Circadian clock component REV-ERBα controls homeostatic regulation of pulmonary inflammation

Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.


Western blotting
Lung tissue and cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Protein lysates from tissues were prepared using a tissue Ruptor (Qiagen) and 2 µl of benzonase nuclease (Novagen) were added. Cell debris were cleared by centrifugation. Lysates were boiled for 10 minutes in SDS loading dye containing 10% β-mercaptoethanol (v/v) (Sigma) and electrophoresed on Mini Protean TGX Precast Gels 4-15% (BioRad). Resolved proteins were transferred to a 0.2 µm pore size Protran nitrocellulose membrane for 70 minutes then rinsed with Tris-HCl pH 7.6 buffered saline (TBS) solution supplemented with 0.1% Tween-20 (TBST). Membranes were blocked (5% skim milk powder in TBST) for 1 hour at room temperature and then incubated with mouse monoclonal GSK6F05 anti-REV-ERBα, anti-HA (Roche, 12CA5) or anti-β-ACTIN (Abcam, ab8227) antibodies overnight at 4°C. Membranes were washed 3 × 10 minutes with TBST and secondary HRP-linked antibodies (GE Healthcare) were incubated for 1 hour. After 3 × 10 minute TBST washes immunoreactive bands were detected using Supersignal West Dura (ThermoScientific) and chemiluminescence visualised on Kodak BioMax MR or XAR Film.

Kinase inhibitors
JNK and p38 inhibitors were purchased from Sigma (SP600125, SP239063) and used at 10µM and 20µM respectively. IRAK1/4 and MEK1 (U0126) inhibitors were purchased from Calbiochem and used at 20µM and 5µM respectively.

Synthesis of GSK1362
Commercially available solvents and reagents were used without further purification. Flash chromatography was carried out using Merck Kieselgel 60 H silica or Matrex silica 60 unless stated otherwise. Analytical thin layer chromatography was carried out using aluminium-backed plates coated with Merck Kieselgel 60 GF254 that were visualised under UV light (at 254 and/or 360 nm). Nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 at 18 °C unless stated otherwise and were reported in ppm; J values were recorded in Hz and multiplicities were expressed by the usual conventions. Automated column chromatography was performed on pre-packed silica gel columns (30-90 mesh, IST) using a Biotage SP4 or on a reverse-phase Sunfire C18 column. LCMS analysis was carried out on H2Os Acquity UPLC instrument equipped with a BEH column (50 mm x 2.1 mm, 1.7 µm packing diameter) and H2Os micromass ZQ MS using alternate-scan positive and negative electrospray. Analytes were detected as a summed UV wavelength of 210-350 nm. In vacuo refers to evaporation at reduced pressure using a rotary evaporator and diaphragm pump, followed by the removal of trace volatiles using a vacuum (oil) pump.

Supplemental figures and tables
Supplemental Figure 1: Specificity of antibody GSK6F05 for REV-ERBα HEK 293 cells were transfected with HA-REV-ERBα and REV-ERBβ-Halotag. Total cell lysates were analysed by Western blotting for REV-ERBα protein levels using mouse monoclonal antibody GSK6F05 and for REV-ERBβ protein levels using rabbit polyclonal antibody (Proteintech, # 13906-1-AP). Non specific band at about 150 kDa detected with the GSK6F05 antibody. Figure 2: Rev-Erbα -/mice possess an intact clock within the lung but exhibit exaggerated pulmonary innate immune responses (A) Q-PCR analysis in whole lung tissues. Data normalized to WT at ZT0 and presented as mean SEM; n=5-6 for WT and n=3-5 for Rev-Erbα -/per time point.

