IL-10 Receptor Neutralization-Induced Colitis in Mice: A Comprehensive Guide

Abstract

Interleukin-10 (IL-10) and its receptor (IL-10R) have been forefront targets to understand inflammatory bowel disease (IBD) pathogenesis. For the past several decades, IL-10 deficient (Il10^−/−) mice were considered one of the best models to study immune-mediated colitis. Several physiologic limitations with this model i.e. delayed and varied disease onset has hindered investigators in testing new clinical therapies for IBD. In this protocol, we provide a comprehensive guideline of IL-10R monoclonal antibody (αIL-10R mAb) neutralization as a superior and an alternative model to study IBD. This article describes the feasibility for αIL-10R mAb to induce chronic colitis (within 4 weeks), perform time-dependent mechanistic studies, and assess the efficacy of IBD therapeutics. This article also delineates protocols for in-house assays to critically assess colitis and associated inflammatory parameters. Overall, we underscore αIL-10R mAb neutralization as a relevant immune-mediated murine colitis model to study human Crohn’s disease.

Basic Protocol 1:

Induction of chronic colitis in mice via αIL-10R mAb neutralization

Basic Protocol 2:

Biochemical evaluation of αIL-10R mAb neutralization-induced chronic colitis

Support Protocol 1:

Stool analysis and scoring

Support Protocol 2:

Swiss roll method

INTRODUCTION

Interleukin-10 (IL-10), originally named cytokine synthesis inhibitory factor, functions as a pleiotropic, inhibitory cytokine against inflammatory mediators secreted from activated immune cells (Fiorentino, Bond, & Mosmann, 1989; Vieira et al., 1991). For appropriate immune balance, IL-10 binds to a corresponding heterodimer receptor (IL-10R) composed of a ligand domain (IL-10Rα) and a signaling transduction subunit (IL-10Rβ), in which IL-10R activation upregulates anti-inflammatory genes that inhibit NF-κB dependent inflammation (Figure 1) (Ding et al., 2003; Kotenko et al., 1997; Shouval, Ouahed, et al., 2014). In the gastrointestinal tract, IL-10 is produced by macrophages, neutrophils, natural killer cells, T cells, and B cells to maintain mucosal homeostasis and conserve immune tolerance during inflammation (Morhardt et al., 2019; Saraiva & O’Garra, 2010; Shouval, Biswas, et al., 2014; Wei, Li, Sun, & Guo, 2019).

Figure 1: Blockade of IL-10R signaling induces colitis.

IL-10 interaction with the heterotetrameric IL-10R complex leads to STAT3 phosphorylation by JAK1 and TYK2. STAT3 then forms a homodimer and undergoes nuclear translocation to bind STAT3-binding elements of IL-10 responsive genes. This induces the expression of anti-inflammatory cytokines that inhibit various inflammatory pathways related to NF-κβ activation. In the healthy gut, IL-10R signaling maintains immunologic tolerance to microbial antigens. Blocking IL-10R signaling by a monoclonal antibody (αIL-10R mAb) impedes STAT3 translocation into the nucleus and promotes NF-κβ-dependent inflammatory responses. As a result, the following gut bacteria dysbiosis and impaired immunologic response toward microbial antigens collectively contribute to colitis progression. STAT3: Signal transducer and activator of transcription 3; JAK1: Janus kinase 1; TYK2: Tyrosine Kinase 2; NF-ĸB: Nuclear factor kappa-light-chain-enhancer of activated B cells; MØ: Macrophage; Neu: Neutrophil; NK: Natural killer cell, Th1: T helper cell 1; Th2: T helper cell 2, IBD: Inflammatory bowel disease.

A defect resulting in the loss of IL-10 production and/or IL-10R signaling is therefore a major risk factor for the development of spontaneous inflammatory diseases (Figure 1). Most notable are single nucleotide polymorphisms of either IL-10 or IL-10R in humans that increase the susceptibility for inflammatory bowel diseases (IBD) like ulcerative colitis, Crohn’s disease, and very-early-onset IBD (Amre et al., 2009; Franke et al., 2008; Glocker et al., 2009; Kotlarz et al., 2012; Lees, Barrett, Parkes, & Satsangi, 2011; Moran, Klein, Muise, & Snapper, 2015; Moran et al., 2013; Zhu, Lei, Liu, & Wang, 2013). This has made mouse models of IL-10 and IL-10R deficiency relevant to study IBD (Bernshtein et al., 2019; Hoshi et al., 2012; Keubler, Buettner, Hager, & Bleich, 2015; Kuhn, Lohler, Rennick, Rajewsky, & Muller, 1993; Spencer et al., 1998). Several limitations (e.g., delayed and heterogenous disease) of these gene knockout models, however, substantiates the need to develop an alternative model of IL-10 deficiency or lack of its signaling for inducing murine colitis.

We outline blockade of IL-10R signaling, by a commercially available monoclonal antibody (αIL-10R mAb), in mice as a clinically relevant approach to research human IBD. This protocol describes how the αIL-10R mAb can be utilized to study transmural, chronic colitis in a gradual manner. Detailed assessment of disease activity index, histopathological grading, immunopathology, and colonic mucosal cytokine levels are addressed for this model.

STRATEGIC PLANNING

All experiments involving live animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must conform to federal regulations for the care and use of laboratory animals. Certain genetically altered mouse strains may develop rectal prolapse, a severe form of irreversible colitis, following treatment with αIL-10R mAb (Figure 2) (Singh, Kumar, et al., 2016; Singh, Yeoh, et al., 2016; Singh et al., 2017). Diligent monitoring is therefore required to check for the chance of rectal prolapse and resulting body weight loss when inducing colitis in genetically altered animals. In accordance with IACUC guidelines and National Institute of Health regulations, live mice with rectal prolapse or mice that lose more than 20% of their body weight must be immediately euthanized. This information must be included in the protocol submitted for IACUC review and approval.

Figure 2: Rectal prolapse develops in severe cases of αIL-10R mAb-induced colitis.

Representative pictures of (A) healthy rectal region from isotype treated mice, (B) rectal prolapse in αIL-10R mAb treated mice. Yellow arrow indicates rectal prolapse.

Colitogenesis development is reported to be distinct in C57BL/6 and BALB/c mice due to their Th1- and Th2-biased immune responses, respectively (Davidson et al., 1998; Gorham et al., 1996; Specht, Arriens, & Hoerauf, 2006). Our prior studies signify that mouse genetic background does not impact the αIL-10R mAb experimental design as mice from either strain showed similar colitis progression (Singh, Yeoh, et al., 2016; Singh et al., 2017). Comparatively, it is well recognized that housing mice either in a conventional or specific pathogen free (SPF) environment can dictate colitis severity with the latter having a reduced or delayed disease phenotype. Two strains of Helicobacter spp. in the gut microbiota, noted as H. hepaticus and H. bilis, have been isolated as bacterial agents that can induce chronic colitis in SPF conditions (Danne & Powrie, 2018; Kullberg et al., 2006; Kullberg et al., 1998). Importantly, both microbes have been identified in institutional and commercial mice (Shames et al., 1995; Taylor, Xu, Nambiar, Dewhirst, & Fox, 2007; Treuting, Clifford, Sellers, & Brayton, 2012). It is therefore recommended to confirm whether the planned experimental mice are positive for either H. hepaticus or H. bilis by sequencing or any other means. If the proposed mice are positive for either Helicobacter spp. mentioned, then the chance increases for body weight loss, robust colitis and even rectal prolapse.

