Environmental-mediated intestinal homeostasis in neonatal mice

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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

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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 <0.05 was regarded as significant.

3.

Results

3.1.

AhR-mediated developmental IgA expression

Serum-IgA levels demonstrated significance at 2 wk with AhR LF (0  0), showing significantly less IgA compared with that of wild type (WT, 571.5  84) and AhR KO (785  213), P value ¼ 0.0054 (Fig. 1A). At 8 wk, the differences of serum IgA between the groups were not significant (Fig. 1B). These results suggest that AhR has a systemic effect of IgA levels during the early immune development. In addition, we wanted to determine the contribution of AhR to serum fecal of IgA. Fecal IgA analysis from the stool of 2ewk-old mice confirmed that AhR KO mice compared with WT and AHR LF mice had significantly less IgA levels in the stool (37  37 mg/mL) compared with WT (2519  807 mg/mL) and AhR LF (3043  764 mg/mL), P value ¼ 0.0393 (Fig. 1C). However, in adult mice, AhR LF mice (1769  369 mg/mL) had significantly less fecal IgA than WT (17,574  4916 mg/mL) and KO (12,553  2666 mg/mL), P value ¼ 0.0077(Fig. 1D).

3.2.

Maternal contribution of IgA

Based on these findings, we sought to determine the maternal contrition of IgA levels in neonates. Gastric contents were used as a proxy to determine IgA levels in breast milk in the three groups of neonatal mice. The amount of IgA from the gastric contents of 2-wk-old mice was not significant between groups, WT (40  14 mg/mL), KO (40  18 mg/mL), and AhR LF (24  5 mg/mL), P value ¼ 0.322 (Fig. 2). Therefore, the differences in IgA between the three groups were not due to different levels in breast milk.

3.3.

Intestinal homeostasis and bacterial translocation

Based on the findings of AhR-mediated differential IgA expression, we next sought to determine AhR’s role in bacterial trafficking from the lumen to the MLN. MLNs were cultured and the CFU’s counted to determine the contribution of AhR signaling to maintenance of intestinal homeostasis in neonatal and adult mice. At 2 wk, AhR KO mice had significantly higher levels of bacterial translocation (158  37 CFU) compared with those of WT (2  1 CFU) and AhR LF (9  5 CFU), P value ¼ 0.0132 (Fig. 3A and B). At 8 wk, AhR LF had significantly higher levels of bacterial translocation (119  57 CFU) compared with those of WT (15  7) and KO (11  10), P value ¼ 0.019 (Fig. 4A and B).

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Fig. 1 e Fecal and serum IgA levels. (A) IgA concentration (mg/mL) of 2-wk-old serum of WT, AhR LF, and KO mice. *WT versus AhR LF, P value [ 0.0024. AhR LF versus KO, P value [ 0.0331, n [ 3e6. (B) Eight-week-old serum IgA levels from WT, AhR LF, and KO mice. Nonsignificant (NS), n [ 3e9. (C) Two-week-old fecal IgA levels from WT, AhR LF, and KO mice. *WT versus KO, P value [ 0.0393. KO versus AhR LF, P value [ 0.0054, n [ 3e6. (D) Eight-week-old fecal IgA levels from WT, AhR LF, and KO mice. *WT versus AhR LF, P value [ 0.0077, n [ 8e16.

3.4.

Lymphocyte and B-cell analysis

Finally, we sought to determine the contribution of AhR stimulation of lymphocyte development. We demonstrated total lymphocytes are reduced in the mucosal lymphoid tissues (gut-associated lymphatic tissue [GALT]), PPs and MLNs, but not peripheral tissues such as the spleen (Fig. 5A). This reduction happens only in the AhR LF mice suggesting that dietary AhR ligands are required for the development of immune cells in the GALT. Next, we determined that total CD19 þ B220þ B cells are reduced (Fig. 5B). Because PPs are the inductive sites for IgA secretion, fewer B cells in these tissues

Fig. 2 e Two-week IgA contents. IgA concentration (mg/mL) of gastric contents from 2-wk-old WT, AhR LF, and KO mice. Nonsignificant (NS), n [ 4e9.

would explain why we see reduced IgA in the fecal pellets in the 8-wk-old mice.