Supplemental
(B,C) Rev-Erbα -/and WT mice were exposed to aerosolised LPS (2 mg/ml) at ZT0 or ZT12 and culled 5 hours later. (B) Neutrophil numbers in BAL samples were determined by flow cytometry. (C) G-CSF protein levels in BAL samples, measured using multiplex assay. Data presented as mean SEM; n=6-10, *P <0.05, **P <0.01, ***P <0.001 (Two-way ANOVA, post hoc Bonferroni). (D-E) Single cigarette smoke exposures was performed between ZT8 and ZT10 and animals were culled 24 hours after exposure. (D) Cell numbers in BAL samples. Data presented as mean SEM, n = 9-10. (E) Q-PCR analysis of Cxcl5 in whole lung. Data normalized to sham wild-type group and presented as mean SEM; n = 9-10, *P <0.05 (Twoway ANOVA, post hoc Bonferroni). Rev-Erbα -/or littermate controls were transplanted into wild-type recipient mice which were then exposed to aerosolised LPS at 2 mg/ml or saline at indicated times for 20 min. Monocytes in blood samples collected 5 hours after challenge were determined by flow cytometry analyses. Data presented as mean SEM; n = 2 (saline) or 5-8 (LPS), ***P <0.001 (Two-way ANOVA, post hoc Bonferroni). (B) Q-PCR analysis in PECs collected at ZT9, seeded into plates and let to attach for 2 hours before lysis. Data normalized to Rev-Erbα fl/fl littermate control group and presented as mean SD; n = 3. ***P <0.001 (Student's t-test). (C) PER2 bioluminescence recordings from Rev-Erbα fl/fl (black) and LysM-Rev-Erbα-DBD m (purple) PECs. Photon counts per minute were normalised to a 24 hour moving average and traces are representative of 3 biological replicates. (D,E) LysM-Rev-Erbα-DBD m and littermate control mice were exposed to aerosolised LPS at 2 mg/ml or saline at ZT4 for 20 min. (D) Neutrophil numbers in the BAL samples collected 5 hours after challenge were determined by flow cytometry analyses. (E) CXCL5 protein levels in BAL samples were assessed by multiplex assay. Data presented as mean SEM; n = 7-9 (Two-way ANOVA, post hoc Bonferroni). (F) LysM-Rev-Erbα-DBD m and littermate control mice were exposed to aerosolised LPS at 2 mg/ml at indicated times for 20 min. Neutrophil numbers in the BAL samples collected 5 hours after challenge were determined by flow cytometry analyses. Data presented as percentage of littermate control neutrophil numbers; n = 4 per time point. (G) LysM-Rev-Erbβ -/and littermate control mice were exposed to aerosolised LPS at 2 mg/ml or saline at ZT4 for 20 min. Neutrophil numbers in the BAL samples collected 5 hours after challenge were determined by flow cytometry analyses. Data presented as mean SEM; n = 8 (Two-way ANOVA, post hoc Bonferroni). (H) Snapshots of PER2 oscillations in bronchioles within precision cut lung slices. Scale bars, 500 µM. Bioluminescence intensity from bronchioles was quantified, normalised to a 24 hour moving average. Traces are representative of 2 biological replicates. (I) Ccsp-Rev-Erbα-DBD m and littermate control mice were exposed to aerosolised LPS at 2 mg/ml or saline at ZT0 or ZT12 for 20 min. Neutrophil numbers in the BAL samples collected at ZT5 or ZT17 were determined by flow cytometry analyses. Data presented as mean SEM; n = 16 at ZT0 and n = 7-8 at ZT12, *P <0.05, **P <0.01 (Two-way ANOVA, post hoc Bonferroni).

new homeostatic circuit linking inflammation and REV-ERBα
Under resting, non-stress conditions, REV-ERBα and its paralog REV-ERBβ act as rhythmic repressors to limit inflammatory activity. Upon inflammatory triggers, such as LPS or cytokine challenges, REV-ERBα is SUMOylated, which drives the protein ubiquitination, allowing the proteosomal degradation of a repressor and therefore development of a full immune response. Treatment with synthetic REV-ERBα ligand GSK1362 blocks this degradation pathway, suggesting potential pharmacological intervention to enhance activity of an inhibitor of inflammation in a context of chronic inflammatory diseases.

REV-ERBs
Inflammatory pathways  Table 1: cytokine/chemokine levels in BAL from wild-type and Rev-Erbα -/mice exposed to LPS at ZT4 Mice were exposed to aerosolised LPS (2mg/ml) at ZT4 for 20 minutes and culled 5 hours later. Cytokine/chemokine levels were measured using magnetic luminex assay. Values are presented as mean SEM, n=8, Student's t test.  Table 2: cytokine/chemokine levels in BAL from wild-type and Rev-Erbα -/mice exposed to LPS at ZT0 or ZT12 Mice were exposed to aerosolised LPS (2mg/ml) at ZT0 or ZT12 for 20 minutes and culled 5 hours later. Cytokine/chemokine levels were measured using magnetic luminex assay. OOR< = Out of Range Below. Values are presented as mean SEM, n=6-10, Student's t test.

Significance WT
Supplemental Table 3: cytokine/chemokine levels in BAL from Rev-Erba fl/fl and Ccsp-Rev-Erbα-DBD m mice exposed to LPS at ZT4 Mice were exposed to aerosolised LPS (2mg/ml) at ZT4 for 20 minutes and culled 5 hours later. Cytokine/chemokine levels were measured using magnetic luminex assay. OOR< = Out of Range Below. Values are presented as mean SEM, n=5-9, Student's t test.  Table 4: cytokine/chemokine levels in BAL from Rev-Erba fl/fl and Ccsp-Rev-Erbα-DBD m mice exposed to LPS at ZT0 or ZT12 Mice were exposed to aerosolised LPS (2mg/ml) at ZT0 or ZT12 for 20 minutes and culled 5 hours later. Cytokine/chemokine levels were measured using magnetic luminex assay. OOR< = Out of Range Below. Values are presented as mean SEM, n=7-9, Student's t test.