It is important to note that αIL-10R mAb does not induce intestinal injury nor colitis directly but rather the neutralization of IL-10R results in an overactive immune response to inflammatory stimuli i.e., gut microbial antigens. This indicates that mice with spontaneous innate immune dysfunction and/or microbiota dysbiosis could be more susceptible to αIL-10R mAb-induced colitis. Due to the genotype- and phenotype-specific nature of αIL-10R mAb-induced colitis, it is strongly recommended to use proper littermates for experimental and control groups. Also, a sufficient number of animals (n=6–10 mice per treatment group) should be used to account for disease variability and power analysis. As αIL-10R mAb-induced colitis is also microbiota dependent, all experimental mouse groups should have a common origin (strain, vendor, colony, sex, age, water, food, etc.). Further, investigators must carefully record the diet (regular or autoclaved) and its composition, water source (acidified/ autoclaved), and type of bedding materials including nestlets for reproducibility purposes.

BASIC PROTOCOL 1

Induction of chronic colitis in mice via αIL-10R mAb neutralization

In this protocol, we describe chronic colitis induction by αIL-10R mAb neutralization in mice as a clinically relevant model to study human Crohn’s disease. In brief, mice (6–8 weeks old; males and females; n=6–10) are treated with four weekly injections of αIL-10R mAb (1.0 mg/mouse, i.p., Bio X Cell) to induce transmural, chronic colitis as performed previously (Saha, Singh, Xiao, Yeoh, & Vijay-Kumar, 2016; Singh, Kumar, et al., 2016; Singh, Yeoh, Carvalho, Gewirtz, & Vijay-Kumar, 2015; Singh, Yeoh, et al., 2016; Singh et al., 2017; Singh et al., 2019). Control mice receive the isotype control (IgG1) antibody (Bio X Cell). After αIL-10R mAb administration, mice are monitored twice weekly for body weight and the presence of mucus in the stool. Onset and progression of disease over time can be monitored by measuring fecal lipocalin 2 (Lcn2), which we had previously shown to be an effective, non-invasive, and real-time biomarker of intestinal inflammation (Chassaing et al., 2012). Mice are euthanized one week post the 4^th αIL-10R mAb injection and colitis-related parameters in Flow Chart 1 are analyzed. Note: Immediately euthanize any mice that exhibit a greater than 20% body weight loss or develop rectal prolapse in accordance with IACUC guidelines.

Materials and reagents

• C57BL/6 or BALB/c mice from 6–8 weeks of age (n=6–10 per experimental group)

• αIL-10R mAb [InVivoMAb, anti-mouse IL-10R (CD210), Bio X Cell, Cat# BE0050]

• Isotype control antibody (rat IgG1, Bio X Cell, Cat# BE0088)

• BD insulin syringes with BD Micro-fine™ IV needle U-100 (Ref# 329410)

• Scale

• 70% ethanol

• Sterile phosphate buffered saline (1 x PBS)

• Sterile 0.5 ml and 1.5 ml microcentrifuge tubes

• Sterile forceps

• Sterile scissors

• BD 1 ml, sterile Sub-Q syringe 26G x 5/8 (0.45mm x 16mm) (Ref# 309597)

• BD Microtainer® tubes with BD Microgard closure (Ref# 365967)

• Ruler

• Graph pad paper

• Cell phone or camera

• Ice

• Dry ice

Preparation of mice for αIL-10R mAb neutralization-induced chronic colitis

• 1Acclimate mice procured from vendors in institute vivarium for 5–7 days before starting the experiment.
• 2Mark each mouse (either by ear punch, numbered tag, or other convenient methods) for identification.
• 3Note the initial Day ‘0’ body weight.

  Average body weight for 6–8 week old mice is 18–20 g but if one mouse deviates from the littermates then it can be considered an outlier and thus, excluded.

• 4Assign each mouse to their appropriate experimental (αIL-10R mAb) and control (isotype control antibody) groups.
• 5Collect stool samples from the experimental and control groups prior to the first αIL-10R mAb administration.

  Place a single mouse in a clean, empty cage without bedding material for 15–30 min and aseptically collect the stool using clean forceps into a sterile microcentrifuge tube. Store at −80°C. These samples will serve as the basal (i.e. Day 0) reference point for monitoring colitis progression in the experimental group(s).

• 6Administer αIL-10R and IgG1 isotype control antibodies (1.0 mg/mouse, i.p.) to the respective experimental and control groups.

  Although 1.0 mg/mouse is the recommended dose for an 18–20 g mouse, the amount of αIL-10R mAb may need to be standardized according to the susceptibility of mice to colitis based on other contributing factors (e.g., genetic variability, microbiota status, or vivarium cleanliness). If mice are going to be challenged with an additional compound, decide the route, dose, volume, vehicle and mode (prophylactic or therapeutic) of administration. If the intervention is water insoluble, the suitable vehicle must be given to the control group in a similar method. It is recommended to limit the volume administered to 0.2 ml (i.p., i.g., i.v. or s.c.) to ease the way of administration.

• 7Place experimental and control mice into separate, clean cages with water and food.

  Do not mix control and αIL-10R mAb given mice in the same cage because microbiota transfer via coprophagy may cause either false positive or negative results (‘cage effect’). If specialized diets (purified or compositionally defined diet, CDD) are planned with αIL-10R mAb-induced colitis experiments, record the diet composition. Our prior study indicates that αIL-10R mAb-treated WT mice on CDD develop greater colonic pathology than when fed a standard grain-based chow diet (Singh et al., 2019).

Monitoring mice for αIL-10R mAb neutralization-induced colitis

• 8Administer αIL-10R and IgG1 isotype control antibodies once a week (total of 4 weeks).
• 9Measure body weight twice a week for the entire 4 week study.

  Generally, mice administered αIL-10R mAb maintain their body weight. If rectal prolapse is observed, then moderate to severe body weight loss occurs and euthanasia is required.

• 10Collect stool twice a week and macroscopically analyze to monitor for colitis severity (See Support Protocol 1).

Euthanizing mice for αIL-10R mAb neutralization-induced chronic colitis

• 11Euthanize the mice one week after the 4^th αIL-10R and IgG1 isotype mAb injections.
• 12Perform terminal bleeding (using suitable method) into BD Microtainer tubes with BD Microgard closure collections.

  Centrifuge tubes at 10,000 rpm for 10 min at 4°C for separating and collecting hemolysis-free serum to be stored in −80°C until further analysis (See Basic Protocol 2).

