SUPPLEMENTAL MATERIAL Flavones Modulate Respiratory ...10.1074/jbc.M116.771949/-/DC1/jbc.M… ·...

Hariri,etal.SupplementalMaterial 1

SUPPLEMENTAL MATERIAL Flavones Modulate Respiratory Epithelial Innate Immunity:

Anti-Inflammatory Activity and Activation of T2R14 Benjamin M. Hariri, Derek B. McMahon, Bei Chen, Jenna R. Freund, Corrine J. Mansfield, Laurel J. Doghramji, Nithin D. Adappa,

James N. Palmer, David W. Kennedy, Danielle R. Reed, Peihua Jiang, and Robert J. Lee

SUPPLEMENTAL FIGURE 1. T2R14 and T2R39 antibody validation to test for antibody cross-reactivity. A, Immunofluorescence for T2R14 and T2R39 in A549 cells over-expressing either T2R14 or T2R39. Signal above background was observed with T2R14 antibody against T2R14-transfected cells, but not with T2R39-transfected cells, as well as with T2R39 antibody against T2R39-transfected cells, but not against T2R14-transfected cells. B, Quantification of raw fluorescence intensity from experiments as in A. Mean ± SEM of 8 fields from 2 separate experiments for each condition shown. Significance determined by one-way ANOVA with Bonferroni posttest; ** p <0.01.


SUPPLEMENTAL FIGURE 2. Differences in cilia localization patterns of T2R4, T2R14, T2R38, and T2R16 in sinonasal epithelial cells. Images show two orthogonal planes (apical side with cilia at the top, basolateral side at the bottom) representative of at least 3 cultures from 3 individual patients (left) and representative line scan of fluorescence intensity (right) taken from the region indicated by dashed line. A, Localization of T2R38 and T2R4, showing proximal T2R38 and distal T2R4 with central overlap. B, Similar pattern was observed for T2R14 (proximal) and T2R4 (distal). C-D, In contrast, expression of T2R38 and T2R14 (C) or T2R16 (D) was more tightly co-localized. Scale bars shown are for x-axis only. Because of potential changes in z-distance traveled vs. step size due to refractive index difference, z distance was not calibrated to microns. E, Representative scatter plots of fluorescence intensity from images as in A-D, showing Pearson’s correlation coefficient (R) for T2R4 and T2R38 co-localization, T2R4 and T2R14 co-localization, and T2R14 and T2R38 co-localization. Plots created using raw 12-bit images and Mander’s coefficient ImageJ plug-in.


SUPPLEMENTAL FIGURE 3. Activation of heterologously-expressed T2R14 by multiple flavones. A-D, HEK293T cells transfected with T2R14 + Gα16/gust44 exhibited calcium responses to apigenin (B), wogonin (C), chrysin (D), and tangeritin (E), while cells transfected with Gα16/gust44 alone exhibited no response to any of the flavones tested. Averages of responding cells from 3-6 experiments are shown.

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Supplemental Fig. 3


SUPPLEMENTAL FIGURE 4. T2R14 and T2R39 respond to flavone stimulation, but T2R4, T2R5, T2R16, and T2R38 do not. A-F, HEK293T cells co-transfected with Gα16/gust44 and T2R14 (A) or T2R39 (B) responded to apigenin, while those transfected with T2R4 (C), T2R5 (D) T2R16 (E), or T2R38 (F) exhibited no responses to flavones but did respond to known cognate ligands parthenolide (T2R4), 1,10-phenanthroline (T2R5), salicin (T2R16), or phenylthiocarbamide (PTC; T2R38). Each trace represents average of ~50 responding cells from one representative experiment (n = 3 experiments each).

