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

Abstract

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 A_2A receptor activation limits inflammation and tissue damage, thereby playing an anti-inflammatory role. However, the role of the adenosine A_2A receptor in the development of disease in SF mice remains unclear. Using a genetic approach, we found that adenosine A_2A receptor deletion in SF mice (SF[Formula: see text]) 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, A_2A receptor deletion completely blocked these beneficial effects of L. reuteri in SF mice. Altogether, these results suggest that although absence of the adenosine A_2A 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 A_2A receptor and its activation may have a role in treating other Treg dysfunction-mediated autoimmune diseases.

Keywords: IPEX; Lactobacillus reuteri; adenosine A2A receptor; autoimmunity; cytokines; probiotic; regulatory T deficiency; scurfy.

Figure 1

Effect of adenosine A_2A receptor deletion on the development of diseases in scurfy (SF) mice. (A) Survival curves of ${\text{A}}_{{\text{2A}}^{\text{-/-}}}$, SF, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice (n = 6–9). (B) H&E staining of representative sections of liver and lung of wild-type (WT), SF, ${\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice (n = 6–9). (C) Quantitation of inflammatory infiltrates in liver and lung of WT, SF, ${\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice (n = 6–9). Data are presented as mean ± SEM. ***p < 0.001. SF vs. WT. ^#p < 0.05. SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ vs. SF.

Figure 2

Effect of adenosine A_2A receptor deletion on T_H1/T_H2 cells in spleen of scurfy (SF) mice. (A) Representative FACS plots of IFN-γ-producing CD4⁺ T (T_H1) cells in spleen of wild-type (WT), SF, ${\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice. (B) Percentage of T_H1 cells in spleen of WT, SF, ${\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice (n = 6–9). (C) Representative FACS plots of IL-4-producing CD4⁺ T (T_H2) cells in spleen of WT, SF, ${\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice. (D) Percentage of T_H2 cells in spleen of WT, SF, ${\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice (n = 6–9). Data are presented as mean ± SEM. ***p < 0.001. SF vs. WT. ns, non-significance.

Figure 3

Effect of adenosine A_2A receptor deletion on pro-inflammatory cytokines in scurfy (SF) mice. Plasma levels of IFN-γ, IL-1β, IL-4, and IL-10 in wild-type (WT), SF, ${\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice were quantified by a mouse multi-spot pro-inflammatory panel kit (n = 6–9). Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001. SF vs. WT. ^#p < 0.05, ^##p < 0.01. SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ vs. SF. ns, non-significance.

Figure 4

Adenosine A_2A receptor deletion blocks effects of Lactobacillus reuteri on scurfy (SF) mice. (A) Survival curves of SF, SF + LR, SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ + LR mice (n = 6–9). (B) H&E staining of representative sections of liver and lung of SF, SF + LR, SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ + LR mice (n = 6–9). (C) Quantitation of inflammatory infiltrates in liver and lung of SF, SF + LR, SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ + LR mice (n = 6–9). Data are presented as mean ± SEM. ***p < 0.001. SF + LR vs. SF. ns, non-significance. ^#p < 0.05.

Figure 5

Effect of Lactobacillus reuteri on T_H1/T_H2 cells in spleen of scurfy (SF) and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice. (A) Representative FACS plots of IFN-γ-producing CD4⁺ T (T_H1) cells in spleen of SF, SF + LR, SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ + LR mice. (B) Percentage of T_H1 cells in spleen of SF, SF + LR, SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ + LR mice (n = 6–9). (C) Representative FACS plots of IL-4-producing CD4⁺ T (T_H2) cells in spleen of SF, SF + LR, SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ + LR mice. (D) Percentage of T_H2 cells in spleen of SF, SF + LR, SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ + LR mice (n = 6–9). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01. SF + LR vs. SF. ns, non-significance.

Figure 6

Effect of Lactobacillus reuteri on pro-inflammatory cytokines in scurfy (SF) and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ mice. Plasma levels of IFN-γ, IL-4, IL-1β, and IL-10 in SF, SF + LR, SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$, and SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ + LR mice were quantified by a mouse multi-spot pro-inflammatory panel kit (n = 6–9). Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001. SF + LR vs. SF. ^##p < 0.01. SF$\cdot {\text{A}}_{{\text{2A}}^{\text{-/-}}}$ vs. SF. ns, non-significance.