• 13Sterilize the mouse by spraying 70% ethanol.
• 14Carefully expose the mouse by ventral midline incision.
• 15Remove and weigh the spleen.

  Usually, increased spleen weight (alias splenomegaly) correlates with the degree of inflammation (Figure 3B). You can optionally plate spleen lysates (100 mg/ml in sterile 1 x PBS) on non-selective agar plates to examine the potential extent of systemic disseminated intestinal bacteria.

• 16Isolate and check the relative size of the mesenteric lymph nodes.

  If pertinent to the study, process the mesenteric lymph nodes for immune cell profile via flow cytometry and/or gut bacteria translocation via plating lysates (100 mg/ml in sterile 1 x PBS) on non-selective agar plates.

• 17Arrange the organs and tissues in the visceral region so that the colon and cecum are visible.
• 18Carefully dissect out the colon and cecum together by separating them from the small intestine at the ileocecal junction and from the anus at the distal end of rectum.

  The use of blunt forceps is highly recommended when separating the colon from the connecting tissue to avoid damaging the organ during excision.

• 19Take representative pictures of the inflamed and healthy intestine samples (from cecum to rectum) on graph paper to visually depict the gross morphology (Figure 3A).
• 20Straighten but do not stretch the colon to measure its length (if necessary disconnect the neighboring, creeping fat).
• 21Separate the proximal colon from the cecum.
• 22Save the entire cecum at −80°C.

  Bacterial content or other metabolite and co-metabolite (e.g. host, bacterial) analysis can be later performed.

• 23Gently dry the colon and weigh the tissue.

  Increased colon weight (i.e. colomegaly) (Figure 3C) is a prominent sign of colitis.

• 24Cut several pieces of the immediate portion of the proximal colon and store at −80°C for later analysis of inflammatory and colitis parameters (See Basic Protocol 2).
• 25Perform colon Swiss roll (See Support Protocol 2) that will be later used for histological analysis (See Basic Protocol 2).

Figure 3: αIL-10R mAb induces chronic colitis in WT mice.

WT mice (n=6, male, 8 weeks old) were treated with αIL-10R mAb (1.0 mg/mouse, 4 weekly injections, i.p.) or isotype control antibody. One week after the 4th injection, mice were euthanized and analyzed for the following parameters. (A) Gross colon and spleen (blue arrows show thickening of proximal colon). Percent (B) Spleen and (C) Colon weights. (D) Fecal Lipocalin 2 (Lcn2) during colitis progression. (E) Serum Lcn2. (F) Colonic Myeloperoxidase (MPO) activity. Histochemical staining for colon sections (G) H&E and Alcian blue. (H) Histologic score assessed by visualizing the entire H&E and Alcian Blue stained colon section for goblet cell loss, epithelial hyperplasia, distorted crypt structure, and mucosal thickening. N.D. = not detected in isotype given WT control mice. Black double headed arrows indicate hyperplasia in colitic colon. Data represented as mean ± SEM. **p<0.01 and ***p<0.001.

BASIC PROTOCOL 2

Biochemical evaluation of αIL-10R mAb neutralization-induced chronic colitis

Once the study is complete, biochemical evaluation at the mRNA, protein and functional levels are critical to analyze. This includes the assessment of routine inflammatory and colitis markers in serum and colonic tissue. Expected results are depicted in Figure 3.

Materials and reagents

• BD Microtainer® tubes with BD Microgard closure (Ref# 365967)

• Carbonate-Bicarbonate buffer (Sigma, C3041)

• Flagellin (Flagellin FliC VacciGrade™, Invivogen)

• Lipopolysaccharide from Escherichia coli O128:B12 (Sigma, Ref# L2755)

• Goat serum (Sigma, Cat# G9023)

• Tween® 20 (Sigma, Cat# P1379)

• Anti-mouse IgG-HRP (produced in goat, Southern Biotech, Cat# 1030–05)

• Anti-mouse IgA-HRP (produced in goat, Southern Biotech, Cat# 1040–05)

• TMB (BD OptEIA™, Cat# 555214)

• 96-well ELISA plate (Costar)

• 96-well plate (Corning)

• Hexadecyltrimethylammonium bromide (Sigma, Cat# H9151)

• Guaiacol (alias 2-methoxyphenol, Alfa Aesar, Ref# A16319)

• Hydrogen peroxide (Sigma, Cat# H1009)

• Radioimmunoassay Precipitation Assay (RIPA) Buffer (Sigma, Cat# R0278)

• Protease inhibitor cocktail (Sigma, Cat# P8340)

• RNAlater (Sigma, Cat# R0901)

• TriZol (TRI reagent, Sigma, Cat# T9424),

• Chloroform (Sigma)

• Iso-propanol (Sigma)

• 75% ethanol

• RNase free water (Sigma)

• Heat block

• qScript cDNA SuperMix (Quanta Bio, Cat# 95048),

• SYBER Green (Quanta Bio)

• Hematoxylin and Eosin (H&E) stain kit (Vector laboratories, Cat# H-3502)

• Alcian blue kit (Vector laboratories, Cat# H-3501)

• 10% neutral buffered formalin (NBF) (Sigma)

• Hanks’ Balanced Salt Solution (HBSS, Sigma Cat# H9394)

• Penicillin & streptomycin (Fisher Scientific, Cat# BP2959)

• Serum-free RPMI1640 medium (Sigma, Cat# R7388)

• ELISA Plate-coating Buffer (R&D Systems, Cat# DY006)

• Bovine serum albumin (Albumin, Bovine, Cohn Fraction V, 98%, Immunoassay Grade, protease and enzyme-free, pH 7.0, Alfa Aesar, Cat# J65731–22)

Fecal lipocalin 2 as a surrogate marker of intestinal inflammation

Fecal samples can be processed and analyzed for lipocalin 2 (Lcn2) as a quantitative measurement of time dependent inflammation (See Support Protocol 1, Figure 3D).

Measurement of serum inflammatory markers

Acute phase proteins such as serum amyloid A (SAA) and/or Lcn2 can be measured to generate a quantitative measurement of inflammation.

• 1a. Collect hemolysis-free serum as described in Basic Protocol 1.

• 2a. Perform enyzme linked immunosorbent assay (ELISA) designed toward SAA and Lcn2 as per manufacturer’s instructions (e.g., DuoSet ELISA Kit, R&D Systems).

Dilute control serum samples 1:100 for SAA and 1:200 for Lcn2. As samples from αIL-10R mAb treated mice exhibit elevated circulating inflammatory markers (e.g. serum Lcn2, Figure 3E), a significantly higher dilution is required and must be titrated according to disease severity.

When conducting this ELISA, we observe these inflammatory proteins to be present at elevated levels (i.e 3–4 fold increase) with αIL-10R mAb treated mice when compared to isotype given mice. While healthy mice generally exhibit a range of 100–250 ng/ml for SAA and Lcn2 in circulation, colitis severity can impact the systemic amount of SAA and Lcn2.