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SUPPLEMENTAL FIGURE 5. Inhibition of flavone-induced [Ca2+]i signals in the presence of 50 µM 5’-fluoro-6-methoxyflavanone. A, 50 µM 5’-fluoro-6-methoxyflavanone elicited no response from TAS2R14+Gα16/gust44-transfected HEK293T cells, while higher concentrations elicited small calcium responses. B, 100 µM 5’-fluoro-6-methoxyflavanone did not elicit responses from TAS2R38+Gα16/gust44 -transfected HEK293T cells, nor did it inhibit the response to 100 µM phenylthiocarbamide (PTC). C, Responses to apigenin in TAS2R14+Gα16/gust44-transfected HEK293T cells were blocked by prior treatment with 5’-fluoro-6-methoxyflavanone (left). Control experiment from non-5’-fluoro-6-methoxyflavanone-treated cells shown on the right. D, Similar inhibition of chrysin-induced [Ca2+]i responses by 5’-fluoro-6-methoxyflavanone was observed. All traces shown are averages of 30-50 responding cells from a single experiment representative of 3 independent experiments.

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SUPPLEMENTAL FIGURE 6. Lack of effect of TAS2R38 genotype on Ca2+ responses to apigenin. A-B, Fura-2 traces of [Ca2+]i during stimulation with 100 µM apigenin in primary human sinonasal ALIs from TAS2R38 PAV/PAV (homozygous taster) or AVI/AVI (homozygous non-taster) individuals. Traces show average of 6 cultures from 3 individual patients (2 per patient). C, Lack of inhibition of apigenin-induced [Ca2+]i elevation in the presence of reported T2R38 inhibitor probenecid [Greene, T.A., Alarcon, S., Thomas, A., Berdougo, E., Doranz, B.J., Breslin, P.A.S., Rucker, J.B. (2011). “Probenecid Inhibits the Human Bitter Taste Receptor TAS2R16 and Suppresses Bitter Perception of Salicin.” PLoS One. 6:e20123]. D, T2R38 agonist phenylthiocarbamide (PTC) induced [Ca2+]i elevations in PAV/PAV but not AVI/AVI cultures. E, Summary of changes in [Ca2+]i from A-D. Concentrations shown are in µM. Signiciance determined by 1-way ANOVA with Bonferroni posttest; ** p <0.01 and n.s. = not significantly different.

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SUPPLEMENTAL TABLE 1: Probabilities for activation of T2R receptors by flavone compounds used in this study according to BitterX (1), as well as experimentally-derived effective concentration (EC) values from prior studies (2) according to BitterDB (3).

Abbreviations: EC, effective concentraiton; N/A, not available; NR, value not returned by software, PR, probability; SMILES, simplified molcular-input line-entry system

References (1) Huang, W., Shen, Q., Su, X., Ji, M., Liu, X., Chen, Y., Lu, S., Zhuang, H., Zhang, J. (2016). "BitterX: a tool

for understanding bitter taste in humans." Sci. Rep. 6:23450. (2) Roland, W.S., van Buren, L., Gruppen, H., Driesse, M., Gouka, R.J., Smit, G., Vincken, J.P. (2013). "Bitter

taste receptor activation by flavonoids and isoflavanoids: modeled structural requirements for activation of hTAS2R14 and hTAS2R39." J Agric Food Chem. 61:10454-10466.

(3) Wiener, A., Shudler, M., Levit, A., and Niv, M.Y. (2012). "BitterDB: a database of bitter compounds." Nucleic Acids Res. 40:D413-419.

Supplementary Table 1: Probabilities for activation of T2R receptors by flavone compounds used in this study, and expermentally-derived effective concentration (EC) values from prior studies.

Compound SMILES(PubChem)Bitter?(BitterX)

