Animals

All experimental protocols were approved by the Institutional Animal Care and Use Committees (IACUC) at Michigan State University (MSU), USA. Mice of both sexes were used at 8–12 weeks of age and were maintained in a temperature-controlled environment (Innovive, San Diego, CA; Innocage system with ALPHA-dri® bedding) on a 12-hour light: dark cycle with access to acidified water and food ad libitum. Transgenic mice were generated on a C57Bl/6 genetic background. Sox10CreERT2 transgenic mice were a gift from Dr. Vassilis Pachnis (The Francis Crick Institute, London, England)54 and their effectiveness for targeting enteric glia has been described in prior work19,40,55. Sox10CreERT2 mice were bred with Adora2bf/f mice (Ozgene, Bentley DC, WA, Australia)14,56,57, to create a conditional ablation of A2BRs in glia (hereafter referred to as Sox10CreERT2+/−; Adora2bf/f). Littermates not expressing Cre were used as background controls (hereafter referred to as control). Cre recombinase was activated 2 weeks before experiments and induced throughout the study by feeding animals chow containing 400 mg/kg tamoxifen citrate (Envigo, Indianapolis, IN, USA). All mice (including controls) were fed with the tamoxifen diet for the same duration to control for the potential effects of tamoxifen. Genotyping was performed by Transnetyx, Inc (Cordova, TN).

Dextran Sodium Sulfate (DSS) induced colitis

Acute colitis was induced by adding 2% DSS (colitis grade, M.W. 36–50 kDa; MP Biomedical, Solon, OH) to drinking water (2% w/v) for 7 days. Mice were euthanized at 1 day and 3 weeks after the DSS treatment to capture effects at the peak (P-DSS) and resolution (R-DSS) of inflammation time-points, respectively. Mice were weighed every other day and macroscopic damage of intestinal tissue was assessed as described previously34. Spleen weights were also obtained and normalized to body weights.

Pellet weights and fluid content

Pellet composition was assessed as previously described27. Animals were individually housed for 1 h in empty cages and their fecal pellets were collected and weighed (wet weight). Pellets were then dried for 24 h at 60 °C and weighed again to obtain their dry weight. Fluid content was calculated as follows: Fluid content (%) = 100 × (wet weight − dry weight) / wet weight.

Slide and whole-mount immunohistochemistry (IHC)

Mouse distal colons were fixed overnight in Zamboni’s fixative at 4 °C or for 2 h in 4% paraformaldehyde at room temperature. After rinsing in PBS, the fixed colons were micro dissected to obtain whole-mount preparations of myenteric plexus or cryoprotected in 30% sucrose dissolved in phosphate buffer (m/v) before preparing 20 µm thick frozen sections in the Investigative Histopathology Laboratory at Michigan State University. Immunolabeling was conducted as described below and antibody details are supplied in Tables S2–3. Briefly, slides and whole mounts were rinsed three times (10 min each) in phosphate-buffered saline (PBS) or PBS containing 0.1% Triton X-100 (PBST) followed by a 30 or 60 min incubation in blocking solution [containing 4% normal goat serum (or 5% normal horse serum), 0.1 or 0.4% Triton X-100 and 1% bovine serum albumin]. Primary antibodies were applied overnight at room temperature (RT) or for 48 h at 4 °C before being rinsed three times with PBS. Secondary antibodies were applied for 1 or 2 h at RT. Whole mounts were rinsed with 0.1 M phosphate buffer and mounted on slides with bicarbonate-buffered glycerol (consisting of a 1:3 mixture of 142.8 mM sodium bicarbonate and 56.6 mM carbonate to glycerol).

Image acquisition and analysis

Epifluorescence images were acquired through the ×20 [(PlanApo, 0.75 numerical aperture (n.a.)] and ×40 (PlanFluor, 0.75 n.a.) objectives of an upright epifluorescence microscope (Nikon Eclipse Ni, Melville, NY) with a Retiga 2000R camera (QImaging, Surrey, BC, Canada) controlled by QCapture Pro 7.0 (QImaging). Confocal images were acquired through the ×40 (UPLFL, 1.3 n.a.) and ×60 (Plan-Apochromat, 1.42 n.a.) oil immersion objectives of an inverted Olympus Fluoview FV1000 microscope (Olympus, Center Valley, PA).

Epifluorescence images were analyzed offline using ImageJ software (National Institutes of Health, Bethesda, MD). Cell counts were performed using the cell counter plug-in of ImageJ software. Enteric neuron and glial cell numbers are presented as ganglionic packing density, calculated by counting the number of HuC/D-immunoreactive neurons or s100β-immunoreactive glia within the defined ganglionic area cell counts, and ganglionic expression data analysis were performed on a minimum of 10 ganglia per animal and averaged to obtain a value for that animal. N values represent the number of animals in each experiment and data is expressed as percent water control or mean intensity units (i.u.). Confocal images were processed by MetaMorph 7.0 software (Molecular Devices, LLC, San Jose, CA, United States) or by FV10-ASW 04.02.02.09 software (Olympus Corporation, Shinjuku, Japan).

