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. 2012;7(2):e32416.
doi: 10.1371/journal.pone.0032416. Epub 2012 Feb 28.

Adora2b adenosine receptor engagement enhances regulatory T cell abundance during endotoxin-induced pulmonary inflammation

Heidi Ehrentraut  1 , Joseph A Westrich, Holger K Eltzschig, Eric T Clambey
Affiliations

Adora2b adenosine receptor engagement enhances regulatory T cell abundance during endotoxin-induced pulmonary inflammation

Heidi Ehrentraut et al. PLoS One. 2012.

Abstract

Anti-inflammatory signals play an essential role in constraining the magnitude of an inflammatory response. Extracellular adenosine is a critical tissue-protective factor, limiting the extent of inflammation. Given the potent anti-inflammatory effects of extracellular adenosine, we sought to investigate how extracellular adenosine regulates T cell activation and differentiation. Adenosine receptor activation by a pan adenosine-receptor agonist enhanced the abundance of murine regulatory T cells (Tregs), a cell type critical in constraining inflammation. Gene expression studies in both naïve CD4 T cells and Tregs revealed that these cells expressed multiple adenosine receptors. Based on recent studies implicating the Adora2b in endogenous anti-inflammatory responses during acute inflammation, we used a pharmacologic approach to specifically activate Adora2b. Indeed, these studies revealed robust enhancement of Treg differentiation in wild-type mice, but not in Adora2b(-/-) T cells. Finally, when we subjected Adora2b-deficient mice to endotoxin-induced pulmonary inflammation, we found that these mice experienced more severe inflammation, characterized by increased cell recruitment and increased fluid leakage into the airways. Notably, Adora2b-deficient mice failed to induce Tregs after endotoxin-induced inflammation and instead had an enhanced recruitment of pro-inflammatory effector T cells. In total, these data indicate that the Adora2b adenosine receptor serves a potent anti-inflammatory role, functioning at least in part through the enhancement of Tregs, to limit inflammation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Adenosine receptor activation enhances the abundance of Tregs following in vitro stimulation of primary mouse T cells.
(A) Adenosine receptor activation enhances the abundance of Tregs following antibody-mediated stimulation of T cells (using anti-CD3 antibody, 1 µg/mL combined with 10 ng/mL of IL-2, in the absence of TGF-β). Samples were either untreated (+Vehicle) or treated with 10 µM of NECA (+NECA), a potent adenosine receptor agonist, analyzed three days post-stimulation for the relative abundance of FoxP3-expressing Tregs. The numbers present in the upper left-hand corner of each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate. Background staining with an isotype control antibody is indicated in the leftmost panel. (B) Quantitation of FoxP3-expressing cells following either control (vehicle treated, white bars) or NECA treated (black bars), with data indicating mean +/− SEM of triplicate cultures done in two separate experiments. (C) Adenosine receptor activation enhances the abundance of Tregs following antibody-mediated stimulation of T cells in the presence of TGF-β, a known inducer of Tregs (using anti-CD3 antibody, 1 µg/mL combined with 10 ng/mL of IL-2, combined with 0.75 ng/mL TGF-β), showing flow cytometric analysis (C) and quantitation (D). The numbers present in the upper left-hand corner of each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate. Relative abundance of Tregs within cultures were defined by flow cytometry, with Tregs defined as live, MHC class II negative, CD8−, CD4+ cells that express the transcription factor FoxP3. Data indicate mean +/− SEM of triplicate cultures, representative of two independent experiments. (E) Tregs generated by TGF-β with NECA (solid black line) relative to Tregs generated by TGF-β treatment alone (indicated in gray) have a comparable cell surface expression of CD25, CD39 and CD73. Results representative of results from three independent cultures, done in two independent experiments. Statistical analysis was performed using unpaired t test, with statistically significant differences as indicated.
Figure 2
Figure 2. Relative expression levels of adenosine receptor genes in T cell subsets.
