Adenosine A2A Receptor Deletion Blocks the Beneficial Effects of Lactobacillus reuteri in Regulatory T-Deficient Scurfy Mice

The lack of a functional Foxp3 transcription factor and regulatory T (Treg) cells causes lethal, CD4+ T cell-driven autoimmune diseases in scurfy (SF) mice and humans. Recent studies have shown that adenosine A2A receptor activation limits inflammation and tissue damage, thereby playing an anti-inflammatory role. However, the role of the adenosine A2A receptor in the development of disease in SF mice remains unclear. Using a genetic approach, we found that adenosine A2A receptor deletion in SF mice (SF⋅A2A-/-) does not affect early life events, the development of a lymphoproliferative disorder, or hyper-production of pro-inflammatory cytokines seen in the Treg-deficiency state. As shown previously, Lactobacillus reuteri DSM 17938 treatment prolonged survival and reduced multiorgan inflammation in SF mice. In marked contrast, A2A receptor deletion completely blocked these beneficial effects of L. reuteri in SF mice. Altogether, these results suggest that although absence of the adenosine A2A receptor does not affect the development of disease in SF mice, it plays a critical role in the immunomodulation by L. reuteri in Treg-deficiency disease. The adenosine A2A receptor and its activation may have a role in treating other Treg dysfunction-mediated autoimmune diseases.


INTRODUCTION
Foxp3 + regulatory T (Treg) cells play a pivotal role in the phenomenon of self-tolerance. In humans, Foxp3 mutations result in immunodysregulation, polyendocrinopathy, and enteropathy, with X-linked inheritance (called IPEX syndrome). Newborn boys with IPEX syndrome have severe enteropathy, eczema, type I diabetes, thyroiditis, hemolytic anemia, and thrombocytopenia; and they die within the first years of life if left untreated (1,2). In the mouse model, Foxp3-deficient scurfy (SF) mice develop a lethal autoimmune disease which closely resembles the IPEX syndrome (3,4). SF mice develop early-onset dermatitis, progressive multiorgan inflammation, and early death within the first month of life due to a lymphoproliferative syndrome. This lethal lymphoproliferative syndrome is predominately mediated by CD4 + T cells in humans and mice (5,6). Consequently, the SF mouse is a valuable model for studying novel therapies for human IPEX syndrome and other autoimmune diseases associated with Treg deficiency. These include IPEX-like syndromes induced by mutations or deficiency in Itchy E3 ubiquitin protein ligase (ITCH), the α-chain of the IL-2 receptor (CD25), signal transducer and activator of transcription 5b, STAT1, or cytotoxic T-lymphocyte-associated protein 4 (7,8).
High levels of the adenosine A 2A receptor are found in the brain, thymus, and spleen, as well as in circulating platelets and leukocytes (9). On the cell membrane of murine T lymphocytes, the adenosine A 2A receptor is highly expressed and is increased by T-cell receptor (TCR) stimulation (10,11). In humans, the A 2A receptor is more highly expressed in CD4 + compared to CD8 + T cells (12). Moreover, numerous studies have highlighted the anti-inflammatory role of the adenosine A 2A receptor (13,14). There have been observations of anti-inflammatory effects of A 2A receptor agonists in vivo and, conversely, enhanced inflammation in A 2A receptor knockout mice (14). However, the function of adenosine A 2A receptor in the development and control of autoimmune diseases remains unclear.
Recently, probiotics have emerged as relatively safe and inexpensive treatments for a number of gastrointestinal conditions. Lactobacillus reuteri strain DSM 17938 (L. reuteri) is a probiotic originally isolated from a Peruvian mother's breast milk (15). This probiotic has been shown to prevent necrotizing enterocolitis (NEC) in newborn animals (16,17) by inhibiting the toll-like receptor 4-mediated NF-κB pathway, facilitating the induction of immune-modulating Foxp3 + Tregs, and lowering the number of pro-inflammatory effector-memory T-cells in the intestinal mucosa. In humans, L. reuteri has been shown to reduce the severity of acute infant diarrhea (18)(19)(20), to prevent NEC in premature infants (21)(22)(23), and to decrease crying time in infants with colic (24,25).
In addition, our recent studies demonstrated that L. reuteri significantly prolongs the survival rate of the SF mouse (from less than 30 days to greater than 4 months of age) by suppression of inflammatory T cells (mainly T H 1 and T H 2) extensively activated in multiple organs of SF mice (7). Mechanistically, L. reuteri modulates the abnormal microbial communities associated with these diseases, stimulating the production of bioactive metabolites involved in immune modulation. We observed that inosine, a downstream metabolite of adenosine, was decreased in the plasma of SF mice compared to wild-type (WT) mice, but was increased by oral administration of L. reuteri to SF mice. Oral administration of inosine by itself prolonged the survival and decreased autoimmunity of SF mice. Inosine was found to be a critical effector molecule of L. reuteri treatment, altering T H 1/T H 2 cell differentiation by activating A 2A receptors, predominately expressed on T cells. Blocking A 2A receptors by an A 2A antagonist reversed the anti-inflammatory effects of both inosine and L. reuteri, indicating that A 2A receptor appears to play a critical role in the beneficial effects of L. reuteri in the SF model (7).
In this study, we produced SF mice with genetically deleted adenosine A 2A receptor (SF·A 2A -/-) to conclusively provide evidence of a central role of A 2A receptor in the actions of L. reuteri. We demonstrate that A 2A receptor gene deletion in SF mice did not accentuate the development of disease, but prevented the inhibitory effects of L. reuteri on autoimmunity. Our study highlights the A 2A receptor as a key mediator of the immunomodulatory mechanism of this probiotic.

