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Environmental-mediated intestinal homeostasis in neonatal mice Courtney Culbreath, BS,a Scott M. Tanner, PhD,b Venkata A. Yeramilli, PhD,c Taylor F. Berryhill, BS,a Robin G. Lorenz, MD, PhD,d and Colin A. Martin, MDa,* a
Department of Surgery, University of Alabama, Children’s of Alabama, Birmingham, Alabama Department of Biological, Earth, and Physical Sciences, Limestone College, Gaffney, South Carolina c Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama d Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama b
article info
abstract
Article history:
Background: Immunoglobulin A (IgA) plays a key role in coating luminal antigens and
Received 26 December 2014
preventing translocation of harmful bacteria. The aryl hydrocarbon receptor (AhR) is a
Received in revised form
basic helix-loop-helix transcription factor that when stimulated activates factors impor-
23 March 2015
tant for barrier function and intestinal homeostasis. We hypothesize that AhR signaling is
Accepted 1 April 2015
critical for establishment of intestinal homeostasis in neonates.
Available online xxx
Material
and
methods:
Mice:
C57BL/6
(B6)
AhRþ/þ
wild
type
(WT),
B6.AhR/
Aryl-hydrocarbon receptor knockout (KO), and B6.AhRþ/þ raised on an AhR ligand-free Keywords:
diet (AhR LF). Enzyme-linked immunosorbent assay was used to measure fecal and
Immunoglobulins
serum IgA levels. Bacterial translocation was measured by culturing the mesenteric lymph
Necrotizing enterocolitis
nodes.
B cells
Results: Two week old KO mice had significantly less fecal IgA compared with WT (and AhR
Intestinal homeostasis
LF, P value ¼ 0.0393. The amount of IgA from the gastric contents of 2-wk-old mice was not significantly different. At age 8 wk, AhR LF mice had significantly less fecal IgA than WT and KO P value ¼ 0.0077. At 2 wk, KO mice had significantly higher levels of bacterial translocation and at 8 wk AhR LF had significantly higher levels of bacterial translocation compared with WT. Conclusions: In neonatal mice, the lack of AhR signaling is associated with loss of intestinal homeostasis, evidenced by decreased levels of IgA and increased bacterial translocation. In adult mice, exogenous AhR ligand and not receptor signaling is necessary for maintenance of intestinal integrity. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Establishment of mutualism between the bacteria inhabiting the intestinal lumen and the mucosa is critical for growth and development. Stimulation from exogenous pollutants,
microbial products, and dietary components (collectively known as xenobiotics) helps to establish factors important for barrier function and intestinal immunity [1]. If the host is unable to establish this symbiotic relationship, there is loss of intestinal homeostasis and a compromised intestinal barrier.
* Corresponding author. Department of Surgery, Children’s of Alabama, University of Alabama at Birmingham, 1600 7th Ave. S., Lowder Building Suite 300, Birmingham, AL 35233. Tel.: þ1 205 638 9688; fax: þ1 205 975 4972. E-mail address:
[email protected] (C.A. Martin). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.04.002
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Lack of development of this critical relationship between the luminal environment and the mucosa early in life is one mechanism that leads to necrotizing enterocolitis (NEC). The incidence of NEC in low birth weight infants in the United States is 13% incurring an economic burden between $500 million and $1 billion annually [2,3]. The exact cause of NEC is unknown, but evidence points to an inability of the immature immune system to maintain intestinal homeostasis and guard against environmental insults such as changes in the composition or virulence of the intestinal microbiota [4], dietary changes [5], and ischemia [6,7]. A large body of work exists on the study of factors that contribute to this delicate balance, yet there is a relative paucity of therapeutic targets that have led to improved outcomes in neonates with intestinal injury. The aryl hydrocarbon receptor (AhR) is a basic helix-loophelix transcription factor that when stimulated by xenobiotics, propagates factors important for barrier function and intestinal immunity [1]. These findings have been confirmed in adult intestinal models of inflammatory bowel disease [8]; however, its role in neonates is not known. 2,3,7,8Tetrachlorodibenzo-p-dioxin, the most studied AhR ligand, exposure during breast feeding has been shown to decrease Bcell migration from the peritoneal cavity to the intestine in mice resulting in decreased fecal immunoglobulin A (IgA) levels [9]. IgA plays several key roles in the innate immune response including antigen entrapment, limitation of bacterial translocation, and microbial sampling to establish host and/or microbe mutualism [10]. At birth, breast milk is the only source of IgA and has been shown to have a protective effect on intestinal immunity. After age 2 wk, maturing B cells have the potential to secrete IgA primarily from an innate B-cell population, B1a cells [11]. In early life, the environmental stimuli that are responsible for a mature immunoglobulin repertoire are not well understood. AhR signaling may be a critical link between the potentially hostile extrauterine environment and the developing neonatal intestinal immune system. Both exogenous (synthetic) and endogenous (natural) ligands exist, which can stimulate AhR activity. Currently, the mechanism as to how potentially harmful extrauterine insults affect the developing immune system is not known. The aim of this study was to determine the developmental contribution of AhR signaling to immunoglobulin development and maintenance of intestinal homeostasis.
