 0.1% bovine serum albumin [BSA], 2μL). This was repeated on day 2 and day 4 of DSS treatment, and the day after termination of DSS administration. Approximately 24h after the last infusion, mice were placed in the elevated plus maze to test for anxiety-related behavior.
Elevated plus maze (EPM)
The EPM was used for assessment of anxiety-related behavior, as we have done previously [19, 42]. Our EPM consisted of two open (6 x 30 cm, 70 lux) and two closed (6 x 30 x 15 cm, 20 lux) arms radiating from a central platform (6 x 6 cm, 55 lux) to form a plus-shaped figure. The maze was elevated 50 cm above the floor. Each mouse was placed on the central platform facing a closed arm and allowed to explore the maze for 5 min. The 5 min test period was recorded by means of a video camera and later analyzed using TopScanTM 2.0 software (Clever Sys Inc., Virginia, USA). This allowed for the calculation of the percentage time spent in the arms as well as the total distance travelled, which were deemed measures of anxiety and locomotion respectively. The maze was thoroughly cleaned before each test.
Data presentation and statistics for EPM
Each data set consists of experiments carried out in control (PBS, with 0.1% BSA) or IL-1ra -treated DSS and control mice from 2 cohorts of animals over a period of 3 months. Data are shown as individual data points as well as mean ± SEM. For statistical analysis, GraphPad Prism was used, and differences compared between control- vs DSS-treated animals by a 2-way ANOVA (factor treatment x drug), followed by Bonferroni post hoc test for % time in the open arms, closed arms and total distance travelled. A total of 37 animals were used in 2 cohorts. One mouse was eliminated due to technical difficulties and lack of video, while 3 outliers were identified using the Grubbs’ test. A P value of ≤0.05 was considered significant.