An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action

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An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action Supplementary Figure : Expression levels of toll-like receptor 4 (Tlr4) in muse lung does not change throughout the day. Data are normalised to CT levels (n=4/time point). Supplementary Figure : Clock control of pulmonary inflammation is not directed by resident macrophages. (a) Bronchoalveolar macrophages lavaged from the lungs of per-luc mice are rhythmic (data representative of n=3 independent trials). (b)targeted deletion of Bmal in LysM-cre positive cells dampens the diurnal rhythms in the clock genes Nrd, Nrd NRD, NRD (Rev-erbα and β) in bronchoalveolar macrophages, (values normalised to littermate controls (WT) CT, n=3 independent trials). (c) Disruption of the macrophage clock has no significant effect on time-of-day gating of neutrophil recruitment and levels of pro-inflammatory BAL cytokines after aerosolised LPS (n=5-8, Two way ANOVA, post hoc Bonferroni, * p<.5; p<.). Supplementary Figure 3: Clock gene rhythms are un-impaired in CCSP-icre +/- Bmal fl/fl mice (=CCSP-Bmal -/- ) as compared to littermate controls (wildtype) in (a) lung and (b) liver as illustrated by Q-PCR analysis in tissue harvested over 4h, values normalised to wildtype CT levels, n=4. (c) Targeted deletion of Bmal in CCSP expressing cells has no effect on the circadian behaviour of these animals in either h light h dark (:) or under constant darkness (DD) as determined by wheel running activity, actograms representative of n=4-5, grey shaded areas represent darkness (DD). Supplementary Figure 4: Expression of Bmal and Nrd (Rev-erbα) in the alveolar parenchyma of the lung as determined by in situ hybridisation. Values were compared using a two way ANOVA with post-hoc Bonferroni tests, n=3-4, ** p<.; p<.. Genotypes are as defined in Supplementary Figure 3. Supplementary Figure 5: Targeting of the bronchiolar clock results in local neutrophilia, but no associated lung damage. (a) Flow cytometric analysis of neutrophil numbers in bronchoalveolar lavage fluid collected from WT and CCSP- Bmal -/- at CT and CT (n=5-6; t-test shows significant time-of-day differences in littermate controls (*p<.5); macrophage numbers did not vary by time-of-day or gentoype. (b) Histological analysis of lung sections cut from month old CCSP-Bmal -/- mice revealed normal morphology compared to littermate controls (images representative of n=3, scale bar=µm) Supplementary Figure 6: Validation of PCR array data. CCSP-Bmal -/- (n=) and littermate control (n=7) mice were challenged with aerosolised LPS at ZT4 and sacrificed h later and neutrophilia in BAL assessed (a). (b) RNA was extracted from lung tissue and used for Q-PCR analysis to quantify expression of target genes highlighted by the array (Supplementary Table, values normalised to littermate levels and compared using t-tests). Supplementary Figure 7: Dose-dependent neutrophil chemotactic response to CXCL5. Recombinant CXCL5 was tested against mouse neutrophils in a Boyden Nature Medicine: doi:.38/nm.3599