In situ production of cAMP

Live whole mounts of myenteric plexus were micro dissected from the distal colon on ice-cold DMEM/Nutrient Mixture F-12 (Life Technologies) supplemented with L-glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Then the preparations were placed in an incubator (37 °C in a 95% O2 / 5% CO2) for 30 min and subsets of preparations received NECA (10 µM), forskolin (10 µM), or vehicle. After a 15 min incubation, samples were fixed with 4% paraformaldehyde (2 h, RT) and stained for cAMP, GFAP, and HuC/D, imaged and analyzed as described above.

Ca2+ imaging

Live samples of colonic myenteric plexus were prepared for Ca2+ imaging as previously described27. Briefly, colonic segments were collected in ice-cold Dulbecco’s modified Eagle medium (DMEM) and transferred to Sylgard-coated open diamond-shaped bath recording chambers. Tissue segments were opened along the mesenteric border and the mucosa, submucosa, and longitudinal muscle were removed by microdissection to expose the myenteric plexus. The resulting circular muscle myenteric plexus (CMMP) preparations were incubated for 15 min at room temperature in an enzyme mixture consisting of 150 U/mL Collagenase type II and 1 U/mL Dispase (Life Technologies) dissolved in DMEM. CMMP were washed 3 times with DMEM and then loaded with 4 μM Fluo-4 AM, 0.02% Pluronic F-127 and 200 μM water-soluble probenecid (Life Technologies) in DMEM for 45 min at 37°C (5% CO2, 95% air). Whole mounts were superfused with Krebs buffer (37 °C) at 2–3 mL min−1 and drugs were bath applied: BAY 60–6583 (10 nM, 1 µM, and 10 µM) or NECA (10 µM), and then ADP (100 µM) as a positive control. Images were acquired every 1 to 2 s through the ×40 water-immersion objective (LUMPlanFI, 0.8 numerical aperture) of an upright Olympus BX51WI fixed stage microscope (Olympus, Center Valley, PA) using IQ2 software or NIS-Elements software (version 4.5) and a Neo sCMOS or an Andor Zyla sCMOS camera (Andor, South Windsor, CT).

Chemicals and reagents

BAY 60–6583, NECA, and PSB 603 were obtained from Tocris (Bio-Techne Corporation, Minneapolis, MN). Other chemicals and reagents, unless otherwise stated, were purchased from Sigma-Aldrich (St. Louis, MO).

Histology

Sections of the distal colon were fixed overnight in Zamboni’s fixative at 4 °C. The tissue was washed with PBS until cleared. Hematoxylin and Eosin (H&E) staining was performed on 20 µm thick cross-sections of the distal colon by the Investigative Histopathology Laboratory at MSU. A disease activity assessment was performed as previously published34 and criteria for disease activity scoring are identified in Table S4.

Cytokine/chemokine multiplex array

Mouse colon tissue samples were homogenized on ice using a tissue grinder in a buffer (pH 7.5) containing: Tris/Tris-HCl (25 mM), NaCl (130 mM), KCl (2.7 mM), Tween 20 (5% v/v), and a tablet with protease inhibitor cocktail (Sigma S8820). The samples were sonicated with 10 pulses of 1 s intervals and centrifuged at 10,000 × g for 10 min at 4 °C. Supernatant protein concentrations were measured on a NanoDrop™ Lite Spectrophotometer (ThermoFisher Scientific) and samples were adjusted to have the same concentration. An aliquot of each sample was further diluted (2–4×) and coded. A mouse 31-Plex cytokine/chemokine array was run by Eve Technologies (Calgary, AB, Canada) who were blinded to the identity of samples, and analytes with concentrations less than 1 pg/ml were excluded from further analysis.

In vivo assay of the intestinal permeability

Mice had ad libitum access to food and water. Between 9 and 11 am (ZG 2–4) mice were weighted, briefly anesthetized, and gavaged with 1% body weight (volume to mass) of a cell-impermeant fluorescein-5-(and-6)-sulfonate (1 mg/ml, 478.32 Da; Life technologies corporation, Carlsbad, CA) or fluorescein isothiocyanate-dextran (FITC-Dextran, 4 kDa, Sigma Cat #46944). Blood was collected 5 h after the gavage by decapitation and left in a refrigerator (+4 C) overnight. Samples were centrifuged in a pre-chilled centrifuge (+4 C) at 3000 g for 15 min. Serum was diluted 1:2 with dd-water (50 µl of each) and fluorescence intensity was measured on a Synergy™ H1 microplate reader (BioTek Instruments, Inc., Winooski, Vermont, USA) using Gen5™ software (BioTek Instruments, Inc.) using 495/520 nm (excitation/emission) wavelengths.