Detection of intestinal permeability

A surrogate approach to assess intestinal barrier permeability is by measuring serum immunoreactivity toward anti-flagellin and anti-lipopolysaccharide (LPS) antibodies (Singh, Yeoh, et al., 2016; Ziegler et al., 2008). Flagellin and LPS are bacterial products that may increase in circulation for αIL-10R mAb-treated mice (data not shown).

• 1b. Collect hemolysis-free serum as described in Basic Protocol 1.

• 2b. Coat 100 ng/well flagellin or 2 μg/well LPS in 9.6 pH biocarbonate buffer (100 μl/well).

• 3b. Incubate overnight at 4°C.

• 4b. Wash each well (200 μl) with wash buffer (0.05% goat serum and 0.01% Tween®20 in 1 x PBS)

• 5b. Add 100 μl of sample diluted in wash buffer (dilution range 1:200–1:500).

• 6b. Incubate at 37°C for 1 h.

• 7b. Wash three times with 200 μl/well of wash buffer.

• 8b. Add 100 μl/well of anti-mouse IgG-HRP or anti-mouse IgA-HRP (1:1000 in wash buffer).

• 9b. Incubate at 37°C for 1 h.

• 10b. Wash three times with 200 μl/well of wash buffer.

• 11b. Add 100 μl/well of TMB

• TMB should be kept at room temperature 1 hr before use.

• 12b. Incubate at room temperature for 5–10 min (avoid light).

• 13b. Add 100 μl/well of TMB stop solution and read OD at 450 nm.

When performing these ELISA’s, we observe serum immunoreactivity to flagellin and LPS to be present at elevated levels( i.e. 2–3 fold increase) with αIL-10R mAb treated mice when compared to isotype given mice. While healthy mice generally exhibit an O.D. range of 0.2–0.4 of immunoreactivity to these two microbial products, confounding factors like gut microbiota composition can affect the fold change of bacterial dissemination for mice with colitis.

Measurement of colonic myeloperoxidase (MPO) activity

Colonic MPO activity positively correlates with the extent of neutrophil infiltration and thus, degree of inflammation (Figure 3F). Measuring the activity can be performed as mentioned in detail from our previous studies (Chassaing, Aitken, Malleshappa, & Vijay-Kumar, 2014; Singh, Yeoh, et al., 2016).

• 1c. Collect ≥50 mg of the most immediate portion of the proximal colon (closest to cecum) and freeze at −80°C.

• 2c. Homogenize fresh or frozen colon samples in 1 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide.

• 3c. Freeze-thaw the samples 3 times, sonicate, and centrifuge (10,000 g, 4°C)

• 4c. Collect the clear supernatants into a fresh tube.

• 5c. In a 96-well plate, add 10 μl of colonic supernatant and 10 μl of 0.1 M phosphate buffer.

• 6c. Start the reaction by adding final concentrations of 50 mM guaiacol and 0.002% H₂O₂.

• 7c. Measure the change in absorbance at 470 nm over a period of 10 mins at 1 min intervals.

One unit of MPO activity is defined as the amount that increases absorbance at 470 nm by OD of 1.0 per minute at 25°C, calculated from the initial rate of reaction using guaiacol as the substrate. Use recombinant MPO (Sigma) as a positive control.

We observe αIL-10R mAb treated mice to exhibit more MPO activity in the colon tissue (i.e. 2–3 fold) increase when compared to isotype given mice. While healthy mice generally exhibit a 0.2–0.4 U/gm range of MPO activity, colitis severity can impact the amount of elevated MPO activity.

Analysis of mucosal cytokine levels

Intestinal inflammation can be determined via cytokine parameters in the colonic tissue.

• 1d. Collect 20–30 mg of the most immediate portion of the proximal colon (closest to cecum) and freeze at −80°C.

• 2d. Fresh or frozen colonic tissue is processed with RIPA buffer containing containing 1x protease inhibitor cocktail (100 mg/ml).

• 3d. Homogenize the samples.

• 4d. Centrifuge for 20 min at 12,000 rpm and 4°C.

• 5d. Collect the clear supernatants and store at −80°C until analysis.

• 6d. Perform ELISA designed toward MPO and IL-1β as per manufacturer’s instructions (e.g., DuoSet ELISA Kit, R&D Systems).

Normalize the results with total protein content via standardized methods (e.g. Bradford assay).

When performing these ELISA’s, we observe αIL-10R mAb treated mice to exhibit more mucosal cytokines i.e., 2–3 and 4–5 fold increase for MPO and IL-1β, respectively, when compared to isotype given mice.

Ex vivo colon culture and analysis

Culturing colon tissue and measuring proinflammatory cytokines in the supernatant is another method to provide information regarding severity of inflammation.

• 1e. Longitudinally cut ~1.0 cm of proximal colon and wash thrice serially in 37°C Hanks’ Balanced Salt Solution (HBSS) with 1.0% antibiotics (penicillin & streptomycin).

• 2e. Place the washed colons in a 24 well plate containing 1.0 ml of serum-free RPMI1640 medium with 1.0% antibiotics (penicillin & streptomycin) and incubate at 37°C with 5.0% CO₂ for 24 h.

• 3e. Collect the culture supernatants and centrifuge for 10 min at 2000 rpm and 4°C, and then store at −80°C until analysis.

• 4e. Perform ELISA designed toward pro-inflammatory cytokines i.e., IL-1β, TNF-α, IFN-γ, IL-12p40, IL-23, and IL-17 as per manufacturer’s instructions. The following can be analyzed by DuoSet ELISA as previously described (Vijay-Kumar et al., 2007; Zhang, Fu, Sun, Li, & Guo, 2014).

We observe αIL-10R mAb treated mice to have increased secretion of the pro-inflammatory cytokines listed here (e.g., TNFα can exhibit 2–3 fold increase), but colitis severity can cause variation.

Transcript analysis of inflammatory genes via quantitative real time PCR

qRT-PCR for the quantification of a mouse cytokine transcripts is a routine assay to analyze inflammation.

• 1f. Collect 50–60 mg of the most immediate portion of the proximal colon (closest to cecum), store in RNAlater, and freeze at −80°C.

• 2f. Remove colons from RNAlater and extract RNA by any standard procedure (e.g. Trizol method) for qRT-PCR [example reference (Rio, Ares, Hannon, & Nilsen, 2010)].

Candidate genes include, but not limited, to IL-1β, TNF-α, IFN-γ, IL-12p40, IL-23, and IL-17.

Thermal profile for the reaction is: initial denaturation at 95°C for 10 min, and 40 cycles of denaturation (95°C for 15 s) and annealing and extension (60°C for 1 min). Relative fold difference between groups is calculated using comparative Ct (2^−ΔΔCt) method. Normalize with a housekeeping gene (e.g., 36B4, 18S). When comparing isotype given mice with a calculated average Ct value of 1, we observe αIL-10R mAb treated mice to have increased transcript expression of the pro-inflammatory genes listed here (e.g., TNFα can exhibit 7–8 fold increase), but colitis severity can cause variation.