Animals
Wild-type C57BL/6, heterozygous B6.Cg-Foxp3 sf /J and adora2a tm1Jfc /J mice were purchased from Jackson Laboratories and allowed to acclimatize for 2 weeks before experimentation. SF mice were bred with adora2a tm1Jfc /J mice to generate adenosine A 2A receptor-deficient SF mice (A 2A -/-SF mice, SF·A 2A -/-). All males were either SF/SF·A 2A -/-double knockouts, the experimental group, or WT/A 2A -/-littermates, used as controls. All mice were housed in the animal facility at UT Health Science Center at Houston. This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (NIH) and The Institutional Animal Care and Use Committee (IACUC). The protocol was approved by the IACUC (protocol numbers: AWC-14-056 and AWC-17-0045).

L. reuteri Treatment of SF Mice
Lactobacillus reuteri DSM17938 (L. reuteri), originally isolated from human breast milk, was provided by BioGaia AB (Stockholm, Sweden) and prepared as described previously (7). Each mouse was given either De Man, Rogosa, and Sharpe agar (MRS) media as a control or L. reuteri (SF + LR or SF·A 2A -/-+ LR) which was given by daily gavage in cultured media (10 7 CFU/day), starting from 8 to 20 days of age for tissue analysis or to infinity for survival.

Histopathology
All tissues of WT, SF, SF + LR, A 2A -/-, SF·A 2A -/-, and SF·A 2A -/-+ LR mice were fixed and stained with hematoxylin and eosin (H&E) for histological evaluation by the Cellular and Molecular Morphology Core Lab (The Texas Medical Center Digestive Diseases Center, Houston, TX, USA). The area of lymphocyte infiltration in liver and lung was assessed in a blinded fashion using Image J morphometry software (NIH, USA).

In vitro Tissue Preparation and Stimulation for Flow Cytometry Analysis
Single-cell suspensions from the spleen were prepared by gently fragmenting and filtering the tissues through 40-μm cell strainers (BD Bioscience) into MACS buffer (1× PBS, 0.5% bovine BSA, and 2 mM EDTA). For in vitro stimulation of splenocytes, cells were stimulated with 50 ng/mL of phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL of ionomycin in the presence of brefeldin A (5 μ/mL) for 4 h to analyze IFN-γ-producing (T H 1) and IL-4producing (T H 2) CD4 + T cells by flow cytometry.

Staining Cells for Flow Cytometry Analysis
For evaluation of T H 1 and T H 2 cells, cells were surface stained by fluorescein-labeled CD4. Intracellular staining was performed with a fixation/permeabilization kit, according to the manufacturer's protocol (eBioscience) and stained with IFN-γ and IL-4 for T H 1 and T H 2 cells, respectively. The data from all samples were acquired on BD FACSCalibur and analyzed using FlowJo software (TreeStar, Inc.).

Plasma Cytokine Assays
Plasma cytokine levels of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-10, and IL-12p70 were assessed using a mouse multi-spot proinflammatory panel kit, and signals were detected by Imager 2400 from Meso Scale Discovery, according to the manufacturer's protocol.