4.

Discussion

Dietary intake and bacterial colonization early in life help shape the developing immune system and microbiota. The ability to sense harmful luminal antigens and maintain the mucosal barrier is critical for health and well-being [12]. Conversely, some signals received from luminal components are vital for epithelial growth and function [13]. Lack of development of this synergistic relationship is one mechanism that leads to intestinal injury early in life. In the present study, we have shown that the AhR signaling plays a vital role in regulating IgA secretion early in life. Furthermore, dietary stimulation and specific environmental cues are required to maintain IgA secretion and intestinal homeostasis during development. These findings describe a novel link between environmental stimuli and the developing immune system in neonates. Immunoglobulin development early in in life is critical for establishment of a competent immune system [11]. Particularly, IgA plays a vital role establishing protection. Secretory IgA from developing B1a are critical for luminal surveillance by relaying critical information to the mucosal lymphatic tissue [14,15]. The factors that are responsible for intestinal bacterial populations being harmful or beneficial are largely dependent of the host’s response and the microbial environment. The new concept of the host’s environment shaping a

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Fig. 3 e Two-week bacterial translocation. (A) Two-week-old bacterial cultures from MLN from WT, AhR LF, and KO mice. Results expressed as CFU. *WT versus KO, P value [ 0.0132. AhR LF versus KO, P value [ 0.0051. (B) Representative agar plates showing CFU, n [ 3e4. (Color version of the figure is available online.)

virulent bacterial phenotype is termed “pathobiome” development [13,16,17]. IgA from either developing plasma cells or from breast milk is critical in preventing pathobiome development. Two week old WT mice used in our experimental design that have the equivalent intestinal development of a 24-wk fetus [11,18], had nearly a 10-fold decrease in fecal IgA levels compared with adult mice. In our neonatal mice, lack of AhR receptor signaling resulted in significantly less fecal IgA compared with the other groups. This decrease of fecal IgA in the 2wk AhR KO mice also corresponded with significantly more bacterial colonization of the MLN. These findings together suggest that lack of AhR signaling in neonatal mice is associated with worse intestinal homeostasis evidenced by increased trafficking of luminal bacteria from the intestinal lumen to the MLN. In addition to patrolling the intestinal lumen, immunoglobulins are also critically involved in inciting an appropriate systemic immune response to luminal antigens. The MLNs are part of the GALT [19]. Luminal antigens that traverse the mucosal defense systems then collect in the MLNs and PP. It is there that the antigen-specific immune response is propagated with secretion of cytokines, chemokines, and upregulation of other immune cells [20e22]. In our study, serum levels of IgA were significantly lower in 2-wk AhR LF free mice

compared with the other groups. This is not congruent with the fecal IgA data. One explanation for this is the relative lack of serum IgA in the 2-wk AhR KO mice leads to more bacteria traversing the mucosal barrier and entering the MLN. More bacteria in the MLN lead to a systemic antibody response resulting in higher levels of IgA in the serum of AhR KO at 2 wk. Another explanation is that absence of AhR ligands during gestation may be detrimental to GALT development thus resulting in decreased baseline systemic antibody levels in neonatal mice. Unpublished data from our laboratory suggest that AhR LF mice have lower total lymphocyte counts in the MLN and PP compared with those in WT and AhR KO. To better understand the contribution of AhR signaling to developmental immunoglobulin secretion and establish of intestinal homeostasis, we used AhR LF mice that have not had any exposure to exogenous AhR ligands. AhR ligands have been found in standard rodent chow and corn-cob bedding; therefore, this experimental group was maintained on food and bedding free of AhR ligands. AhR is a highly conserved receptor that is stimulated by a variety of exogenous and endogenous compounds [23e26]. 2,3,7,8-Tetrachlorodibenzop-dioxin, an environmental pollutant, is the most studied exogenous AhR ligand. However, there is convincing evidence that AhR has physiologic functions, and several endogenous

