Research ArticleImmunology

Desynchronization of the molecular clock contributes to the heterogeneity of the inflammatory response

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Science Signaling  05 Mar 2019:
Vol. 12, Issue 571, eaau1851
DOI: 10.1126/scisignal.aau1851
  • Fig. 1 Heterogeneity in BMDM cytokine production is bimodal and unstable.

    (A) BMDMs were stimulated with the indicated concentrations of LPS for 7 hours (top) or 3 hours (bottom) in the presence of brefeldin A (BFA) and then analyzed by intracellular flow cytometry for IL-12p40 (top) or TNF (bottom). Numbers within flow cytometry plots indicate the percentages of cytokine-positive cells. Count (y axis) refers to the number of cells with a given amount of cytokine measured by fluorescence (x axis). (B) Concentration-time response curves for BMDMs stimulated with LPS for the indicated times and then analyzed by intracellular flow cytometry for IL-12p40 or TNF. Graphs indicate the percentage of the total population of cells that were positive for the indicated cytokine at each time point. Data in (A) and (B) are representative of two independent experiments. (C) BMDMs were stimulated with 10-ng LPS/ml for 10 hours. BFA was added after 4 hours of stimulation. Cells were then analyzed by flow cytometry for iNOS and IL-12p40. Data are representative of three independent experiments. (D) Day 6 BMDMs from IL-12p40 (yet40) YFP reporter mice were stimulated with 10-ng LPS/ml for 6 hours and subsequently sorted by flow cytometry into YFP+ and YFP populations. Cells were returned to culture for 4 days and on day 10 were left unstimulated or were restimulated with 10-ng LPS/ml for 6 hours in the presence of BFA before being subjected to intracellular staining for IL-12p40. Data are representative of two independent experiments.

  • Fig. 2 Clock genes Nfil3 and Dbp oppositely control the fraction of LPS-induced BMDMs that are IL-12p40+.

    (A) Intercellular heterogeneity can be generated through desynchronization of cell-intrinsic oscillators. (B) Schematic representation of the primary and secondary loops of the molecular clock. (C) Flow cytometry plots of IL-12p40 in BMDMs of the indicated genotype unstimulated or stimulated with 10-ng LPS/ml for 6 hours. Data are representative of two independent experiments. The numbers within the flow cytometry plots indicate fraction of cells positive for IL-12p40. (D) BMDMs retrovirally transduced with Nfil3, Dbp, or empty expression vectors containing an IRES-hCD2 to allow for identification of successfully transduced cells. BMDMs were either unstimulated or stimulated with 10-ng LPS/ml for 6 hours. Flow cytometry plots for IL-12p40 in transduced BMDMs are shown. Data are representative of three independent experiments. (E) Relationship between the amount of gene overexpression and the fraction of LPS-induced cells that were IL-12p40+. Data are representative of two independent experiments. (F) Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) for Il12b primary transcripts in BMDMs stimulated with 10-ng LPS/ml for the indicated times. Error bars represent mean with SD. Graph is representative of two independent experiments. (G) ChIP for NFIL3 at the Il12b enhancer at time 0 (no stim), 2 hours, and 6 hours after BMDM stimulation with 10-ng LPS/ml. (H) ChIP for FLAG at the Il12b enhancer at the indicated times in Dbp-Flag retrovirally transduced BMDMs after stimulation with 10-ng LPS/ml. For (G) and (H), the percent input represents enrichment of Il12b enhancer DNA relative to DNA immunoprecipitated from an unrelated region of the genome (negative control). Data are representative of three independent experiments (fig. S3, A and B).

  • Fig. 3 Nfil3 and Dbp regulate BMDM inflammatory response to LPS.