Paracellular permeability of the distal colon

Intestinal barrier function was assessed using Ussing chambers as described previously19. Briefly, segments of distal colon were mounted in Ussing chambers (aperture 0.3 cm2; EasyMount Ussing Chamber system, Physiologic Instruments, San Diego, CA, USA), equilibrated for 20 min, and a cell-impermeant fluorescein-5-(and-6)-sulfonate (478.32 Da; Life technologies corporation, Carlsbad, CA) was added to the mucosal chamber (0.05 mg ml−1). Samples from the serosal chamber were taken before the dye was added and every 20 min for 2 h (100 μL duplicates and buffer was replenished). Fluorescence intensity was measured on a Synergy™ H1 microplate reader using Gen5™ software using 495/520 nm (excitation/emission) wavelengths. Intestinal permeability was assessed from the slope of fluorescence values of the last four time points. Experiments were performed in a blinded fashion where the experimenter was unaware of the genotype, DSS treatment, or neutralization antibody injections.

In vivo neutralization

One day after the completion of the DSS treatment, mice received an intraperitoneal injection of 100 µg of either a rat anti-mouse CXCL1 (R and D Systems Cat# MAB453, RRID: AB_2087696) or a rat IgG2A isotype control (R and D Systems Cat# MAB006, RRID: AB_357349). Intestinal barrier function was assessed 3 weeks after the treatments using Ussing chambers as described above.

Flow cytometry of mouse colon lamina propria

Leukocytes from the colon lamina propria were isolated using a modification of a published protocol58. Briefly, colons were harvested and flushed with ice-cold Epithelial Removal Buffer (ERB) composed of HBSS without Ca2+ or Mg2+ (Cat. No. 14185, Thermo Fisher Scientific) and supplemented with 10 mM HEPES and 5 mM EDTA. After removing the remaining mesentery, colon tissues were cut into smaller pieces, placed into a conical tube, and incubated with the fresh aliquot of the ERB for 10 min at 4 °C with occasional mixing. The tissues were transferred into another fresh aliquot of the ERB and the process (incubation for 10 min at 4 °C, vortexing, and the tissue transfer) was repeated for additional 2 times. Then, preparations were rinsed twice with a regular HBSS (Cat. No. 14065, Thermo Fisher Scientific) substituted with 10 mM HEPES and digested in this buffer substituted with Liberase TM (0.13 Wünsch units/ml) and DNAse I (100 Kuntz units/ml) for 30 min at 37 °C with occasional mixing. A gentleMACS Dissociator and gentleMACS C tubes were used for cell dissociation. The enzyme reaction was stopped by an ice-cold ERB substituted with 10% fetal bovine (FBS, Denville Scientific, Inc). Cell suspensions were filtered through 100 µm and 40 µm cell strainers. Filtered cell suspensions were spun down (10 min at 350 × g and 4 °C), resuspended, and aliquoted in 1.5 ml centrifuge tubes containing 1 ml of PBS at RT. Cells were resuspended in 100 ul PBS containing Zombie Yellow™ Fixable Viability dye (BioLegend cat # 423103, diluted 1:500) for 15 min at RT, then washed with 1 ml of PBS and spun down at 400 × g for 5 min at 4 °C (following washing/centrifugation steps had the same conditions). Cells were incubated with flow cytometry buffer (FCB, filtered 1% bovine serum albumin in PBS) containing TruStain FcX™ (anti-mouse CD16/32 antibody, Biolegend cat # 101319, 1:100) for 15 min at 4 °C. Primary conjugated antibodies (Table S5) were added and incubated for 1 h at 4 °C. Cells were washed twice with FCB, resuspended in 300 µl of FCB, and analyzed on a BD LSR II [Becton Dickinson (BD) Biosciences, San Jose, CA] run by BD FACSDiva v8.0.1. Of note, a regular AmCyan bandpass filter was exchanged for 585/42. Compensation was performed using unstained cells and single-stained beads. UltraComp eBeads™ Compensation Beads (Invitrogen™ cat # 01-2222-42) and ArC™ Amine Reactive Compensation Bead Kit (Thermo Scientific cat # A10346)were used for conjugated antibodies and Zombie NIR ™ Fixable Viability dye. Each fully stained sample was run acquiring 20,000 events. Fluorescence minus one (FMO) controls were run for proper gating. Post-hoc analysis was performed using FCS Express 7 Research Edition (Win64) v7.01.0018.