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

BitterXPr

BitterDBEC

Apigenin C1=CC(=CC=C1C2=CC(=O)C3=C(C=C(C=C3O2)O)O)O

Yes61.587 N/A 59.18 N/A NR N/A NR N/A 77.119 8 NR N/A NR N/A 79.32 1 62.173 N/A 58.419 N/A NR N/A

Chrysin C1=CC=C(C=C1)C2=CC(=O)C3=C(C=C(C=C3O2)O)O

Yes62.482 N/A 59.01 N/A NR N/A NR N/A 77.46 63 NR N/A NR N/A 80.49 16 65.441 N/A 58.491 N/A NR N/A

Wogonin COC1=C(C=C(C2=C1OC(=CC2=O)C3=CC=CC=C3)O)O

Yes64.253 N/A 60.05 N/A NR N/A 56.653 N/A 76.567 N/A NR N/A NR N/A 80.91 N/A 62.164 N/A 72.073 N/A 60.956 N/A

Tangeritin COC1=CC=C(C=C1)C2=CC(=O)C3=C(O2)C(=C(C(=C3OC)OC)OC)OC

Yes58.542 N/A 58.12 N/A 65.114 N/A NR N/A 73.954 N/A 62.402 N/A 51.679 N/A 78.13 N/A 59.824 N/A 65.244 N/A 59.114 N/A

Abbreviations:EC,effectiveconcentraiton;N/A,notavailable;NR,valuenotreturnedbysoftware,PR,probability;SMILES,simplifiedmolcular-inputline-entrysystem

ReferencesBitterX Huang,W.,Shen,Q.,Su,X.,Ji,M.,Liu,X.,Chen,Y.,Lu,S.,Zhuang,H.,Zhang,J.(2016)."BitterX:atoolforunderstandingbittertasteinhumans".Sci.Rep.6:23450.BitterDB Wiener,A.,Shudler,M.,Levit,A.,andNiv,M.Y.(2012)."BitterDB:adatabaseofbittercompounds."NucleicAcidsRes.40:D413-419.ECvalues Roland,W.S.,vanBuren,L.,Gruppen,H.,Driesse,M.,Gouka,R.J.,Smit,G.,Vincken,J.P.(2013)."Bittertastereceptoractivationbyflavonoidsandisoflavanoids:modeledstructuralrequirementsforactivationofhTAS2R14andhTAS2R39."JAgricFoodChem.61:10454-10466.

