Thymocyte respiration. Figure 1 shows a typical experiment involving thymocyte respiration. First, we establish the ‘drift rate’ in the reaction mixture (PBS + albumin + Pd phosphor) without thymic tissue. This ‘noise level’ was ≤ 10% of the signal (Panel A). A newly collected thymic fragment (see Methods) was then added, and oxygen consumption was monitored as a function of time. As expected, the rate of thymocyte respiration slowed down, confirming the process was driven by the intracellular nutrients (Panel B, before the addition of glucose). At this point, the addition of glucose provided an external source of metabolic fuel, which established a steady-state rate of thymocyte respiration. These consecutive steps provided an insight into the thymocyte energy conversion (storage) pools. To avoid an irreversible injury from intracellular nutrient depletion (e.g., cell swelling as a result of ATP depletion), glucose must be added when the rate of respiration begins to slow down (see Panel B, after the addition of glucose). The addition of glucose oxidase tested the availability of dissolved oxygen and sets the numerical value of ‘zero oxygen concentration’ (Panel B). Respiration was inhibited by the addition of cyanide, confirming oxygen reduction occurred in cytochrome oxidase (Panel C).
The overall rate of thymocyte respiration (mean ± SD, in µM O2 min− 1 mg− 1) was 0.046 ± 0.011 (median = 0.043, range = 0.028 to 0.062, n = 10). It is important to note the variability of thymocyte respiration, a limitation that prohibited establishing a valid reference range. Therefore, these experiments should be performed in littermates, or in at least age-matched mice. The drug experiments described below were performed in littermates.
In vitro effects of sirolimus on thymocyte respiration. Runs similar to the above were then performed in the presence of 270 µM sirolimus or 10 µL of the vehicle DMSO. As shown in Fig. 2, the rate of thymocyte respiration (kc, in µM O2 min− 1 mg− 1) with DMSO was 0.067 and with sirolimus was 0.050 (25% lower). The effect of sirolimus was notable after approximately 60 min. Overall, the values of kc (mean ± SD, in µM O2 min− 1 mg− 1) with DMSO was 0.063 ± 0.009 (median = 0.064, range = 0.040 to 0.078, n = 12) and with sirolimus was 0.058 ± 0.015 (median = 0.054 [16% lower, P = 0.4248], range = 0.042 to 0.077, n = 6). Similarly, dose-response curves for sirolimus with or without the addition of ‘response modifier(s)’ could be similarly investigated.
In vitro effects of ozanimod on thymocyte respiration. Figure 3 shows an inhibitory effect of ozanimod on thymocyte respiration, similar to that of sirolimus. The runs were in the presence of 0.68 µM ozanimod or 10 µL diluent (5% DMSO, 5% Tween-20, and 90% 0.1 N HCl). The rate of thymocyte respiration (in µM O2 min− 1 mg− 1) with the diluent was 0.059 and with ozanimod was 0.039 (34% lower). Overall, the values of kc (µM O2 min− 1 mg− 1) in the presence of the diluent was 0.055 ± 0.011 (median = 0.059, range = 0.040 to 0.062, n = 3) and in the presence of ozanimod was 0.045 ± 0.009 (median = 0.042 [29% lower, P = 0.4], range = 0.039 to 0.055, n = 3).
In vivo effects of ozanimod on thymocyte respiration. We then examined the effects of an in vivo treatment with ozanimod (0.6 mg/kg/day, or 0.57 µmol/kg/day) on thymocyte respiration at the completion of 30-consecutive days of administration. As shown in Fig. 4A, this treatment caused a sustained weight loss. Nevertheless, thymocyte respiration was similar in mice given ozanimod compared to those given the diluent (Fig. 4B). Overall, the values of kc (µM O2 min− 1 mg− 1) in the presence of the diluent was 0.041 ± 0.010 (median = 0.041, range = 0.026 to 0.056, n = 7) and in the presence of ozanimod was 0.047 ± 0.016 (median = 0.043, range = 0.028 to 0.079, n = 8; P = 0.6), Fig. 4C.