Primary cultures of mouse enteric glia

Enteric glia were isolated and cultured as described elsewhere34. Briefly, colons were flushed with ice-cold DMEM/Nutrient Mixture F-12 (Life Technologies) supplemented with L-glutamine and HEPES, the remaining mesentery was removed, and colons were cut in half to yield two 3–4 cm segments. The segments were individually placed on plastic rods (≈2 mm in diameter) and cotton swabs wetted with the ice-cold DMEM were used to remove the myenteric plexus and longitudinal muscle layers. Preparations were rinsed twice with HBSS substituted with HEPES (10 mM) and digested in 2.5 ml of HBSS/HEPES buffer substituted with Liberase TM (0.13 Wünsch units/ml) and DNAse I (100 Kuntz units/ml) for 30 min at 37 °C with occasional mixing. A gentleMACS Dissociator and gentleMACS C Tubes were used for cell dissociation. The enzyme reaction was stopped by an ice-cold complete medium composed of DMEM/Nutrient Mixture F-12 supplemented with L-glutamine and HEPES, penicillin and streptomycin (100 U/mL and 100 µg/mL), and 10% fetal bovine serum (FBS, Denville Scientific, Inc). Cell suspensions were filtered through a 100 µm cell strainer and plated in 24-well plates on coverslips coated with poly D-Lysine (100 µg/ml) and laminin (25 µg/ml). Cell suspensions were initially seeded in the complete medium and plates were placed in an incubator (37 °C, 95% O2/5% CO2). New media composed of DMEM/Nutrient Mixture F-12 supplemented with L-glutamine and HEPES, and substituted with antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL), Gibco™ N-2 Supplement (0.2%), Gibco™ G-5 Supplement (0.2%), and mouse NGF-β (0.05%) was exchanged every 2 days. Cells reached 50–80% confluency with 2 weeks. Experimental drugs including IL-1β (1 ng/ml), PSB 603 (1 µM), NECA (10 µM), PSB 603 and NECA, PSB 603 and IL-1β, or vehicle were added to media the day before collection for RNA isolation and analysis by quantitative PCR (qPCR) as described below.

Isolation of RNA and RT–qPCR

TRIzol™ Reagent (Thermo Fisher Scientific, 15596026) or RLT buffer were used for RNA extraction from frozen tissue and cultured glia, respectively, in concert with RNeasy Mini Kit (Cat. No. 74104, QIAGEN) and DNase I (Cat. No. 79254, QIAGEN) following manufactures instructions. The concentration of the isolated RNA was determined on NanoDrop™ Lite Spectrophotometer (ThermoFisher Scientific). The same amount of total RNA per experiment was used for generation of cDNA utilizing High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368814, ThermoFisher Scientific) and Bio-Rad T100 Thermal Cycler running the following program: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min and hold at 4 °C. Diluted cDNA (1:5) was used as a template for quantitative PCR (qPCR) reactions utilizing Power SYBR™ Green PCR Master Mix (Cat. No. 4367659, ThermoFisher Scientific) and primer sets (Tables S6–7) purchased from Integrated DNA Technologies, Inc. The reactions were run in the Research Technology Support Facility Genomics core at MSU on a QuantStudio 7 Flex qPCR instrument with QuantStudioTM Real-Time PCR Software v1.1. All reactions following cycling conditions: 1× (50 °C for 2 min), 1× (95 °C for 10 min), 40× (95 °C for 15 s and 60 °C for 1 min), and hold at 4 °C. All reactions also had a control ROX dye and the ROX fluorescence was unchanged in all reactions. Dissociation curves were performed at the end and data with 2 or more peaks were not included in analysis performed by QuantStudioTM Real-Time PCR Software v1.7.1. Obtained Ct values were normalized to Cdh17 or 18 S rRNA for the expression of tight junction genes in whole colon tissue or immune mediator transcript abundance in cultured glia, respectively, and presented as a relative expression from the median control sample using the standard ΔΔCt method.

Statistical analysis

Data were analyzed using Prism 7 (GraphPad Software, San Diego, CA) and are shown as mean ± standard error of the mean (SEM) unless stated otherwise such as mean ± standard deviation (SD), median ± range, and median ± 95% confidence interval. The DSS-treated group was assigned by splitting the same genotype and sex littermates into two cohorts and treating one of them with DSS. The DSS-treated cohort had an extra member if the total of the same genotype and sex littermates was an odd number. Male and female data were pooled and tested for normality using D’Agostino and Pearson or Shapiro–Wilk tests. Outliers were excluded using the ROUT method (Q = 5%). Sample sizes were decided based on prior published studies. Randomization and confounders were not controlled. Data were analyzed by two-way analysis of variance (ANOVA) followed by Tukey, Bonferroni, or Sidak post hoc tests or when applicable a two-tailed Welch’s t-test and one-tailed one-sample t-test. A P value of less than 0.05 was considered significantly different from the null hypothesis. Statistical details of experiments such as the statistical tests used, the exact value of n and what n represents, the definition of center and dispersion/precision measures (e.g., mean ± SD) can be found in the figure legends, figures, and Results.