Histological staining and scoring of colonic tissue

Hematoxylin & Eosin (H&E) and Alcian blue staining are recommended in colon Swiss roll sections. This will help to detect colonic pathology i.e., severity of inflammation, epithelial hyperplasia, crypt elongation, immune cell infiltration, and goblet cell depletion (Figure 3G). It is also advisable to perform Ki67 staining to confirm the epithelial hyperplasia and measure epithelial cell proliferation.

• 1g. Perform colon Swiss roll (See Support Protocol 2).

• 2g. Place the Swiss roll in a cassette and then temporarily in 10% neutral buffered formalin for 24 h to fixate the sample.

• 3g. Transfer and store samples in 70% ethanol until paraffin embedded and serial sectioned (5 μm) for staining.

• 4g. Stain colonic sections by H&E and Alcian Blue according to manufacturer’s instructions (see Materials and Reagents).

• 5g. Score stained colonic sections in a blinded fashion with a pathologist in all experimental groups of mice.

Histologic scoring can be performed on H&E stained colonic tissue by microscopically visualizing multiple sections of the slide. Each whole section is assessed on a four-scale scoring system based on the extent of immune cell infiltration in mucosa/submucosa and the corresponding number of crypt abscesses, epithelial hyperplasia, mucosal thickness, goblet cell loss, and distorted crypt structure (e.g. elongation). A score of ‘0’ denotes no inflammation with only a few infiltrating immune cells and large presence of goblet cells; ‘1’ denotes a mild lesion corresponding to a one-cell-thick layer in the lamina propria and minor loss in goblet cells; ‘2’ depicts an average two-layer thickness of mucosa and immune cells, including lymphocytes and neutrophils, and a considerable decrease in goblet cell number; ‘3’ implies mostly a three-to-four-cell thickness in lamina propria and severe goblet cell loss; and ‘4’ indicates to a more-than-four-cell thickness or the presence of severe lesions, such as crypt abscesses, strong immune cell infiltration, and depletion of goblet cells (Figure 3H). The average scores for control and αIL-10R mAb-treated groups can then be tabulated to represent the severity of colonic inflammation. Entire colon section results is presented as the mean score ± standard error mean (SEM) with 6–10 mice/group.

SUPPORT PROTOCOL 1

Stool analysis and scoring

Lipocalin 2 (Lcn2) is an acute phase protein generally upregulated in conditions of inflammation. Measuring fecal Lcn2 via ELISA serves as a cost-effective, non-invasive method with a dynamic range broad enough to reflect classic robust gut inflammation (Chassaing et al., 2012). It is recommended to collect fecal samples for Day 0 and twice a week post first αIL-10R mAb injection as this will allow the tracking of colitis severity over time.

Materials and reagents

• Phosphate buffered saline (PBS)

• Tween® 20 (Sigma, Cat# P1379)

• Sterile forceps

• Sterile 0.5 ml microcentrifuge tubes

• DuoSet murine Lcn2 ELISA kit (e.g. R&D Systems)

Fecal sample collection and scoring

• 1Place a single mouse in a clean, empty cage without bedding material for 15–30 min.

  The time for mouse defecation may be delayed (>30 minutes) when mice experience severe intestinal inflammation.

• 2Aseptically collect the stool using clean forceps into a sterile microcentrifuge tube.
• 3Examine the fecal samples and apply a score.

  Stool consistency formed (and hard): 0; Mild soft: 1; Soft: 2; Mucous: 3; Mild diarrhea: 4. In some cases of severe colitis, it is plausible to observe occult blood in the stool, which can be confirmed with the Hemoccult Dispensapak Plus from Beckman Coulter (Fisher Scientific, Cat# 61130).

• 4Store at −80°C until analysis.

Fecal sample processing

• 5Reconstitute freshly collected or frozen fecal samples in sterile 1 x PBS containing 0.1% Tween® 20 (100 mg/ml).
• 6Homogenize the samples to get a fecal suspension.

  If there is occult blood in the feces, suspension appears red in color.

• 7Centrifuge for 20 min at 12,000 rpm and 4°C, collect the supernatant, and store at −80°C until analysis.

Fecal Lcn2 analysis via ELISA

• 8Follow instructions laid by the manufacturer for the DuoSet murine Lcn2 ELISA kit.

  Basal feces samples are diluted 1:10, but colitic fecal sample dilution may need to be higher and standardized according to degree of inflammation.

• 9Expected result is a positive correlation between elevated fecal Lcn2 levels and colitis severity over time (Figure 3D).

SUPPORT PROTOCOL 2

Swiss roll method

A well established approach to analyze the histomorphometric changes caused from colitis is by performing the ‘Swiss roll’. This method generates a colon spiral with enough 3D dimension to microscopically analyze an entire colon when sectioned and stained.

Material and reagents

• 5–10 ml syringe with feeding needle (18G-3” Straight 2.25 mm ball, Braintree Scientific Inc)

• 10% neutral buffered formalin (NBF, Fischer Scientific, SF100)

• 70% ethanol

• Shandon Tissue Cassettes (Fisher Scientific, Cat# B1000729WH)

• Cotton ear swab

Steps

1. Fill sterile 1 x PBS into a 5–10 ml syringe with feeding needle (18G-3” Straight 2.25 mm ball, Braintree Scientific Inc) and flush the colon to remove fecal particles.

   If studying mucous layers and bacterial encroachment, abstain from flushing the colon and immerse a piece of the colon section with feces in Carnoy’s solution (60% v/v dry methanol + 30% v/v chloroform + 10% v/v glacial acetic acid) as reported previously (Johansson et al., 2010) to preserve the mucus architecture.

2. Place the cleaned colon on a clean surface.

3. Longitudinally cut and open out the colon.

4. Using the short rod end of a cotton swab wetted with PBS, wrap the colon inward from proximal to distal end.

   It is recommended to begin the Swiss roll at the proximal side because this section should be the interior part for better histologic view and analysis.

5. Put the now Swiss roll into a cassette and temporarily store in 10% NBF for 24 h.

6. Transfer and store the cassette in 70% ethanol until paraffin embedding and sectioning for histological analysis (See Basic Protocol 2).

COMMENTARY

BACKGROUND INFORMATION

There is a wide range of methods used to induce colitis in mice, for which IL-10 and IL-10R have been either directly or indirectly incorporated as mechanistic factors. Below, we briefly describe three well-known experimental designs of immune-mediated murine colitis and their connection to the IL-10 signaling pathway. Following, we describe how αIL-10R mAb was first generated and utilized as a co-colitogenic agent. Lastly, we delineate the merits of αIL-10R mAb as a fairly new approach to induce colitis in mice.