chamber assay, representative of 3 independent trials (-way ANOVA and posthoc Bonferroni test; *p<.5; p<.). Supplementary Figure 8: Co-expression of Cxcl5 mrna and CCSP protein in lung. Cxcl5 mrna was quantified by ISH and tissue slices subsequently subjected to immunohistochemical staining for CCSP protein. CCSP positive cells (around the bronchioles) were visualised with DAB (brown), sections were counterstained with haematoxylin. A primary antibody negative condition was utilised as a control. Supplementary Figure 9: Cxcl5 promoter does not respond to circadian CLOCK or BMAL. (a) RAT- cells stably transfected with Cxcl5-LUC promoter were incubated in recording medium and luciferase output tracked over time (representative of n=4). (b) Effects of over-expression of BMAL and CLOCK on Cxcl5-LUC promoter activity in transiently transfected RAT- cells (n=4). Supplementary Figure : Adrenalectomy affects systemic inflammatory responses, but not the rhythmic expression of clock genes in the lung. (a) IL6 response of intact and ADX mice to an intraperitoneal dose of LPS. Levels of IL6 in serum were measured by ELISA, (intact: n=4 ADX: n=6). (b) Effect of adrenalectomy (ADX) on clock gene expression in pulmonary tissue sampled 5h after exposure to aerosolised saline (vehicle) at the indicated times, (Intact CT n=6, intact CT n=3, ADX CT n=5, ADX CT n=4). Values were compared using Two Way ANOVA with post-hoc Bonferroni, *p<.5; **p<.; p<.). Supplementary Figure : Expression and quantification of Glul mrna in the mouse lung (bronchioles and alveolar parenchyma) as assessed by in-situ hybridisation (n=4 two-way ANOVA and post hoc Bonferonni; **p<.; p<.). Supplementary Figure : Expression of glucocorticoid receptor in CCSP- Bmal -/- lung. (a) Immunohistochemical staining of lung tissue collected at CT and CT from CCSP-Bmal -/- and littermate controls (wildtype) mice for glucocorticoid receptor (GR). Brown (DAB) positive staining with a haematoxylin counterstain. Images representative of n=3-4, scale bar µm. (b) Western blot showing protein expression of GR in lung tissue collected at CT from CCSP-Bmal -/- (-/-) and littermate controls (wildtype); α tubulin was used as a loading control, n=3. Supplementary Figure 3: Intra-peritoneal DEX engages target genes in the lung. Mice injected with vehicle and DEX (mg/kg), n=3/treatment), and lungtissue analysed for GR-target gene expression h later. (p<., t-test) Supplementary Figure 4: Suppressive effect of DEX pre-treatment on cytokine production in the lung after aerosolised LPS. 5 cytokines/chemokines were measured in BAL fluid collected 5h after challenge at either ZT or ZT by a bioplex or ELISA (CXCL, CXCL, CXCL5), n=7-9 Values were compared at each time point by a t-test to determine significant effects of DEX on cytokine production. (IL4 and IL5 were below the limit of detection; IL, IL9, IL, CCL, IFNγ, CCL5, CXCL and CXCL were not significantly repressed by DEX at either time point). Only DEX responsive cytokines are shown here (p<.5). None of these cytokines showed a time-of-day variation in response. Nature Medicine: doi:.38/nm.3599

Tlr4 (RQ).5..5. Time of challenge Nature Medicine: doi:.38/nm.3599 Supplementary Figure

Neutrophils x 5 /ml BAL G-CSF (ng/ml) Bioluminescence (cpm) Rev-erb α (RQ) Rev-erb β (RQ) a 3 b 8 6 * CT CT 4 3 * 4 Period: 4.9 ±. 4 48 7 Time (hours) c 6 5 4 3 WT LysM-bmal-/- Bmal -/- * d.5..5 CT CT Time of challenge. CT CT Time of challenge Nature Medicine: doi:.38/nm.3599 Supplementary Figure :

Time (days) Time (days) Period (h) Per (RQ) Bmal (RQ) Rev-erb α (RQ) Cry (RQ) Per (RQ) Bmal (RQ) Rev-erb α (RQ) Cry (RQ) a Lung 8.5 3 4.5 6 b Liver 6 8 Time (h) 6 8 Time (h) 6 8 Time (h) 6 8 Time (h) wildtype.5 3 5 5 CCSP-bmal-/- Bmal -/-.5 9 6 3 6 8 Time (h) 6 8 Time (h) 6 8 Time (h) 6 8 Time (h) c Circadian time (CT) Circadian time (CT) 4 L:D D:D 3.8 3.6 3.4 3. wildtype CCSP-Bmal -/- 3 Nature Medicine: doi:.38/nm.3599 Supplementary Figure 3 :

Bmal (AU) Rev-erb α (AU) Alveolar parenchyma..8 Bmal ** CT CT Rev-erb α 8.4 4 Wildtype CCSP-bmal-/- Bmal -/- Wildtype CCSP-bmal-/- Bmal -/- Nature Medicine: doi:.38/nm.3599 Supplementary Figure 4:

