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. 2015 Jun 9;11(9):1339-49.
doi: 10.1016/j.celrep.2015.04.058. Epub 2015 May 21.

The Cell-Intrinsic Circadian Clock Is Dispensable for Lymphocyte Differentiation and Function

Saskia Hemmers  1 , Alexander Y Rudensky  2
Affiliations

The Cell-Intrinsic Circadian Clock Is Dispensable for Lymphocyte Differentiation and Function

Saskia Hemmers et al. Cell Rep. .

Abstract

Circadian rhythms regulate many aspects of physiology, ranging from sleep-wake cycles and metabolic parameters to susceptibility to infection. The molecular clock, with transcription factor BMAL1 at its core, controls both central and cell-intrinsic circadian rhythms. Using a circadian reporter, we observed dynamic regulation of clock activity in lymphocytes. However, its disruption upon conditional Bmal1 ablation did not alter T- or B-cell differentiation or function. Although the magnitude of interleukin 2 (IL-2) production was affected by the time of bacterial infection, it was independent of cell-intrinsic expression of BMAL1. The circadian gating of the IL-2 response was preserved in Bmal1-deficient T cells, despite a slight reduction in cytokine production in a competitive setting. Our results suggest that, contrary to the prevailing view, the adaptive immune response is not affected by the cell-intrinsic clock but is likely influenced by cell-extrinsic circadian cues operating across multiple cell types.

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Figures

Figure 1
Figure 1. Dynamic Regulation of Circadian Reporter Expression During Lymphocyte Development but T Cell-Specific Deletion of Bmal1 has no Effect on T Cell Development
(A) PER1Venus expression in thymocytes was analyzed by flow cytometry. The gated populations in the left plot correspond to the histogram overlay in the right plot (DN: red line; DP: blue line; CD4SP: green line; CD8SP: orange line). (B) Data as in (A) plotted for individual mice (n= 2–3 animals per time point) analyzed at four time points (ZT1, ZT7, ZT13, ZT19). (C) Bmal1 expression analysis in FACS purified thymocyte populations from three individual mice. Expression is shown relative to the DN population. (D) BMAL1 protein levels were assessed by Western blotting in lysates from total thymus of WT or Bmal1T-KO mice. Equal loading was confirmed by β-actin expression. (E) Thymocyte development is unperturbed in Bmal1T-KO mice. (F, G) IFNγ and IL-17A production in CD4+ T cells after αCD3/αCD28 stimulation in spleen (F) and large intestine LPL (G) is not affected by BMAL1-deficiency. (H) Frequencies of RORγt expressing CD4+ T cells in small and large intestine LPL are unchanged in Bmal1T-KO mice. (B) Error bars represent mean ± SEM. (C, E–H) Error bars represent mean ± SD. All data are representative of at least three individual experiments. See also Figure S1, 3.
Figure 2
Figure 2. T Cell-Specific BMAL1-Deficiency has no Impact on Experimental Autoimmune Encephalomyelitis Disease Susceptibility
(A) Disease severity was scored on a daily basis starting at day 7 after immunization. Mean score ± SEM is plotted (WT = 9 mice; Bmal1T-KO = 7 mice). Mice were analyzed on d22 post immunization. (B) Immune infiltration of the CNS (pooled brain and spinal chord) was assessed by counting CD45+ lymphocyte infiltrates at d22 post immunization. (C) Frequencies of CNS-infiltrating CD4+, CD8+ and Treg cells are comparable between WT and Bmal1T-KO mice. (D) Representative FACS plot of cytokine production of CD4+Foxp3− T cells infiltrating the CNS (αCD3/αCD28 stimulation). (E, F) IFNγ- and IL-17A- production by CNS-derived T cells upon αCD3/αCD28 (E) or MOG peptide stimulation (F). Frequencies are represented in the upper panels, total numbers in the lower panels. (B, C and E, F) Error bars represent mean ± SD. All data are representative of two individual experiments.
Figure 3
Figure 3. Circadian Oscillation of the Molecular Clock in T Cells is Minor Compared to Liver
Liver (left panel), thymus (middle panel) and CD4+ splenocytes (right panel) were harvested from control and Bmal1T-KO mice at the indicated time-points. RNA was processed, reverse transcribed and analyzed by quantitative PCR. Expression (2−ddCt) of Bmal1 (A) and Rev-Erbα (B) is shown relative to the WT 7AM time-point. Expression of Rev-Erbα is significantly lower in thymus and CD4+ splenocytes at all time-points analyzed. To visualize oscillation of the transcripts independent of gene expression, the data from (A) and (B) was calculated as % of mean for each individual mouse and time-point for Bmal1 (C) and Rev-Erbα (D). Data are comprised from 3–6 mice per group. Error bars represent mean ± SEM. See also Figure S4.
Figure 4
Figure 4. Circadian Time of Infection has a Minor Effect on Acute Antiviral T cell Response
(A) Total splenocyte counts on d7 post LCMV-Armstrong infection at ZT1 and ZT13 in WT or Bmal1T-KO mice. (B) Representative FACS plots of the antiviral CD8+ T cell response assessed by gp33-specific tetramer staining at ZT1 and ZT13. (C) The magnitude of the virus-specific CD8+ T cell response is comparable between WT and Bmal1T-KO cells and not affected by time of infection. (D) Virus-specific IFNγ-production by CD8+ (left panel) and CD4+ T cells (right panel) is comparable in all groups analyzed. (E) The time of infection impacts the CD8+ T cell specific TNFα response independent of BMAL1-expression in T cells. *p<0.05, **p<0.01; significantly different from ZT1 as per unpaired t-test. (F) The CD8+ T cell specific TNFα response in uninfected mice is not affected by circadian time of analysis. (A and C–F) Error bars represent mean ± SD. All data are representative of at least two individual experiments. See also Figure S5.
Figure 5
Figure 5. L. monocytogenes-Elicited IL-2 Production is Sensitive to Circadian Time of Infection but Independent of Bmal1 Expression in T Cells
(A) Representative FACS plots of IL-2 production by CD4+ (upper panels) and CD8+ T cells (lower panels) at d7 post Lm-OVA infection at ZT2 versus ZT14. (B) Summary of data from (A). Cytokine response was measured after αCD3/αCD28 stimulation ex vivo. (Upper panel: CD4+ T cells; lower panel: CD8+ T cells). (C–D) Paired analysis of co-transferred WT (black squares) or Bmal1T-KO OTI cells (blue squares) at d7 post Lm-OVA infection after re-stimulation with OVA-peptide. (C, E) Paired comparison of co-transferred cells showed significant differences in IFNγ and TNFα production (paired t-test). (D) Pairwise comparison revealed significant differences in IL-2-production at ZT2 (paired t-test), as well as significant differences in IL-2 production at ZT2 versus ZT14 (unpaired t-test). (F–H) Single-transfer of WT or Bmal1T-KO OTI cells were analyzed at d7 post Lm-OVA infection for IFNγ (F), IL-2 (G), and TNFα production (H). IL-2 production was significantly different at ZT2 versus ZT14 (unpaired t-test). (B–H) Error bars represent mean ± SD. All data are representative of at least two individual experiments. ***p<0.001, ****p<0.0001. See also Figures S6–7.
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