Genetically Engineered:

Since 1993 and 1998, respectively, disruption of either the IL-10 or IL-10Rβ (alias CRFB4) loci to cause its loss-of-function has been a prevalent model for murine colitis research (Kuhn et al., 1993; Spencer et al., 1998). Accordingly, IL-10 deficient (Il10^−/−) mice served as a colitis model to test rescue therapeutics like IL-10-secreting Lactococcus lactis for IBD alleviation (Steidler et al., 2000). Il10^−/− mice have also been utilized to uncover the anti-inflammatory mechanism(s) of IL-10 with a recent report demonstrating the cytokine to mitigate inflammasome activation via inducing mitophagy and suppressing reactive oxidative species in macrophages (Ip, Hoshi, Shouval, Snapper, & Medzhitov, 2017). A detailed review by Keubler et al. delineates how Il10^−/− mice have advanced our scientific knowledge on colitis development and its plausible immunologic, genetic, and gut microbiota dependent mechanisms (Keubler et al., 2015). It is impressive that Il10^−/− mice would eventually become a widely established model of immune-mediated colitis for scientists over the last 30 years, and even leading to the generation of nearly 50 different double knockout models that have addressed the protective or pathological involvement of other interactive candidate genes in IBD.

Throughout the 1990s and early 2000s, IBD research in mice revealed immune dysfunction and colitis progression to be contextualized to the genetic strain. Sundberg et al. characterized C3H/HeJBir mice (a sub strain of C3H/HeJ) to be inheritably susceptible toward spontaneous colitis (Sundberg, Elson, Bedigian, & Birkenmeier, 1994). Quantitative trait locus (QTL) mapping has pinpointed the cytokine deficiency-induced colitis susceptibility 1 allele as the candidate gene for increased IBD susceptibility in C3H/HeJBir mice (Beckwith, Cong, Sundberg, Elson, & Leiter, 2005; Bleich et al., 2010) and other murine colitis models (Borm et al., 2005; Ermann et al., 2011) including Il10^−/− mice (Farmer et al., 2001; Mahler et al., 2002). Impressively, Berg et al. found colitis severity in Il10^−/− mice to be Th1 and Th2 immune dependent as the degree of disease worsened from C57BL/6J < BALB/c < 129/SvEv mouse strains (Berg et al., 1996). Bristol et al. expanded this by showing the most severe lesions with Il10^−/− mice on the C3H/HeJBir background (Bristol et al., 2000). Further details on the genetic mechanisms associated with Il10^−/− murine colitis can be found here (Keubler et al., 2015).

Pathogen Induced:

Similar groundbreaking work from the late 1990s identified two microaerobic bacteria named H. hepaticus and H. bilis as infectious agents that induce IBD in severe combined immunodeficiency (SCID) mice (Cahill et al., 1997; Shomer, Dangler, Schrenzel, & Fox, 1997). These Helicobacter spp. are now widely utilized as surrogate pathogens to understand the role of gut microbiota in mucosal inflammatory responses [in-depth discussion (Fox, Ge, Whary, Erdman, & Horwitz, 2011)]. Noteworthy, the connection between gut microbiota and murine IBD can be traced back to the original Kühn et al. (1993) study that introduced Il10^−/− mice. The authors found colitis concentrated within the small and large intestines of conventionally raised Il10^−/− mice, and this became more localized to the colonic region when mice were rendered in a SPF environment (Kuhn et al., 1993). Subsequent studies of Il10^−/− mice in germ-free conditions or treated with antibiotics confirmed the gut microbiota to be required for immune dysfunction and enterocolitis (Madsen et al., 2000; Sellon et al., 1998). Importantly, Kullberg et al. identified H. hepaticus as the single bacterial agent that can induce chronic colitis in SPF-reared Il10^−/− mice (Kullberg et al., 1998). QTL mapping has interestingly found the Hiccs gene to regulate H. hepaticus-induced colitis in Il10^−/− mice and other IBD mouse models (Boulard, Kirchberger, Royston, Maloy, & Powrie, 2012). Further details on the gut microbiota-dependent mechanisms associated with Il10^−/− murine colitis can be found here (Keubler et al., 2015).

Adoptive T Cell Transfer:

In 1993, Powrie et al. established that the adoptive transfer of naïve CD4⁺ T cells with high CD45RB expression (CD45RB^hi) into immunocompromised SCID mice induced colitis (Powrie, Leach, Mauze, Caddle, & Coffman, 1993). The same group uncovered that the Th1 dominant immune response post adoptive transfer could be abrogated with the co-transfer of either low CD45RB expression (CD45RB^low) cells or recombinant IL-10 (Powrie et al., 1994). Both interventions resulted with complete protection against colitis in SCID mice. After it was reported that IBD in Il10^−/− mice was T cell dependent and B-cell independent (Davidson et al., 1996), a series of adoptive transfer experiments decrypted that IL-10 produced by CD11b⁺ myeloid cells interact with IL-10Rβ to sustain anti-inflammatory Foxp3 expression in regulatory T cells and this was imperative for protection against colonic inflammation (Murai et al., 2009; Shouval, Biswas, et al., 2014). A modified adoptive transfer model by Ikenoue et al. finds that the introduction of spleen and mesenteric lymph node cells from Il10^−/− mice to SCID mice recapitulated colitis found in Il10^−/− mice (Ikenoue, Tagami, & Murata, 2005). As the mouse recipients in this study developed colitis much earlier than their donor mice, Il10^−/− cell transfer was suggested as an alternate murine colitis model (Ikenoue et al., 2005).

Monoclonal Antibodies:

Solid phase radioimmunoadsorbent assay was used by Mosmann et al. to isolate monoclonal antibodies specific for IL-10 (Mosmann et al., 1990). Anti-IL-10 antibodies were groundbreaking in its in vitro application to confirm the origin of IL-10 production from Th2 cells and its inhibitory effects on acute inflammatory responses (de Waal Malefyt, Abrams, Bennett, Figdor, & de Vries, 1991; Fiorentino, Zlotnik, Mosmann, Howard, & O’Garra, 1991). Not long after, O’Farrell et al. generated a neutralizing monoclonal antibody against IL-10R (αIL-10R mAb) to elucidate how IL-10 activity impacted immune responses and cell proliferation (O’Farrell, Liu, Moore, & Mui, 1998). Another major significance for αIL-10R mAb was to validate the IL-10R complex originally predicted from computer modelling (Liu et al., 1997; Reineke, Sabat, Volk, & Schneider-Mergener, 1998; Zdanov, Schalk-Hihi, & Wlodawer, 1996). The first implication of αIL-10R mAb in murine colitis was when Asseman et al. uncovered that this mAb negated the protective effects of CD45RB^low cells in the adoptive T cell transfer model (Asseman, Mauze, Leach, Coffman, & Powrie, 1999). Similar results were later found in the combined H. hepaticus CD4⁺ adoptive T cell transfer model (Kullberg et al., 2006; Kullberg et al., 2002), and αIL-10R mAb was observed to aggravate H. hepaticus-induced colitis (Kullberg et al., 2006; Morrison et al., 2015). It was not until 2003 when Asseman et al. discovered that administration of αIL-10R mAb alone is ample to induce colitis in adult mice (Asseman, Read, & Powrie, 2003). This observation, however, remained relatively unnoticed in literature, and very little consideration placed toward αIL-10 mAb as a potential murine model of IBD.