Statistical Analysis
Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA corrected for multiple comparisons with Tukey and Dunnett's posttests. The statistical analysis was performed using Prism version 4.0 (GraphPad Software). A p-value <0.05 was considered to indicate statistical significance.

Adenosine A 2A Receptor Deletion Does Not Affect Early Death in SF Mice
To determine the role of the adenosine A 2A receptor in the pathogenesis of autoimmunity in the SF mouse, we bred female (Foxp3 sf/+ ) mice with adora2a gene knockout (A 2A -/-) mice. The male adenosine A 2A receptor-deficient SF (SF·A 2A -/-) mice died between 21 and 25 days of age ( Figure 1A). Our data show that A 2A receptor deletion does not enhance or reverse the effect of the lethal autoimmune disease as it relates to lifespan in the SF mouse.

Adenosine A 2A Receptor Deletion Regulates Organ-Specific Inflammation in SF Mice
Scurfy mice develop severe inflammation in several tissues, including liver, lung, ear, tail, intestine, and colon (26). To examine whether adenosine A 2A receptor deletion alters the autoimmune damage in these tissues, we measured the area of inflammatory cell infiltration in H&E-stained tissues sections from WT, SF, A 2A -/-, and SF·A 2A -/-mice at the 20 days of age. There were no inflammatory infiltrates in the liver, lung, ear, tail, and intes- Figure S1 in Supplementary Material). Indeed, the area of inflammatory cell infiltration in most organs studied (liver, ear, tail, and intestine) in SF·A 2A -/mice was similar to the inflammatory cell infiltrate in SF mice (Figures 1B,C; Figure S1 in Supplementary Material). However, the inflammatory cell infiltration of the lung was slightly reduced in SF·A 2A -/-mice compared to SF mice. These results demonstrate that the A 2A receptor deletion does not have a major impact on inflammation in SF mice.

Adenosine A 2A Receptor Deletion Does Not Reduce T H 1/T H 2 Cells in SF Mice
The lethal lymphoproliferative syndrome in SF mice is predominantly caused by CD4 + T cell-induced pathology (27,28).

Adenosine A 2A Receptor Deletion Alters the Majority of Pro-inflammatory Cytokines in SF Mice
After TCR stimulation, CD4 + T cells from SF mice produce high levels of cytokines, including IFN-γ, IL-2, IL-4, IL-10, and TNF-α (29,30). To examine whether these pro-inflammatory cytokines reached higher levels in SF·A 2A -/-mice compared to SF mice, we measured the concentration of pro-inflammatory cytokines in plasma (Figure 3; Figure S2 in Supplementary Material). Our results demonstrated that the levels of IFN-γ, IL-2, IL-4, IL-5, and IL-10 were increased in SF mice compared to WT mice. Conversely, the levels of IL-1β and IL-12p70 were not increased in SF mice compared to WT mice. However, A 2A receptor deletion increased the levels of pro-inflammatory IL-1β and anti-inflammatory cytokine IL-10 in SF·A 2A -/-mice compared to SF mice. Together, our results show that the majority of proinflammatory cytokines contribute to the development of disease in SF mice.

Adenosine A 2A Receptor Deletion Reverses the Effect of L. reuteri on Lifespan in SF Mice
Previous studies have suggested that L. reuteri increases survival in SF mice by restoring plasma levels of the nucleotide inosine, which is an adenosine A 2A receptor agonist (7), which represents a novel mechanism of action of probiotics. However, the effect

Adenosine A 2A Receptor Deletion Negates the Effect of L. reuteri on Inflammation in SF Mice
We next asked whether A 2A receptor deletion could inhibit the beneficial effect of L. reuteri on multiorgan inflammation in living SF mice. Therefore, we fed SF and SF·A 2A -/-mice with a daily dose of L. reuteri, starting from 8 to 20 days. H&E-stained tissue sections from SF, SF + LR, SF·A 2A -/-, and SF·A 2A -/-+ LR groups were scored. Inflammatory cell infiltration of liver and lung was reduced in SF + LR mice compared to SF mice. However, this infiltration was not reduced in SF·A 2A -/-+ LR mice compared to SF·A 2A -/-mice (Figures 4B,C). These results demonstrate that A 2A receptor activation contributes to the inhibition by L. reuteri of inflammation in the SF mouse.