Specialty Products, Milford, NJ (#ADT60). Light cycles were alternated at 12 h on and 12 h off. Animals were bred and maintained under specific pathogen-free conditions in Thoren Isolator racks (Hazleton, PA) under positive pressure. Specific pathogen-free conditions at the University of Alabama at Birmingham include absence of the following organisms, as determined by serological screening: mouse parvoviruses, including mouse parvovirus-1 (MPV-1), MPV-2, and minute virus of mice; mouse hepatitis virus, Theiler murine encephalomyelitis virus; mouse rotavirus (epizootic diarrhea of infant mice), Sendai virus; pneumonia virus of mice; reovirus; Mycoplasma pulmonis; lymphocytic choriomeningitis virus; mouse adenovirus; ectromelia (mousepox) virus; K polyoma virus; and mouse polyoma virus. Testing and other methods were as described at uab.edu/Sites/ComparativePathology/ surveillance/. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
2.2. Feces and serum collection for immunoglobulin analysis Fresh fecal pellets were collected from individual mice at age 2 and 8 wk and kept on ice after weighing the contents in microcentrifuge tubes. Sterile phosphate-buffered saline (PBS) supplemented with 0.05% NaN3 and 10-mL/mL mammalian protease inhibitor (SigmaeAldrich, St. Louis, MO) was placed in the tube per 10 mL/mg of feces then vortexed. After centrifuging at 12,000 rpm for 10 min in the microcentrifuge tube, the supernatant collected was stored at 20 C for later analysis. Blood was collected by cardiac puncture. Serum samples were stored at room temperature for 1 h. Serum was separated from blood cells by centrifuging at 12,000 rpm for 5 min. Serum was stored a 20 C until further analysis.
2.3.
Bacterial translocation to mesenteric lymph nodes
The mesenteric lymph nodes (MLN’s) were harvested from mice at age 2 and 8 wk and homogenized in PBS. Two hundred microliters of sample was plated on Schaedler gel plates Thermo Oxoid Remel, Waltham, MA (R454522) and cultured under aerobic conditions at 37 C. After 4 d, the colonies were counted to determine colony-forming units (CFU).
2.4. Gastric contents collection for immunoglobulin analysis
2.
Materials and methods
2.1.
Mice
C57BL/6J were purchased from Jackson Laboratories (Bar Harbor, ME). C57BL/6 AhRþ/- were made available to our laboratory by Dr Chris Bradfield, University of Wisconsin. Mice were created from Ahrþ/- AhRþ/- breeders to generate AhR KO mice and were fed autoclaved NIH-31 rodent diet (Harlan Teklad, Madison, WI) and sterile drinking water ad libitum. AhR ligand-free (AhR LF) mice were generated from C57BL/6J mice that were provided an AhR LF diet (Harlan Teklad [#TD130959]) as well as being housed with AhR LF paper bedding Shepard
For analysis of gastric contents, 2-wk-old mice were sacrificed, stomachs dissected, and all contents removed. The samples were then weighed and resuspended in 1-mL enzyme-linked immunosorbent assay sample buffer (50 mM Tris, pH 7.4, 0.14 M NaCl, 1% bovine serum albumin, 0.05% Tween 20). All samples were stored at 20 C until analysis.