    (A) RNA-seq of wild-type (WT) and Nfil3 knockout (KO) BMDMs stimulated with 10-ng LPS/ml for 0 (no stim), 4, or 24 hours. Two independent samples for each condition were sequenced. Genes differentially expressed greater than twofold (Padj < 0.01) in WT or Nfil3 KO BMDMs during at least one of the assayed times are shown. Differential expression is plotted in log2. Blue indicates enriched in WT BMDMs; red indicates enriched in Nfil3 KO BMDMs. Il12b is shown but was only 1.8× enriched in Nfil3 KO at 4-hour LPS. IFN, interferon. (B) RT-qPCR for selected transcripts in WT and Nfil3 KO BMDMs under the indicated conditions (expressed as relative to WT for the indicated conditions). Error bars represent mean with SD from three technical replicates. Data are representative of three independent experiments. (C) RT-qPCR for selected transcripts in BMDMs either transduced with a Dbp-overexpressing or empty vector (EV) under the indicated conditions. Expressed as relative to empty vector for the indicated conditions. Error bars represent mean with SD from three technical replicates. Data are representative of two independent experiments. (D) Ccr2 expression in BMDMs sorted by CCR2 abundance (normalized to Ccr2 expression in CCR2-low cells). Data are representative of three independent experiments. (E) IL-12p40 production in BMDMs sorted by CCR2 abundance and immediately stimulated with LPS (10 ng/ml) with BFA for 6 hours. Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t test.

  • Fig. 4 Resident peritoneal macrophage response to LPS is time of day dependent and controlled by the molecular clock.

    (A) RT-qPCR analysis of clock genes Nfil3 and Dbp from peritoneal cells isolated from C57BL/6 mice (n = 3 per time point) at ZT 0, ZT 6, and ZT 12. ZT 0 indicates the time at which the lights turn on, and ZT 12 indicates the time at which lights turn off in the animal facility. The horizontal bar above each graph indicates the light/dark cycle. Error bars represent mean with SD from three technical replicates. (B) IL-12p40 production in peritoneal macrophages (CD11b+ F4/80+) isolated from C57BL/6 mice at ZT 0 or ZT 12 and stimulated in vitro under the indicated conditions for 4 hours in the presence of BFA. Numbers within boxes indicate fraction of macrophages that are IL-12+ as measured by flow cytometry. (C) Fraction of peritoneal macrophages from Bmal fl/fl;LysMcre (Bmal WT) and Bmal fl/fl;LysMcre+ (Bmal MKO) mice that were IL-12p40+ after stimulation with LPS at either ZT 1 or ZT 12. Cells were stimulated as described in (B). Each circle represents one mouse. Error bars represent mean with SD. (D) Dbp and (E) Nfil3 expression in Bmal fl/fl;LysMcre (Bmal WT) and Bmal fl/fl;LysMcre+ (Bmal MKO) mice at either ZT 1 or ZT 12. Error bars represent mean with SD. (F) Proposed mechanism of why clock phase may control IL-12 production only in a fraction of cells. Each oscillation represents the molecular clock in an individual cell with respect to a “threshold” for IL-12 production. All data are representative of at least three independent experiments. ***P < 0.001, **P < 0.01. n.s., not significant by Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/571/eaau1851/DC1

    Fig. S1. IL-12p40 production by BMDCs depends on Nfil3.

    Fig. S2. The surface abundance of hCD2 correlates with the extent of retroviral gene overexpression.

    Fig. S3. NFIL3 and DBP dynamically interact with the Il12b enhancer after LPS stimulation.

    Fig. S4. Peritoneal macrophages exhibit circadian oscillation of clock gene expression.

    Table S1. RT-qPCR primers used in this study.

  • This PDF file includes:

    • Fig. S1. IL-12p40 production by BMDCs depends on Nfil3.
    • Fig. S2. The surface abundance of hCD2 correlates with the extent of retroviral gene overexpression.
    • Fig. S3. NFIL3 and DBP dynamically interact with the Il12b enhancer after LPS stimulation.
    • Fig. S4. Peritoneal macrophages exhibit circadian oscillation of clock gene expression.
    • Table S1. RT-qPCR primers used in this study.

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