H and E Masson Trichrome cdb a Neutrophils x 3 /mouse Macrophages x 4 /mouse Neutrophils Macrophages CT: WT CT: WT 5 4 3 CT CT 8 6 CT CT * 4 CT: CCSP-Bmal -/- CT: CCSP-Bmal -/- Ly6G b WT CCSP-Bmal -/- Nature Medicine: doi:.38/nm.3599 Supplementary Figure 5

Neutrophils (x 5 /ml BAL) Cxcl3 (RQ) Itgb (RQ) Cxcl (RQ) a..8 * b..5...4..5.... Nature Medicine: doi:.38/nm.3599 Supplementary Figure 6

Neutrophils (% of upper chamber) 4 * 3.5 5 5 rcxcl5 (ng/ml) Nature Medicine: doi:.38/nm.3599 Supplementary Figure 7

ISH: Cxcl5 Post ISH staining: CCSP (Brown, DAB) Post ISH staining: Primary negative control Nature Medicine: doi:.38/nm.3599 Supplementary Figure 8

Bioluminescence (cpm) Cxcl5-LUC a 3 5 5 Cxcl5-LUC reporter b 7 6 5 4 3 Empty BMAL CLOCK BMAL/CLOCK BMAL/CLOCK 5 3 4 5 Time (days) Control + LPS Nature Medicine: doi:.38/nm.3599 Supplementary Figure 9

Per (RQ) Bmal (RQ) Rev-erb α (RQ) IL6 (pg/ml serum) a 5 5 5 Intact ADX b 5 4 3 CT ** CT..8.6.4. *.5.5 Intact ADX Intact ADX Intact ADX Nature Medicine: doi:.38/nm.3599 Supplementary Figure

Glul (AU) Glul (AU) wildtype CT CT Bronchioles Alveolar Parenchyma 3 ** CT CT CCSP-Bmal -/- Wildtype CCSP-bmal-/- Bmal -/- Wildtype CCSP-bmal-/- Bmal -/- Nature Medicine: doi:.38/nm.3599 Supplementary Figure

a CT CT Wildtype Wildtype CCSP-Bmal -/- CCSP-Bmal -/- IgG control b 5 75 WT -/- WT -/- WT -/- α GR (M) 5 α tubulin Nature Medicine: doi:.38/nm.3599 Supplementary Figure

Gilz (RQ) Dusp (RQ) Tnf α (RQ) Il6 (RQ) 8 6.5.5 4.5.5 Nature Medicine: doi:.38/nm.3599 Supplementary Figure 3

ZT ZT and ZT ZT G-CSF GM-CSF CT ZT CT ZT CCL CCL3 CXCL5 ILa ILb IL3 IL6 IL3 CCL4 TNF alpha ILp4 ILp7 IL7 4 6 8 % of LPS treated Nature Medicine: doi:.38/nm.3599 Supplementary Figure 4

Supplementary Table : BAL cytokines levels in wildtype and LysM-Bmal -/- measured in mice 5h after challenge with aerosolised LPS at either CT or CT, n=5-8. Values were compared using Two Way ANOVA and post-hoc Bonferroni time of day difference within genotype P<.5, * time of day difference within genotype P<.5. Wild-type LysM-Bmal -/- CT CT CT CT IL6 67. ± 34.9 73.±.7 35. ± 9.5 54.9 ± 3.6* TNF α 86.8± 5.3 49.8±44.3 8.5± 58.8 74.6 ± 4. CXCL 9.6±.7 577.9± 7.8 76.± 8.9 7.3 ±.7 CXCL 838.±33.4 58.8±. 544.4±5. 7.4±5. CXCL5 778.9±6.3 98.6±79.9 967.8±43.5 5.6±397. G-CSF 84. ± 75.3 543.3± 8. 979.5± 54.9 66.7 ± 36.3 Nature Medicine: doi:.38/nm.3599

