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. 2013 Nov 8;342(6159):727-30.
doi: 10.1126/science.1243884.

TH17 Cell Differentiation Is Regulated by the Circadian Clock

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Free PMC article

TH17 Cell Differentiation Is Regulated by the Circadian Clock

Xiaofei Yu et al. Science. .
Free PMC article

Abstract

Circadian clocks regulate numerous physiological processes that vary across the day-night (diurnal) cycle, but if and how the circadian clock regulates the adaptive immune system is mostly unclear. Interleukin-17-producing CD4(+) T helper (T(H)17) cells are proinflammatory immune cells that protect against bacterial and fungal infections at mucosal surfaces. Their lineage specification is regulated by the orphan nuclear receptor RORγt. We show that the transcription factor NFIL3 suppresses T(H)17 cell development by directly binding and repressing the Rorγt promoter. NFIL3 links T(H)17 cell development to the circadian clock network through the transcription factor REV-ERBα. Accordingly, TH17 lineage specification varies diurnally and is altered in Rev-erbα(-/-) mice. Light-cycle disruption elevated intestinal T(H)17 cell frequencies and increased susceptibility to inflammatory disease. Thus, lineage specification of a key immune cell is under direct circadian control.

Figures

Figure 1
Figure 1. NFIL3 suppresses Th17 cell development in a T cell-intrinsic manner
Intestinal Th17 cell frequencies were analyzed in wild-type (wt) and Nfil3−/− mice by intracellular staining of IL-17A and IFNγ (A,B) and nuclear staining of RORγt and Foxp3 (C,D). Representative flow cytometry plots are shown in (A) and (C) and combined data from multiple mice are shown in (B) and (D). (E) Splenic Th17 and Th1 cell frequencies in wild-type and Nfil3−/− mice. (F) Naïve wild-type CD4+ T cells were transduced by lentivirus encoding EGFP only or EGFP-tagged NFIL3, followed by culture under Th17-polarizing conditions. Th17 cell frequencies were compared between transduced (EGFP+) and non-transduced (EGFP) cell populations in the same well. (G) Naïve wild-type and Nfil3−/− CD4+ T cells were transferred intravenously into Rag1−/− mice and LPLs were analyzed 4 weeks later. Data are from two independent experiments. Groups were plotted as mean ± SEM and compared by two-tailed student’s t-test (B, D, E, H) or one-way ANOVA (F). *,p<0.05; **,p<0.01; ***, p<0.001; ****,p<0.0001; ns, not significant.
Figure 2
Figure 2. NFIL3 represses Rorγt transcription by binding directly to its promoter
(A) LPLs from wild-type and Nfil3−/− mice were analyzed by nuclear staining of RORγt and mean fluorescence intensities (MFI) were plotted. (B) ChIP analysis of CD4+ T cells using IgG or anti-NFIL3 antibody. Enrichment of the Rorγt promoter was calculated as the ratio of the anti-NFIL3 to the IgG control pull-down. (C) EMSA with nuclear extracts of HEK293T cells transfected with an empty vector or an NFIL3-encoding vector. A 30-bp DNA fragment encompassing the NFIL3-binding site from the Rorγt promoter was synthesized as a wild-type probe. The mutant probe has the same sequence except that the NFIL3 binding site was mutated. NFIL3 binding specificity was demonstrated by competition with non-radioactively labeled probes and supershift with the anti-NFIL3 antibody. (D) Luciferase reporter assay. A 1018 bp (from −1013 to +5) fragment of the Rorγt promoter was fused with firefly luciferase and position 8 of the NFIL3 binding site was mutated from A to T in the mutant reporter. Jurkat T cells were transfected with reporters and an empty vector or an NFIL3-encoding vector. Luciferase activity was normalized to cells transfected with vector-only controls. Groups were plotted as mean ± SEM and compared by two-tailed student’s t-test. *,p<0.05; ns, not significant.
Figure 3
Figure 3. Th17 cell development is regulated by the circadian clock transcriptional network
(A) Nfil3 expression levels were quantified in activated CD4+ T cells by real-time PCR. (B) Naïve wild-type and Rev-erbα−/− CD4+ T cells were polarized to Th17 cells in vitro. (C,D) Small intestinal Th17 cell frequencies in wild-type and Rev-erbα−/− mice. Representative flow cytometry plots are shown in (C) and combined data are shown in (D). (E) Nfil3 expression in activated wild-type and ClockΔ19/Δ19 CD4+ T cells. (F) Naïve wild-type and ClockΔ19/Δ19 CD4+ T cells were cultured under Th17-polarizing conditions. (G,H) Small intestinal Th17 cell frequencies of wild-type and ClockΔ19/Δ19 mice. Representative flow cytometry plots are shown in (G) and combined data are shown in (H). Data are plotted as mean ± SEM and statistics were performed with the two-tailed student’s t-test. *,p<0.05; **,p<0.01; ***,p<0.001; ns, not significant.
Figure 4
Figure 4. Diurnal regulation of Th17 cell development
(A) Nfil3 and (B) Rorγt expression in activated CD4+ T cells during the circadian cycle. (C) Diurnal NFIL3 binding to the Rorγt promoter as determined by ChIP assay. (D,E) Relative percentage of IL-17A-producing CD4+ T cells after in vitro polarization of naïve wild-type (D) or Nfil3−/− (E) CD4+ T cells. (F) Wild-type, Rev-erbα−/− or Rev-erbα/β−/− double knockout mice were maintained under a normal light cycle (left) or were subjected to perturbed light cycles (6 hour phase advance every 4 days). Age-matched mice were co-housed prior to the experiment to minimize microbiota differences between mice in each group. (G) Intestinal Th17 cell frequencies in wild-type, Rev-erbα−/−, and Rev-erbα/β−/− mice subjected to light cycle perturbation. Th17 cell frequencies were calculated relative to the age-matched, co-caged controls in each experiment. (H-J) Mouse weight loss during DSS treatment of mice previously subjected to normal or perturbed light cycles. Mice were treated with anti-IL17A or an isotype control along with DSS challenge. Disease severity on Day 5 was assessed by weight loss (H,I) and colon shortening (J). (K) Schematic diagram summarizing how NFIL3 links the circadian clock circuitry to Th17 cell development. Data are plotted as mean ± SEM and statistical analysis was performed with two-tailed student’s t-test (A-E,G) or two-way ANOVA (H-J). *,p<0.05; **,p<0.01; ***,p<0.001; ns, not significant.

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