Real-time PCR analysis of mRNA for the four adenosine receptor genes in FACS purified CD4 T cell subsets of naïve CD4 T cells or Tregs, with Tregs isolated from FoxP3GFP or DEREG mice. Values were standardized to bulk spleen mRNA, with each value showing expression relative to actin. High level expression of FoxP3 mRNA is consistent with a highly purified population of Tregs. Data representative of two to three independent experiments, analyzing at least three independently isolated populations for both naïve CD4 T cells and Tregs. Data depict mean ± SEM for each transcript. Statistically significant differences were calculated by unpaired t test comparing expression in naïve CD4 T cells relative to Tregs, as indicated.
Figure 3
Figure 3. An Adora2b-specific agonist enhances Treg abundance in vitro following activation of murine T cells.
Bulk splenocytes from either Adora2b+/+ (C57BL/6J) or Adora2b−/− mice were cultured with soluble anti-CD3 for three days with or without the Bay60-6583 compound, at which time the relative abundance of Tregs was assessed. (A) Fold change in Treg abundance relative to Adora2b+/+ cultures without the Bay compound control, where Tregs were defined as viable, CD4+ FoxP3+ cells by flow cytometry. (B) Flow cytometry plots shown from Adora2b+/+ cultured splenocytes. Bay60-6583, an Adora2b-specific agonist was added to a final concentration of 4 nM. Data from two independent experiments, each containing 1–3 independent replicates. The numbers present on each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate, with FoxP3 expressing cells defined relative to isotype control-stained samples (not shown). Statistically significant differences are indicated and were calculated by one-way ANOVA followed by Bonferroni's post-test correction. ns indicates a comparison that is not statistically significantly different.
Figure 4
Figure 4. Adora2b-deficient mice have increased inflammation with impaired induction of Tregs after LPS-induced lung injury.
LPS-induced inflammation was initiated in 9–12 wk-old male Adora2b+/+ (C57BL/6J) or age and weight matched Adora2b−/− mice by intratracheal instillation of LPS or saline. Bronchoalveolar lavage (BAL) fluids were collected 1 d post treatment. A) Leukocyte cell counts of BAL fluid were determined utilizing a cellometer (mean ± SEM, n = 2–4). B) Protein content of BAL fluid was determined with BCA assay. Fold increase of protein concentration in LPS treated BAL samples over respective saline BAL samples is displayed (mean ± SEM, n = 3–10). C) Analysis of cell infiltration into the lung airspace (BAL) of mice exposed to inhaled LPS. Mice (Adora2b+/+ or Adora2b−/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and BAL was harvested. Cells were then subjected to flow cytometric analysis to identify neutrophils (highly granular, Gr1+ cells). D) Analysis of FoxP3 gene expression in lungs of mice exposed to inhaled LPS. Mice (Adora2b+/+ or −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and perfused lungs were harvested. Total RNA was harvested from lungs, cDNA prepared and analyzed by real-time PCR. Data depict mean (± SEM) fold changes in indicated genes. Fold change was calculated based on primer efficiencies, standardized to changes in actin, with Adora2b+/+ Saline mean value defined as 1. E) Fold change in cell number of either regulatory T cells (lymphocyte size, CD3+ CD4+ FoxP3+) in the lung airspace (BAL) of mice exposed to inhaled saline or LPS as above. Data are from 2–6 mice; 2 independent experiments. F) Relative abundance of CD4 effector T cells (Teffs, defined as lymphocyte size, CD3+ CD4+ CD44high cells) to Tregs based on cell counts from either Adora2b+/+ or Adora2b−/− mice, with pie charts depicting mean cell count for the indicated cell populations from 2–6 mice; 2 independent expts. Analysis of total cellular infiltration into airways and protein leakage (panels A–B) was done in animals treated with intratracheal LPS. Analysis of neutrophil infiltration and Tregs (panels C–F) was done in animals treated with inhaled LPS. Statistically significant differences are indicated and were calculated either by one-way ANOVA (A) or by unpaired t test (B–E) with a focus on whether Adora2b−/− LPS treated animals were statistically different from either saline treated or Adora2b+/+ LPS treated mice.
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