Supplementary Table : A PCR array was utilised to screen a panel of inflammatory genes for differences in expression between lung tissue collected from LPS challenged Wildtype and CCSP-Bmal -/- mice. One sample was utilised per genotype. Fold change is the normalised gene expression in the test sample (CCSP-Bmal -/- ) divided by the normalised gene expression in the control sample (WT). Genes which showed substantial up-regulation are shown in bold text. Gene Fold change Gene Fold change Gene Fold change Gene Fold change Abcf. Ccl9.5 Cxcr3. Ilrg. Bcl6. Ccr.99 Ccr. Il3.99 Cxcr5.93 Ccr. Ifng.3 Il4. C3. Ccr3. Il.99 Il5ra. Casp.3 Ccr4. Ilra. Il6ra.99 Ccl. Ccr5. Ilrb. Il6st. Ccl.5 Ccr6.5 Il. Cxcr.99 Ccl. Ccr7. Il3. Itgam. Ccl7. Ccr8. Il3ra. Itgb 4.3 Ccl9. Ccr9.5 Il5. Lta. Ccl. Crp. Il6. Ltb. Ccl 4.4 Cx3cl. Il7b.46 Mif. Ccl. Cxcl. Il8. Aimp. Ccl4.95 Cxcl.5 Ila. Spp. Ccl5.99 Cxcl 8.7 Ilb. Tgfb. Ccl3. Cxcl. Ilf6. Tnf. Ccl4. Cxcl3 8.3 Ilf8. Tnfrsfa. Ccl5.3 Cxcl5. Ilr. Tnfrsfb. Ccl6. Pf4. Ilr. Cd4lg. Ccl7. Cxcl5 8.5 Il. Tollip.5 Ccl8 4. Cxcl9.5 Ilrb. Xcr. Nature Medicine: doi:.38/nm.3599

Supplementary Table 3: BAL cytokines levels in wildtype and CCSP-Bmal -/- mice 5h after challenge with aerosolised LPS at either CT or CT, n=7-. Values were compared using Two Way ANOVA and post-hoc Bonferroni ** time of day difference within genotype P<., * time of day difference within genotype P<.5. and P<.5 genotype difference within time point Wild-type CCSP-Bmal -/- CT CT CT CT IL6 453±84 86±3** 37±36 3±43 G-CSF 98±53 34±8** 789±3 7±56* CXCL 34±576 646±39 56±34 936±59 CXCL 978±67 774±9 85±597 54±3 CXCL5 385±746 68±383 8453±57 6653±545* TNF α 3867±66 48±59 65±44 54±548 Nature Medicine: doi:.38/nm.3599

Supplementary Table 4: Nucleotide sequences of Q-PCR primers and probes utilised Gene Sense Anti sense Probe (FAM-TAMRA) Bmal CCAAGAAAGTATGGACACAGACAA GCATTCTTGATCCTTCCTTGGT TGACCCTCATGGAAGGTTAGAATATGCAGAA A Per GCCTTCAGACTCATGATGACAGA TTTGTGTGCGTCAGCTTTGG ACTGCTCACTACTGCAGCCGCTCGT Rev-erbβ AGT AGG TGG ATG TTC TCA GAC TGA GA ATG GAG ACT TGC TCA TAG GAC ACA C CAG AAA TAG TTA CCT GTG CAA CAC TGG AGG GAG Tlr4 AAA CTT GCC TTC AAA ACC TGG C ACC TGA ACT CAT CAA TGG TCA CAT C CAC GTC CAT CGG TTG ATC TTG GGA GAA Cry CTGGCGTGGAAGTCATCGT CTGTCCGCCATTGAGTTCTATG CGCATTTCACATACACTGTATGACCTGGACA β-actin AGGTCATCACTATTGGCAACGA CACTTCATGATGGAATTGAATGTAGTT TGCCACAGGATTCCATACCCAAGAAGG 36B4 (SYBR green) GCT GAT GGG CAA GAA CAC CA CCC AAA GCC TGG AAG AAG GA - Nature Medicine: doi:.38/nm.3599

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