5'-AMP impacts lymphocyte recirculation through activation of A2B receptors

Abstract

Natural hibernation consists of torpid phases with metabolic suppression alternating with euthermic periods. Induction of torpor holds substantial promise in various medical conditions, including trauma, major surgery, and transplantation. Torpor in mice can be induced pharmacologically by 5'-AMP. Previously, we showed that during natural torpor, the reduction in body temperature results in lymphopenia via a reduction in plasma S1P. Here, we show that during torpor induced by 5'-AMP, there is a similar reduction in the number of circulating lymphocytes that is a result of their retention in secondary lymphoid organs. This lymphopenia could be mimicked by engagement of A(2B)Rs by a selective A(2B)R agonist (LUF6210) in the absence of changes in temperature and prevented by A(2B)R antagonists during 5'-AMP-induced torpor. In addition, forced cooling of mice led to peripheral blood lymphopenia, independent of A(2B)R signaling. The induction of torpor using 5'-AMP impacted the migration of lymphocytes within and between secondary lymphoid organs. During torpor, the homing into LNs was impaired, and two-photon intravital microscopy revealed that cell motility was decreased significantly and rapidly upon 5'-AMP administration. Furthermore, the S1P plasma concentration was reduced by 5'-AMP but not by LUF6210. S1P plasma levels restored upon arousal. Likely, the reduced migration in LNs combined with the reduced S1P plasma level substantially reduces lymphocyte egress after injection of 5'-AMP. In conclusion, 5'-AMP induces a state of pharmacological torpor in mice, during which, lymphopenia is governed primarily by body temperature-independent suppression of lymphocyte egress from LNs.

Keywords: anesthesiology; hibernation; inflammation; metabolism; suspended animation.

Figure 1.. 5′-AMP-induced torpor results in the reversible reduction in the number of circulating T and B lymphocytes as a result of the activation of A_2BR and independently of changes in body temperature.

Body temperature of mice prior to and following torpor induction by 5′-AMP (7.5 mmol/kg) or injection of the A2b agonist LUF6210 (3 mg/kg; A). The number of circulating total lymphocytes (B), T lymphocytes (C), or B lymphocytes (D) in mice at the indicated time following administration of 5′-AMP, the specific A_2BR antagonist MRS1754 (12.5 mg/kg), and/or the A2b agonist LUF6210. Bars represent mean ± sem; */** = P < 0.05/0.01, respectively (n=6–9 animals/group).

Figure 2.. 5′-AMP-induced peripheral blood lymphopenia occurs independent of hypothermia.

Body temperature prior to (0 h) and 4 h following torpor induction by 5′-AMP administration in mice kept at room temperature (∼22°C) or at the higher T_a of ∼30°C (A). Total lymphocyte (B), T lymphocyte (C), and B lymphocyte (D) counts in peripheral blood of mice kept at T_a of ∼22°C or 30°C prior to and 4 h after 5′-AMP administration. Mice kept at T_a = 30°C were given two doses of 5′-AMP at the start of the experiment (t=0; 7.5 mmol/kg) and 2 h (1.9 mmol/kg). Bars represent mean ± sem; */** = P < 0.05/0.01, respectively (n=3–9 animals/group).

Figure 3.. Forced hypothermia leads to a decrease in the number of circulating lymphocytes.

The peripheral blood lymphocyte (A), T lymphocyte (B), and B lymphocyte (C) counts were measured in euthermic controls (EU), 5′-AMP-treated mice, or anesthetized mice, whose body temperature was lowered to ±24°C. Mice in forced hypothermia were injected with saline or with an A_2BR antagonist (1.25 mg/kg and 12.5 mg/kg MRS1754), 30 min prior to cooling. Bars represent mean ± sem. Circles represent data from individual mice; */** = P < 0.05/0.01, respectively (n=7–9 animals/group where bars are shown). df, Degrees of freedom.

Figure 4.. Restoration of lymphocyte counts during return to euthermia after torpor requires lymphocyte egress from secondary lymphoid organs.

Plasma S1P levels in mice prior to (0 h), 4 h, or 10 h following torpor induction with 5′-AMP (7.5 mmol/kg) or at 4 h after administration of the A_2BR agonist LUF6210 (3 mg/kg; A). Peripheral blood lymphocyte counts (B), T lymphocyte counts (C), and B lymphocyte counts (D) in mice during torpor (4 h post-5′-AMP administration) or following return to euthermia in mice (10 h post-5′-AMP administration) that were given the S1P₁-specific antagonist W146 (10 mg/kg) compared with untreated controls. Bars represent mean ± sem; */** = P < 0.05/0.01, respectively (n=6–9 animals/group).

Figure 5.. 5′-AMP-induced torpor reduces lymphocyte recirculation rates and causes retention of lymphocytes in secondary lymphoid organs.

LN homing of lymphocytes was blocked by the administration of anti-α4- and anti-αL-integrin antibodies, 2 h prior to 5′-AMP (7.5 mmol/kg) or saline injection and the number of total lymphocytes (A), T lymphocytes (B), or B lymphocytes (C) measured in the peripheral blood. To assess the change in homing efficiency of T cells during torpor, congenic CD45.1+ T cells were adoptively transferred to recipients given 5′-AMP and the number of CD45.1+ cells counted, 2 h post-transfer in inguinal (iLN), axillary (aLN), and mesenteric (mLN) LNs; the homing efficiency of adoptively transferred T cells is shown as the percent of cells relative to saline controls (D). Bars represent mean ± sem; */** = P < 0.05/0.01, respectively (n=4–6 animals/group).

Figure 6.. In vivo decrease in motility of T lymphocytes within the LN following 5′-AMP-induced torpor.

Tracks of 75 CD4 T cells in the inguinal LN, prior to and following torpor induction by 5′-AMP (7.5 mmol/kg) administration in mice kept at a body temperature of ∼35°C. x and y coordinates (given in μm) are shown for each cell, normalized such that all starting coordinates are set at 0,0 (left). Summary of mean velocity of CD4 and CD8 T cells in mice prior to and following 5′-AMP injection. Data points represent mean of 80–670 T cell tracks from four mice and two independent experiments (right); *P < 0.05.