T2R38 T2R39 T2R41 T2R43 T2R46T2R16T2R1 T2R5 T2R7 T2R10 T2R14


Raw Data Values From Main Text Figures FIGURE 2. A, Muc5AC/phalloidin ratios (right bar graph) were 0.51 ± 0.01 (control) and 1.2 ± 0.07 (PMA-stimulated; p <0.05 vs. control). Apigenin, chrysin, and wogonin reduced PMA-stimulated Muc5AC/phalloidin ratio to 0.70 ± 0.03, 0.88 ± 0.07, and 0.66 ± 0.02, respectively (all values p <0.05 vs. PMA only). B, iNOS/α-tubulin ratio was 0.1 ± 0.003 (control) and 1.0 ± 0.04 (PMA-stimulated cells; p <0.01 vs. control). Apigenin, chrysin, wogonin, and the flavone mix reduced the PMA-stimulated iNOS/α-tubulin ratio to 0.29 ± 0.01, 0.46 ± 0.03, 0.26 ± 0.02, and 0.44 ± 0.02, respectively (all values p <0.05 vs. PMA alone). FIGURE 3. A, Baseline unstimulated IL8 (control) was 370 ± 195 pg/ml and increased to 1996 ± 225 pg/ml after 42 hrs. PMA (p < 0.01 vs unstimulated). IL8 was 1134 ± 27 pg/ml and 1355 ± 205 pg/ml with PMA + apigenin or chrysin, respectively (both p <0.05 vs. PMA alone). Unstimulated GCSF was 4.7 ± 0.9 pg/ml and increased to 72 ± 6 pg/ml with PMA (p <0.01 vs. unstimulated); GCSF was 11 ± 2 and 34 ± 6 with PMA + apigenin or chrysin, respectively (p < 0.01 vs PMA only). Unstimulated GMCSF was 6 ± 2 pg/ml and increased to 38 ± 9 pg/ml with PMA (p <0.01 vs unstimulated). GMCSF with PMA + apigenin or chrysin was 12 ± 3 pg/ml and 19 ± 4 pg/ml, respectively (p <0.05 vs. PMA only). Significance determined by 1-way ANOVA (Tukey-Kramer posttest). C and D, Baseline IL-8 was 162 ± 25 pg/ml (16HBE) and 340 ± 58 pg/ml (primary sinonasal) and increased to 2326 ± 258 pg/ml (16HBE) and 2770 ± 279 pg/ml (primary sinonasal) with TNFα (both p <0.01 vs. baseline). IL-8 was 492 ± 52 pg/ml (16HBE) and 926 ± 114 pg/ml (primary sinonasal) with TNFα + wortmanin (both p <0.01 vs. TNFα alone). IL-8 with apigenin or chrysin alone was 188 ± 26 pg/ml and 236 ± 27 pg/ml, respectively, in 16HBE ALIs (n.s. vs. unstimulated; p <0.01 vs TNFα) and 236 ± 38 pg/ml and 300 ± 87 pg/ml, respectively, in primary sinonasal ALIs (n.s. vs. unstimulated; p <0.01 vs TNFα). TNFα-stimulated IL-8 secretion in 16HBE ALIs was 1122 ± 142 pg/ml, 980 ± 152 pg/ml, and 684 ± 99 pg/ml in the presence of 0.1, 1, and 10 µM apigenin, respectively, and 1680 ± 191 pg/ml, 1300 ± 77 pg/ml, and 978 ± 137 pg/ml in the presence of 0.1, 1, and 10 µM chrysin, respectively (all p <0.01 vs. TNFα alone). Apigenin and chrysin (10 µM) also reduced IL-8 secretion in primary sinonasal ALIs (1306 ± 93 pg/ml and 909 ± 153 pg/ml, respectively; both p <0.01 vs. TNFα alone). Significance determined by 1-way ANOVA, Bonferonni posttest. E, Baseline IL-8 was 319 ± 75 (16HBE), 920 ± 166 (Calu-3), and 243 ± 55 (primary sinonasal). Pyocyanin-induced IL-8 was 3120 ± 377 (16HBE), 5350 ± 332 (Calu-3), and 2360 ± 441 (primary sinonasal). Apigenin, wogonin, and tangeritin reduced pyocyanin-induced IL-8 to 606 ± 3030 (16HBE apigenin), 1500 ± 266 (Calu-3 apigenin), 394 ± 93 (primary sinonasal apigenin), 581 ± 140 (16HBE wogonin), 1020 ± 332 (Calu-3 wogonin), 410 ± 124 (primary sinonasal wogonin), 936 ± 202 (16HBE tangeritin), 108 ± 29 (Calu-3 tangeritin), 1106 ± 255 (primary sinonasal tangeritin); all values p <0.01 vs. pyocyanin alone. Chrysin did not significantly decrease pyocyanin-induced IL-8: 2370 ± 342 (16HBE), 4066 ± 761 (Calu-3), 2126 ± 685 (primary sinonasal); all values n.s. vs. pyocyanin alone. FIGURE 6. C, Resting intracellular Ca2+ concentration ([Ca2+]i) was 86 ± 5 nM (n = 11 ALIs from 3 separate patients), which was elevated to 135 ± 16 nM and 121 ± 17 nM after 5 min stimulation with 30 µM apigenin (n = 9 ALIs from at least 3 separate patients) or 120 µM chrysin (n = 8 ALIs from 3 separate patients), respectively (both values p <0.05 vs resting [Ca2+]i). D, [Ca2+]i elevation (above baseline) was 52 ± 4 nM (apigenin), 11 ± 10 (apigenin + 4’-fluoro-6-methoxyflavanone), 287 ± 16 (ATP after apigenin only), and 290 ± 7 nM (ATP after 4’-fluoro-6-methoxyflavanone). FIGURE 7. DAF-FM fluorescence increases (in relative fluorescence units; n = 5-11 ALIs from at least 3 patients for each condition) were 640 ± 64 with apigenin, 13 ± 24 with apigenin + L-NAME (p <0.01 vs. apigenin only), 520 ± 96 with apigenin + D-NAME (p <0.01 vs apigenin + L-NAME; n.s. vs. apigenin only), 49 ± 12 with apigenin + U73122 (p < 0.01 vs. apigenin only), 510 ± 199 with apigenin ± U73343 (p <0.01 vs. U73122; n.s. vs apigenin only), 737 ± 166 with chrysin (n = 6 ALIs from 3 patients), and 11 ± 18 with chrysin + L-NAME (p <0.01 vs. L-NAME only).

SUPPLEMENTAL MATERIAL Flavones Modulate Respiratory ...10.1074/jbc.M116.771949/-/DC1/jbc.M… ·...

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