αIL-10R mAb neutralization:

Considering the history of immune-mediated colitis models until now, αIL-10R mAb neutralization represents the continual progression to further advance the experimental rigor, reproducibility and feasibility for studying IL-10 dependent IBD. In comparison to the prolonged time lag of 3–4 months for colitis development in global Il10^−/− mice, for instance, αIL-10R mAb-induced chronic colitis happens within 4 weeks and can be induced in younger mice (e.g., 4 weeks old). As αIL-10R mAb mediated colitis develops in a progressive fashion, this model is also practical for time-dependent studies and capable to research acute colitis. The disease incidence and severity following αIL-10R mAb colitis is not variable like seen in Il10^−/− mice and there is very low risk for rectal prolapse development. Since this procedure only requires the intraperitoneal injection technique, bacterial culturing and special housing conditions that would normally be implemented for H. hepaticus-induced colitis are unnecessary from the αIL-10R mAb model. There is also no need for cell sorting via flow cytometry that is obligatory for the adoptive T cell transfer and Il10^−/− cell transfer models. Investigators can certainly employ multiple groups to test disease susceptibility between genetic backgrounds without needing to employ either SCID or recombination activating 1 deficient (Rag1^−/−) mice. In addition, the time consuming and expensive generation of double knockout models as done previously with Il10^−/− mice is exempted from the αIL-10R mAb model. From all these advantages with the αIL-10R mAb model, evaluating the efficacy of prophylactic and therapeutic strategies becomes achievable. Collectively, our protocol supports αIL-10R mAb neutralization as one of the better approaches to induce gut inflammation in mice that resembles human IBD.

CRITICAL PARAMETERS AND TROUBLESHOOTING:

The successful reproducibility of αIL-10R mAb-induced colitis depends on maintaining consistency with genetic variation and strain, vendor source, antibody dose, study duration, age, sex, and microbiota composition (Nell et al., 2010). The environment of the vivarium (i.e. water source, food, bedding material and nestlets) also plays a crucial role in the development of αIL-10R mAb-induced colitis. Researchers should first determine whether C57BL/6, BALB/c or C3H/HeJBir is the most relevant mouse strain for both control and experimental groups. To accurately and precisely differentiate disease pathogenesis, αIL-10R mAb-induced colitis must be gradual. Therefore, our prior experience supports C57BL/6 and BALB/c mice as appropriate strains whereas the high susceptibility in C3H/HeJBir mice may negate the gradual progression needed for αIL-10R mAb-induced colitis.

In line with this, it is important to identify mice positive for either H. hepaticus or H. bilis because the presence of either microbe can accelerate colitis progression. Therefore, if the bacteria are present in the mouse colony, it might be considered to adjust the αIL-10R mAb dose and/or duration. As a general rule of thumb, it is proper to use mice from the same vendor or to compare experimental groups with WT littermates harboring similar gut microbiota. Based on our prior studies, genetically altered mice can have a shift in microbiota composition and altered immune responses, both of which may impact colitis onset. As the microbiome environment and mucosal responses impact each other, tracking the microbiota composition prior and post colitis would be significant if the study demands such analysis.

Once the mouse groups are confirmed, next step is to standardize the optimal αIL-10R mAb dosage and duration in which colitis develops gradually. In this protocol, we have found that a 1.0 mg/mouse (18–20 g mouse) dose and 4 week study (weekly injections) to be suffice in inducing chronic colitis. There is no fixed guideline for the number of αIL-10R mAb injections needed to induce colitis, rather it is at the investigator’s discretion to titrate according to colitis severity based on body weight loss (>20%) and occurrence of rectal prolapse in mice. Under situations for when colitis severity is too drastic to perform a chronic study, it is recommended to perform an acute study with just 1 injection of the αIL-10R mAb. Keep in mind that the source of αIL-10R mAb cited in this protocol does not contain stabilizers nor preservatives (buffer: PBS + 0.01% Tween®, pH 6.5). If researchers want to use αIL-10R mAb from a different vendor, make sure it doesn’t have any carrier proteins and preservatives like sodium azide. Even though the αIL-10R mAb described in this study also contains endotoxin levels at <0.002EU/μg, we found this minimal amount does not impact the colitis phenotype. Irrespective, follow the manufacturers instructions on antibody storage for which the stock concentration is generally stored at 4°C (never freeze). When prepping αIL-10R mAb for injection, place the stock on ice to avoid any abrupt temperature shifts. Be cautious of the injection site as it’s essential to perform i.p. and not subcutaneous. In line with αIL-10R mAb, it is important to determine the volume, dose, vehicle and route of administration for special treatment groups (e.g., drug compound). Everyday administration of a treatment (i.p., i.v., i.g.) is not advisable as such practice may cause severe stress that confounds the study. It is recommended to limit the volume administered to 0.2 ml (i.p., i.g., i.v. or s.c.) to ease the way of administration.

Once the study commences, quantification of fecal Lcn2 is a useful approach to non-invasively track intestinal inflammation as described in Support Protocol 1 (Chassaing et al., 2012). Make sure that stool samples are collected aseptically in case gut microbiota profiling via 16S sequencing is required for the study. Note that the chronic colitis model requires twice a week fecal collection whereas daily stool samples are needed for the acute model. Irrespective, Day ‘0’ feces serve as the basal, reference point for monitoring colitis progression in the αIL-10R mAb-treated experimental group(s). It is also important to be on the lookout for rectal prolapse (Figure 2), another indicator of severe colitis. This observation would initiate the troubleshooting advice mentioned previously on gauging αIL-10R mAb dosage and study duration.

After euthanasia, the gross colon and spleen provides several macroscopic critical parameters of αIL-10R mAb colitis features, such as emptying of the colon (irregular stool formation), inflamed and thickened colon at the proximal region and splenomegaly. Compared to some other murine models of colitis, colon shortening is not prominent in αIL-10R mAb-induced colitis but rather colomegaly is a distinct feature. Rectal prolapse, if observed, is associated with greater colomegaly and splenomegaly. Note: In the case where rectal prolapse develops but there are no indicators of colitis, a defect in the enteric nervous system should be considered.

To obtain correct histologic sectioning, the colon must be rolled from proximal to distal end and the ‘inner rolls’ must face upward for 5 μm horizontal sliced sections. Histologic analysis of the colon further reveals epithelial hyperplasia, focal crypt epithelial distortion, mucin depletion via loss of goblet cells, and immune cell infiltration (e.g., neutrophils, macrophages). Importantly, epithelial erosion is absent in αIL-10R mAb-induced colitis and therefore, measuring antibodies against flagellin and LPS can serve as surrogate markers for bacterial dissemination.