Adenosine A 2A Receptor Deletion Inhibits L. reuteri-Mediated Reduction of T H 1/T H 2 Splenocytes in SF Mice
Our studies have shown that L. reuteri reduces T H 1/T H 2 cells in SF mice (7). To explore whether genetic deletion of A 2A receptor contributes to the inhibition of L. reuteri of T H 1/T H 2 cell differentiation in SF mice, we measured the frequency of T H 1/T H 2 cells in the spleen from SF, SF + LR, SF·A 2A -/-, and SF·A 2A -/-+ LR mice ( Figure 5). L. reuteri-treated SF mice had reduced T H 1/T H 2 cells when compared to SF mice at 20 days of age, consistent with our previous studies (7). Interestingly, L. reuteri treatment did not reduce the percentage of T H 1/T H 2 cells in SF·A 2A -/-mice, demonstrating that the activated A 2A receptor plays an important role in L. reuteri-mediated immunoregulation in SF mice.

Adenosine A 2A Receptor Deletion Reverses the Effect of L. reuteri on Pro-inflammatory Cytokines in SF Mice
To test whether cytokine production regulated by L. reuteri treatment depends on the A 2A receptor in SF mice, we examined plasma cytokines from SF, SF + LR, SF·A 2A -/-, and SF·A 2A -/-+ LR mice (Figure 6; Figure S3 in Supplementary Material). L. reuteritreated SF mice had reduced levels of IFN-γ and IL-4 and increased the levels of IL-12p70, but they had no changes in the levels of IL-1β, IL-2, IL-5, and IL-10, when compared to SF mice.

