Short-Term Fasting Induces Cell Cycle Arrest of Immature Hematopoietic Cells and Increases the Number of Naive T Cells in the Bone Marrow of Mice

Calorie restriction has long been studied not only as a way to prolong longevity but also as an approach to improve cancer prevention and treatment. Dietary restriction may prevent senescence of the immune and hematopoietic systems. In addition, short-term fasting before chemotherapy can reduce hematological toxicity in cancer patients. We studied the influence of fasting on immune cells, hematopoietic stem cells, and progenitor cells in the bone marrow and spleen of mice.

Six-to-twelve-week old C57BL/6 mice were starved for 48 hours before analysis. We collected bone marrow and splenic cells from starved and control mice. After 48 hours of fasting, the body weight significantly decreased by an average of 24.1% compared to that of normal control mice. Calorie restriction caused a significant decrease in peripheral white blood cell count by an average of 48.3%, but hemoglobin level and platelet count were less affected.

The averaged total number of bone marrow cells in the fasting group was significantly lower than that in the normal control group (2.45×10⁷ versus 3.14×10⁷, P < 0.01). In fasted mice there was a significant reduction in the hematopoietic stem cell count, using detection based on the lineage^- c-kit⁺ Sca-1⁺, compared to control mice (0.83×10⁵ versus 1.24×10⁵, P < 0.05). In contrast, there was no significant difference for progenitor cells detected based on the lineage^- c-kit⁺ Sca-1^- (6.81×10⁵ versus 7.75×10⁵, P = 0.11). We performed colony assays with bone marrow cells from fasted and control mice. There was no difference between the two groups for not only the primary colony assay but also for the secondary and tertiary assays. Annexin V and 7-AAD analysis by flow cytometry showed that the rates of early and late apoptosis were almost identical in hematopoietic stem cells and progenitor cells, on comparing fasted and control mice. Furthermore, DNA cell cycle analysis of progenitor cells showed that short-term fasting caused a significant increase in the percentage in G0/G1 phase (83.1% versus 70.7%, P < 0.05) and decreases in the S and G2/M phases. These results imply that immature bone marrow cells retained their proliferative capacity by maintaining cell cycle arrest after short-term fasting, a finding that may account for the protective effect of starvation against chemotherapy in cancer patients.

Calorie restriction caused a significant decrease in B cells in bone marrow (5.38×10⁶ versus 8.1×10⁶, P < 0.05) and especially in the spleen (6.65×10⁶ versus 33.0×10⁶, P < 0.001), and also significantly decreased T cells in the spleen (3.91×10⁶ versus 14.5×10⁶, P < 0.01). To our surprise, we detected a remarkable increase in the number of T cells in the bone marrow of fasted mice (1.25×10⁶ versus 0.91×10⁶, P < 0.01). Of greatest significance CD44^- naive CD8⁺ T cells were dramatically increased in fasted bone marrow (1.74×10⁶ versus 0.47×10⁶, P < 0.01), and CD44^- naive CD4⁺ T cells were also increased (0.23×10⁶ versus 0.07×10⁶, P < 0.05). In contrast, the numbers of CD62L^- CD44⁺ effector memory and CD62L⁺ CD44⁺central memory T cells were not substantially changed after starvation. The increased naive T cells had no activated markers and appear to have migrated into bone marrow in a resting state without proliferating there.

Short-term fasting decreased the number of hematopoietic stem cells but progenitor cells remained in a relatively quiescent state, with a prolonged DNA cell cycle and retention of colony-forming capabilities. The number of T cells in the bone marrow of fasted mice also increased dramatically, especially naive CD8⁺ T cells which probably migrated in from other lymphoid tissues. These findings imply that immature hematopoietic cells and some lymphoid cells can survive starvation while maintaining their function. The mechanisms by which T lymphoid cells promptly accumulate in bone marrow during starvation are under investigation.