When testing αIL-10R mAb-induced colitis in genetically altered mice or in mice challenged with a new compound, assume that these experiments will result in aggravated disease. This will initiate for continuous and extra observations of the mice for body weight loss, mucousy stool, and/or rectal prolapse. From our previous studies and this detailed protocol, we have characterized αIL-10R mAb-induced murine colitis as a relevant model of human Crohn’s disease. Importantly, αIL-10R mAb-induced murine colitis is relatively mild in WT mice but exacerbated in specific innate immune compromised mice (Table 1). Our best reference is toward mice deficient of either Toll-like receptor 5 (Tlr5^−/−) or Lcn2 who exhibit severe gut microbiota-dependent intestinal inflammation (Carvalho et al., 2012; Singh et al., 2015; Singh, Yeoh, et al., 2016; Singh et al., 2017).

Table 1:

αIL-10R mAb colitis susceptibility in genetically altered mice reported by our group

Gene Deficiency	αIL-10R mAb Colitis Susceptibility	Reference
Il1r1 −/−	Protective	Carvalho et al., 2012
Tlr4 −/−	Protective	Carvalho et al., 2012
Apoe −/−	Susceptible	Singh et al., 2016
Lcn2 −/−	Susceptible	Singh et al., 2016 Singh et al., 2017 Saha et al., 2016
Nlrc4 −/−	Susceptible	Carvalho et al., 2012
Tlr5 −/−	Susceptible	Singh et al., 2015
Tlr5−/−/Il1r1−/−	Protective	Carvalho et al., 2012
Tlr5−/−/Nlrc4−/−	Susceptible	Carvalho et al., 2012
Tlr5−/−/Tlr4−/−	Susceptible	Carvalho et al., 2012

Ilr1, Interleukin 1 Receptor Type 1; Tlr4, Toll-like receptor 4; Apoe, Apolipoprotein E; Lcn2, Lipocalin 2; Nlrc4, NLR Family CARD Domain Containing 4; Tlr5, Toll-like receptor 5.

STATISTICAL ANALYSIS:

All data can be represented as mean ± SEM. The statistical significance between two groups is calculated using unpaired, two-tailed t-test. Data from more than two groups are compared using a one-way ANOVA followed by Tukey’s multiple comparison tests (when to compare the mean of each column with the mean of every other column). p values <0.05 are considered statistically significant and are denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

UNDERSTANDING RESULTS:

αIL-10R mAb neutralization-induced colitis illustrates transmural inflammation and distinctive features of human Crohn’s disease. This colitis model induces gradual inflammation, making it feasible for time-dependent studies and studying acute (1 injection) and chronic (4 weekly injections) conditions. Upon examining the early events of intestinal inflammation like in acute colitis, there is no significant loss in body weight nor impact on gross colon appearance (data not shown). Nonetheless, there are elevated levels of the inflammatory markers SAA, serum KC, and Lcn2 (in both serum and feces) (data not shown). The histological analysis would show moderate levels of acute colonic inflammation in αIL-10R mAb-treated mice (data not shown). Similar to acute colitis, the chronic model does not have severe diarrhea, weight loss, nor mortality risk. The absence of well-formed stools corresponds to the increasing degree of inflammation. αIL-10R mAb-treated WT mice show classic gross features of chronic colitis such as thickened colon (especially at the proximal region) (Figure 3A), splenomegaly (Figure 3B), colomegaly (Figure 3C), and enlarged mesenteric and linguinal lymph nodes (data not shown). Fecal, circulating, and gut inflammatory markers like Lcn2 (Figures 3D-E) and colonic MPO (Figure 3F) are elevated in αIL-10R mAb-treated WT mice. Notably, histologic analysis reveals the typical discontinuous and transmural inflammatory lesions found with αIL-10R mAb-induced chronic colitis (Figure 3G). The proximal colon also displays extensive areas of epithelial hyperplasia, focal crypt epithelial destruction, mucin depletion via loss of goblet cells, and mononuclear infiltration (Figure 3G-H). Transepithelial migration of neutrophils and extensive migration of neutrophils into the crypt lumen (crypt abcess) is also evident in αIL-10R mAb-pathology (data not shown). Increased systemic immunoreactivity toward bacterial products LPS and flagellin is observed in αIL-10R mAb-treated mice indicating increased dissemination of microbial products (data not shown). Further, the blockade of IL-10 signaling alters the gut microbiota composition (Carvalho et al., 2012; Matharu et al., 2009; Sellon et al., 1998; Singh et al., 2015; Singh, Yeoh, et al., 2016; Vijay-Kumar et al., 2007). Importantly, we have found all these parameters from WT mice to be further aggravated in certain genetic deficient mice (such as Tlr5^−/−, Figure 4), including the development of rectal prolapse after the 4^th injection (Carvalho et al., 2012; Singh, Yeoh, et al., 2016; Singh et al., 2017). An additional observation we have seen with genetically altered mice includes cecum shrinking and mild diarrhea (no well-formed stool) (Figure 4A).

Figure 4: αIL-10R mAb triggers severe, chronic colitis in Tlr5^−/− mice.

Tlr5^−/− mice (n=6, male, 8 weeks old) were treated with αIL-10R mAb (1.0 mg/mouse, 4 weekly injections, i.p.) or isotype control antibody. One week after 4th injection, mice were euthanized and analyzed for colitis parameters. (A) Gross colon and spleen (Blue arrows showing thickening of proximal colon and shrunken ceca). Percent (B) Spleen and (C) Colon weights. (D) Fecal Lcn2. (E) Colonic MPO activity. Histochemical staining in colon sections (F) H&E and Alcian blue. (G) Histologic score evaluated by imagining the entire H&E and Alcian Blue stained colon section for loss of goblet cells, epithelial hyperplasia, distorted crypt structure, and mucosal thickening. N.D. = not detected for isotype treated Tlr5^−/− control mice. Black double headed arrows indicate hyperplasia in colitic colon. Data represented as mean ± SEM. **p<0.01 and ***p<0.001.

TIME CONSIDERATIONS:

This model reproducibly recapitulates the transmural inflammation and typical features of human Crohn’s disease. αIL-10R mAb can induce both acute (single injection) and chronic colitis (4 weekly injections). In ideal conditions, chronic disease induction occurs within 3–4 weeks following αIL-10R mAb administration depending on variation in mouse genetic background and gut microbiota composition. The preferable age to begin αIL-10R mAb-induced colitis is 6–8 weeks old for both male and female mice, but colitis can be induced as early as 4 weeks of age. This is a significant advantage in comparison to Il10^−/− mice whose onset of disease occurs between 12 to 16 weeks of age and varies among facilities. αIL-10R mAb-induced colitis develops in a progressive fashion, making it achievable for time-dependent studies. Investigators can certainly employ multiple groups to test disease susceptibility between genetic backgrounds and evaluate the efficacy of prophylactic or therapeutic strategies. As αIL-10R mAb-induced colitis is microbiota-dependent, this model is also feasible for gnotobiotic studies. For experienced researchers, the analysis of αIL-10R mAb-induced colitis can be completed within 2 weeks post animal euthanasia, including the evaluation of feces, serum, and colon culture supernatants for pro-inflammatory cytokine, biomarkers, as well as histologic examination.