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Fig. 4 e Eight-week bacterial translocation. (A) Eight-week-old bacterial cultures from MLN from WT, AhR LF, and KO mice. Results expressed as CFU. *WT versus AhR LF, P value [ 0.0199. (B) Representative agar plates showing CFU, n [ 5e9. (Color version of the figure is available online.)

ligands have been identified [27]. Many endogenous ligands are tryptophan metabolites that are broken down by bacterial species in the intestine [28,29]. In this study, the AhR LF

neonatal mice had equivalent fecal IgA levels with WT mice. The finding is likely explained by equivalent contribution of maternal IgA before weaning, as evidenced by similar levels of

Fig. 5 e (A) Total lymphocyte count. (B) Total B-cell count. (A) Percentages and counts of CD19D B220D B cells as determined by flow cytometry. Cells were isolated and stained as described in the methods section. n [ 8e14 animals. For spleen, PP, and MLN. *P value <0.001, n [ 8e14.

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IgA in 2-wk-old gastric contents. However, in adult mice (8 wk) after weaning, the AhR LF group has significantly less IgA. These findings suggest that environmental stimulation of AhR receptor is needed to maintain intestinal homeostasis after weaning. There is a growing body of literature on the function of AhR on intestinal immune development. Kiss et al. [30] demonstrated that AhR-deficient mice were more susceptible to Citrobacter rodentium infection secondary to underdeveloped innate lymphoid follicles and T-cell-dependent mechanisms. In addition, Li et al. [8] demonstrated that AhR deficiency resulted in decreased T-cell receptor intraepithelial lymphocyte development. Collectively, these studies demonstrate that AhR stimulation has a T cellmediated immune protective phenotype. The results from our analysis complement the previously mentioned studies. Two-week AhR KO mice had decreased levels of fecal IgA suggesting a protective effect of AhR signaling in early B-cell development. However, in adults, mice environmental AhR stimulation and not receptor signaling is necessary to maintain intestinal homeostasis. These findings in context with prior studies on AhR immune development suggests a presently unknown mechanism of AhR ligand stimulation in maintenance of IgA homeostasis. Our data should be interpreted with the understanding that AhR affects many immune regulatory cells and compartments. For example, AhR signaling is essential for interleukin 22 secretion by Th-17 cells (3) and maintenance of intraepithelial lymphocytes (2). These findings may be secondary to AhR affects these or other immune systems. However, there is a relative paucity of mechanistic data of AhR’s effect of developing B-cells function. Future studies will further clarify this mechanism B analyzing B cell-specific AhR knockout mice in our experimental modeling.

5.

Conclusions

In conclusion, AhR signaling is necessary for the establishment of intestinal homeostasis in neonatal mice. Lack of AhR receptor signaling leads to increased bacterial trafficking in neonatal mice. However, environmental stimulus of the AhR receptor is needed to maintain intestinal mutualism after the neonatal period. This mechanism of loss of intestinal homeostasis mediated by decreased B-cell secretion of IgA may be one presently unknown mechanism of NEC. Future studies will clarify the role of environmental AhR ligands in immune development and test these findings in other intestinal disease models.

Acknowledgment This study was supported by John W. Kirklin Research Foundation (grant number 3115299.000.453115299.311850000.0000), Kaul Pediatric Research Institute (grant number 321558.01.01.2011670.10), and Association of Academic Surgery Joel J. Roslyn Faculty Research (grant number 324219.01.01.2013075.10) Award.

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Authors’ contributions: C.A.M. conceived the study concept and design. T.F.B. provided the mice used. C.C performed the experiments then collected and analyzed the data. C.A.M. and C.C. prepared the article. R.G.L., V.A.Y, and S.M.T. provided technical support and conceptual advice. All authors discussed the results and implications and edited the article.

Disclosure The authors have no financial and personal relationships with other people or organizations that could potentially and inappropriately influence (bias) their work and conclusions to disclose.

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