DISCUSSION
This study demonstrated a central role of the adenosine A 2A receptor in mediating the protection of probiotic L. reuteri against inflammation in the Treg-deficient SF mouse (a model of human IPEX syndrome), evidenced by the observation that SF mice with an A 2A receptor deletion continued to have systemic inflammation which was unresponsive to L. reuteri treatment.
It is well known that the lethal lymphoproliferative syndrome characterizing SF mice is predominately mediated by T H 1 and T H 2 cell-induced pathology (27,28). The key to Treg suppression of T effector cells (T H 1/T H 2/T H 17) is an interaction between adenosine produced by Tregs (mediated by a CD39-CD73 pathway) and the A 2A receptor expressed on nearby T effector cells (31). Lymphocytes predominately express A 2A receptors (10-12, 32, 33). However, during Treg deficiency in SF mice or human IPEX syndrome, T H 1 and T H 2 cells lose their regulation by adenosine A 2A -mediated signaling, resulting in T H 1 and T H 2 cell-induced pathology. Studies by Csoka et al. showed that an agonist of A 2A receptors inhibited the proliferation and effector functions of CD4 + T cells isolated from WT mice but failed to block these of cells obtained from A 2A knockout mice (33), indicating that the activated adenosine A 2A receptor plays a critical role in the suppression of T H 1 and T H 2 cells.
Our previous study demonstrated that Treg deficiency induces gut microbial dysbiosis dynamically over the first 22 days of life, an effect which could be reprogrammed by oral administration of L. reuteri. L. reuteri suppressed T H 1 and T H 2 cells in SF mice, as evidenced by lower circulating levels of IFN-γ (T H 1) and IL-4 (T H 2) and reduced numbers of IFN-γ and IL-4-expressing lymphocytes in spleen and mesenteric lymph nodes of SF mice. Metabolites produced by L. reuteri or L. reuteri-modulated bacteria are known to promote or suppress immune cell function (34)(35)(36). We discovered that the purine metabolite inosine, a metabolite of adenosine, is severely decreased in SF mice, while increased after oral administration of L. reuteri (7). Inosine has been proved to be a functional agonist of the A 2A receptor which has an antiinflammatory effect (37)(38)(39)(40)(41)(42)(43). Our previous experiments by using adenosine receptor knockout mice to study the suppression of inosine on naïve CD4 + T cell differentiation into T H 1 and T H 2 in vitro strongly suggested that the effects of inosine are dependent on the A 2A receptor on T cells (7). In addition, an in vivo study showed that an A 2A receptor antagonist blocks the anti-inflammatory effects of both inosine and (L. reuteri DSM 17938) on T H 1 and T H 2 suppression and multiorgan lymphocyte infiltration in SF mice (7). In summary, the A 2A receptor mediates the beneficial biological effects of L. reuteri and inosine in SF mice. In this study, we further confirmed a critical role of A 2A receptor-mediated effects by genetic deletion of A 2A in SF mice (SF·A 2A -/-mice).
Mechanistically, how L. reuteri results in increased serum level of inosine is not fully understood. When we compared L. reuteri cultures to MRS broth (culture media without L. reuteri) after 16 h of anaerobic growth, L. reuteri did not generate significant amounts of purines or inosine in culture. Our previous studies indicated that enterally feeding L. reuteri is associated with recovery of the plasma levels of inosine and hypoxanthine to levels similar to WT, at the same level that inosine levels decreased in the stool of these mice (7). We hypothesized that, most likely, L. reuteri promotes inosine absorption in the intestine by improving overall gut health through multiple mechanisms (for example, by improving villus length) and/or by modulating the gut microbial community. We measured the small intestinal villi in SF mice compare with SF mice after oral feeding L. reuteri and showed that orally feeding L. reuteri improves the length of villi and depth of crypts. Furthermore, an increased expression of equilibrative nucleoside transporter transporters after L. reuteri feeding was found, which could contribute to produce improved absorption. The best method to confirm enhanced absorption would be to orally feed labeled inosine after administration of L. reuteri and quantify the labeled inosine in the circulation. However, the labeling approach for small molecules like inosine is much more difficult than for amino acid or proteins. In the meantime, we could not rule out that in vivo the gut environment could activate the enzymes such as adenosine deaminase (ADA) and 5 ′ -nucleotidase generated by L. reuteri to produce inosine. But it is difficult to distinguish the ADA activity in the intestinal tissue lysates from the activity of L. reuteri or other microbes, because ADA activity is very high in the intestine (44). The direct links between L. reuteri and the metabolites required further exploration.
We also noticed that A 2A receptor appears to be expressed in other organs besides lymphocytes (11,45). In liver, the A 2A receptor is expressed in Kupffer cells, hepatocytes, and hepatic stellate cells (46)(47)(48). Some studies suggested that the A 2A receptor plays a role not only in regulating inflammation but also in maintaining liver function in general (39). Previous studies also revealed that it is more highly expressed in spleen, lymph nodes, liver, and lung than that in the small intestine or adrenal gland, supporting a functional role of this receptor in the regulation of the immune response in peripheral lymphoid tissues (11). It has been reported that A 2A receptor activation confers tissue protection in peripheral organs (49,50). While the mechanism of L. reuteri in regulating inflammation in SF mice clearly involves T cell modulation, we cannot rule out that A 2A receptor expression in these organs may also contribute to the beneficial effects of L. reuteri in SF mice. Therefore, A 2A receptor expression on both immune cells and other cells and their interaction may determine the overall impact of A 2A receptor deletion on beneficial effects of L. reuteri.
The role of the T cell and its expression of A 2A modulated by L. reuteri or highly related metabolites such as inosine could be further studied by using a T cell knockout mouse model by adoptive transfer of CD4 + T cells isolated from WT, SF, A 2A -/-, or SF·A 2A -/-with/without L. reuteri or inosine treatment, which is currently under investigation.
In summary, our study demonstrates that adenosine A 2A receptor deletion does not inhibit the development of autoimmune disease in the SF mouse. However, adenosine A 2A receptor deletion reverses the inhibition of L. reuteri on autoimmunity induced by Treg-deficiency in SF mice. Our results support the concept that activated adenosine A 2A receptors are linked to L. reuteri effects in vivo. They also suggest that the activated A 2A receptor by L. reuteri or other agonists may represent a useful therapeutic strategy for preventing lethal outcomes in Foxp3 deficency-or dysfunction-induced autoimmune diseases.

ETHICS STATEMENT
This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (NIH) and The Institutional Animal Care and Use Committee (IACUC). The protocol was approved by the IACUC (Protocol number: AWC-14-056 and AWC-17-0045).

AUTHOR CONTRIBUTIONS
BH, YL, and JR conceived and designed the experiments. BH, TH, and YL performed all experiments and analyzed the data. BH, YL, DT, and JR wrote the paper and edited the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS
The authors thank Pamela Parsons (Cellular and Morphology Core Lab at Texas Medical Center Digestive Diseases Center) for histological technical assistance and Dr. Eammon Connolly (Biogaia AB, Stockholm, Sweden) for providing Lactobacillus reuteri DSM 17938.