System A amino acid transporters regulate glutamine uptake and attenuate antibody-mediated arthritis

Abstract

Proliferation of rapidly dividing bone marrow-derived cells is strongly dependent on the availability of free glutamine, whose uptake is mediated through different amino acid transporters. The sodium-coupled neutral amino acid transporter (SNAT) family was previously reported to be associated with the development of collagen-induced arthritis in mice. Here, we tested the hypothesis whether impairment of SNAT proteins influences immune cell function and in turn alters arthritis development. The 2-(methylamino)isobutyric acid (MeAIB), a SNAT-specific substrate, was used to modulate the function of SNAT proteins. We demonstrate that glutamine uptake by murine naive lymphocytes, and consequent cell proliferation, is strongly associated with system A transporters. Physiological impairment of SNAT proteins reduced the antibody-initiated effector phase of arthritis, mainly by affecting the levels of circulating monocytes and neutrophils. MeAIB was also shown to affect the proliferation of immortalized cells, through trans-inhibition of SNAT proteins. Based on our observations, we conclude that SNAT proteins regulate the initial stages of lymphocyte activation by regulating glutamine uptake, and that the effector phase of arthritis can be affected by non-metabolized SNAT substrates. Most probably, metabolically active cells within both the adaptive and the innate immune systems are regulated by SNAT proteins and play a role in modifying arthritis development.

Keywords: 2-(methylamino)isobutyric acid; amino acid transporter; arthritis; glutamine; sodium-coupled neutral amino acid transporter.

Figure 1

T‐cell activation up‐regulates the expression of sodium‐coupled neutral amino acid transporter 1 (SNAT1) and SNAT2. (a) Expression of Slc38a1 and Slc38a2 genes in different immunological organs. (b–e) Expression of Slc38a1, Slc38a2, Mtor and Mapk3 genes after anti‐CD3/28 stimulation of naive splenocytes in the absence (Dulbecco's modified Eagle's medium; DMEM) or presence of 10 mm 2‐(methylamino)isobutyric acid (MeAIB) (n = 5). Data were normalized to the expression levels of non‐stimulated cells. Data shows mean ± SEM and are representative of three independent experiments, with a total of 18 mice used per group. Kruskal–Wallis test was used and data were considered significant when P < 0·05 for a 95% confidence interval. *P < 0·05.

Figure 2

Glutamine‐dependent T‐cell proliferation is associated with system A family of amino acid transporters. (a) Cytokine secretion by splenocytes at different concentrations of glutamine after anti‐CD3/28 stimulation (n = 5). Glutamine uptake in the presence (b) or absence (c) of Na⁺ after T‐cell stimulation with or without 2‐(methylamino)isobutyric acid (MeAIB) in the culture medium (n = 4). [³H]Thymidine incorporation (d) and cytokine secretion (e) by stimulated lymphocytes in the presence of MeAIB (n = 6). Data shows mean ± SEM and are representative of four independent experiments, with a total of 20 to 22 mice used. Kruskal–Wallis (a, d and e) and two‐way analysis of variance (b and c) tests were used and data were considered significant when P < 0·05 for a 95% confidence interval. *P < 0·05; **P < 0·01; ***P < 0·005.

Figure 3

Administration of 2‐(methylamino)isobutyric acid (MeAIB) attenuates collagen antibody‐induced arthritis (CAIA) by affecting circulating polymorphonuclear cells. Mice were given 3 mg/150 μl of MeAIB intraperitoneally daily from day –7 until day 18. At day 7, 25 μg of lipopolysaccharide (LPS) was given intraperitoneally to all mice. Development of arthritis was evaluated by visual scoring of the inflamed joints (a) and prevalence (b). (c) Circulating levels of neutrophils (CD11b⁺ Ly6G⁺) and monocytes (CD11b⁺ Ly6C^lo) in the peripheral blood of mice at day 11 after anti‐collagen II (CII) antibody transfer. The data shown are representative of two independent experiments using a total of 26 PBS‐treated mice and 25 MeAIB‐treated mice. Gating strategies are presented in the Supplementary material (Fig. S1). (d) Effect of MeAIB on NO production by in vitro stimulation of naive peritoneal macrophages with LPS. Data shows mean ± SEM. Kruskal–Wallis (c and d), two‐way analysis of variance (a) and Fisher's exact test (b) were used and data were considered significant when P < 0·05 for a 95% confidence interval. *P < 0·05; **P < 0·01; ***P < 0·005; n.s., not significant.

Figure 4

System A substrates affect proliferation of immortalized cell lines: 2·5 × 10⁵ HEK293T cells were seeded per 25‐cm² culture flask in Dulbecco's modified Eagle's medium (DMEM) supplemented with different concentrations of 2‐(methylamino)isobutyric acid (MeAIB). After 72 hr, the total number of cells was assessed (a) and total RNA samples were collected for quantitative expression of Slc38a1 and Slc38a2 genes (b, c; n = 4). (d) Visualization of HEK293T cells under normal culture conditions or after addition of 50 mm MeAIB. (e) Interleukin‐2 (IL‐2) production by HCQ3 T‐cell hybridomas stimulated with rat CII for 48 hr in different concentrations of MeAIB (n = 5). The data shows mean ± SEM and are representative of three independent experiments. Kruskal–Wallis test was used and data were considered significant when P < 0·05 for a 95% confidence interval. *P < 0·05; **P < 0·01.