2.5. Enzyme-linked immunosorbent assay for immunoglobulin synthesis Samples were stored at 20 C until analysis. A 96-well plate (Immunlon; Krackeler Scientific, Lenexa, Kansas; 4 Assay
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Plate) was coated with goat anti-mouse Ig (IgA; Southern Biotech, Birmingham, AL at 1 mg/mL) and allowed to incubate overnight at 4 C. The mixtures were aspirated and washed with washing buffer three times. Subsequently, 200 mL of blocking buffer was placed into each well and incubated for 2 h at room temperature. After washing each well three times with washing buffer, 50 mL of alkaline phosphataseelinked goat anti-mouse IgM or IgA (Southern Biotech) was added to each well and incubated for 2 h at room temperature. After the final three washes of washing buffer, 100 ml of pNPP substrate solution (Sigma #N-2770) was added to each well and incubated in the dark at room temperature for 30 min Fifty microliters of 3N sodium hydroxide was added to stop the reaction. The plate was read at 405 nm using a Roche Molecular Diagnostics, Pleasanton, CA microplate reader. Concentrations of IgA were determined based on a standard concentration curve and analyzed using SoftMax Software, Molecular Devices, Sunnyvale, CA (version, and so forth).
2.6.
B Cell Isolation
Spleen, MLN, and Peyer patches (PP) were removed and a single cell suspension prepared by mechanical disruption from 8-wk mice. Intestinal tissue was collected and cells isolated using the Miltenyi Biotec Mouse Lamina Propria Dissociation Kit (Miltenyi Biotec, Auburn, CA) following the manufacturer’s protocol. Briefly, PP and fat was removed, and tissue was opened longitudinally. Tissue was then cut into 0.5-cm pieces. To isolate intraepithelial lymphocytes, the intestinal tissue was then incubated twice for 30 min at 37 C with gentle shaking in Hank balanced salt solution containing 5% newborn calf serum (NCS; HyClone, Logan, UT), 5-mM ETDA (SigmaeAldrich), and 1-mM DTT (SigmaeAldrich). Media containing intraepithelial lymphocytes s were collected, washed, and further purified and collected at the interface of a 40%/75% Percoll (GE Healthcare Life Sciences, Fairfield, CT) gradient. Remaining tissue was then incubated with the components of the Miltenyi Biotec Lamina Propria Dissociation Kit in Hank balanced salt solution containing Ca2þ and Mg2þ and 5% NCS for 30 min at 37 C with gentle shaking. After incubation, tissue was dissociated using the m_intestine_01 program on the Miltenyi Biotec gentleMACS Dissociator (Miltenyi Biotec), releasing lamina propria cells. Cells were washed and further purified and collected at the interface of a 40%/75% Percoll (GE Healthcare Life Sciences) gradient. Cells were washed in PBS supplemented with 2.5% NCS and prepared for downstream experiments. Peritoneal cells were collected by peritoneal cavity lavage using 2.5% NCS in PBS.
2.7.
FACS staining
Cells isolated from spleen, MLN, PP, small intesinal lamina propria, colonic lamina propria, and peritoneal cavity were stained with CD19 (Clone 1D3; BD Biosciences, Franklin Lakes, NJ), anti-B220 APC (Clone RA3-6B2; BD Biosciences), CD11b PerCP-Cy5.5 (Clone M1/70; BioLegend, San Diego, CA), and CD5 Biotin (Clone 53-7.3; BD Bioscience) þ Streptavidin FITC (BD Biosciecnes). All samples were then run on a BD FACSCalibur
3
flow cytometer and analyzed with FlowJo Software (Tree Star, Inc, Ashland, OR).
2.8.
Statistical analysis
GraphPad Prism version 6.0 software was used for data analysis and all figures (GraphPad Software Inc, La Jolla, CA). Oneway analysis of variance with Tukey posttest analysis was used to analyze continuous variables. Results were expressed as the mean the standard error of the mean. A P value of