Limits...
Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen

View Article: PubMed Central - PubMed

ABSTRACT

Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available ‘cromones’ are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo. These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease.

No MeSH data available.


Related in: MedlinePlus

Isopimpinellin accelerates inflammation resolution in vivo by inducing neutrophil apoptosis. (A) Inflammation resolution assay in mpx:GFP larvae treated with varying doses of isopimpinellin at 6 hpi. Isopimpinellin significantly reduces neutrophil numbers at the wound at 12 hpi in a dose-dependent manner (one-way ANOVA with Dunnett's multiple-comparison post-test; **P<0.01, ***P<0.001; n=18, performed as three independent experiments). Dotted line at y=18.5 indicates mean neutrophil number at wound in DMSO control larvae. (B) Total neutrophil number measured in mpx:GFP larvae treated with DMSO or 25 μM isopimpinellin for 24 h. Isopimpinellin did not affect total neutrophil number (unpaired t-test; P=0.8696; n=18, performed as three independent experiments). (C) Reverse-migration assay in mpx/Kaede larvae treated with DMSO or 25 μM isopimpinellin from 4 hpi. Neutrophils at the site of injury were photoconverted at 6 hpi and the numbers of photoconverted cells that moved away from the wound were quantified over 5 h. Neutrophils migrated away from the wound at a slower rate in isopimpinellin-treated larvae compared to DMSO control larvae. (D) Representative image of isopimpinellin-treated mpx/Kaede larvae at 8 hpi (scale bar: 70 μm). Solid white line in the left panel indicates the outline of the tail-fin, and the boxed area is magnified in the right-hand panel. White arrows in magnified view indicate neutrophils that appear apoptotic. (E,F) FRET assay in Tg(mpx:FRET)sh237 larvae treated with DMSO or 25 μM isopimpinellin from 4 hpi and imaged from 6 hpi. Cleavage of the caspase-3 target site results in separation of the fluorophores and loss of the FRET signal (red, F). Acceptor (neutrophil) fluorescence (green, F) persists for a further 10-20 min before cell death and loss of fluorophore integrity. Time is shown as hours:minutes. Scale bar: 50 μm. Number of observable apoptotic events was increased in isopimpinellin larvae (unpaired t-test; ***P<0.001; n=18, performed as three independent experiments). (G) TUNEL assay in mpx:GFP larvae treated with DMSO or 25 μM isopimpinellin from 6 hpi and fixed at 12 hpi. Numbers of TSA-positive neutrophils and TSA/TUNEL double-positive apoptotic neutrophils at the site of injury were measured to calculate percentage neutrophil apoptosis, which was increased in isopimpinellin-treated larvae (unpaired t-test; ***P<0.001; n=115, performed as two independent experiments). (H) Larvae were treated with DMSO, 100 μM Z-VAD-FMK (zVAD), 25 μM isopimpinellin (Iso) or in combination (Iso+zVAD) from 4 hpi and imaged from 6 hpi. Number of observable apoptotic events was increased with isopimpinellin alone but the effect was lost with the addition of Z-VAD-FMK (one-way ANOVA with Bonferroni's multiple-comparison post-test to compare selected columns; *P<0.05; ns, non-significant; n=14, performed as three independent experiments). (I) Larvae were treated with DMSO, 20 μM roscovitine or 50 μM pyocyanin from 4 hpi and imaged from 6 hpi. Number of observable apoptotic events was increased with pyocyanin (one-way ANOVA with Bonferroni's multiple-comparison post-test to compare selected columns; *P<0.05; n=18, performed as three independent experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920152&req=5

DMM024935F3: Isopimpinellin accelerates inflammation resolution in vivo by inducing neutrophil apoptosis. (A) Inflammation resolution assay in mpx:GFP larvae treated with varying doses of isopimpinellin at 6 hpi. Isopimpinellin significantly reduces neutrophil numbers at the wound at 12 hpi in a dose-dependent manner (one-way ANOVA with Dunnett's multiple-comparison post-test; **P<0.01, ***P<0.001; n=18, performed as three independent experiments). Dotted line at y=18.5 indicates mean neutrophil number at wound in DMSO control larvae. (B) Total neutrophil number measured in mpx:GFP larvae treated with DMSO or 25 μM isopimpinellin for 24 h. Isopimpinellin did not affect total neutrophil number (unpaired t-test; P=0.8696; n=18, performed as three independent experiments). (C) Reverse-migration assay in mpx/Kaede larvae treated with DMSO or 25 μM isopimpinellin from 4 hpi. Neutrophils at the site of injury were photoconverted at 6 hpi and the numbers of photoconverted cells that moved away from the wound were quantified over 5 h. Neutrophils migrated away from the wound at a slower rate in isopimpinellin-treated larvae compared to DMSO control larvae. (D) Representative image of isopimpinellin-treated mpx/Kaede larvae at 8 hpi (scale bar: 70 μm). Solid white line in the left panel indicates the outline of the tail-fin, and the boxed area is magnified in the right-hand panel. White arrows in magnified view indicate neutrophils that appear apoptotic. (E,F) FRET assay in Tg(mpx:FRET)sh237 larvae treated with DMSO or 25 μM isopimpinellin from 4 hpi and imaged from 6 hpi. Cleavage of the caspase-3 target site results in separation of the fluorophores and loss of the FRET signal (red, F). Acceptor (neutrophil) fluorescence (green, F) persists for a further 10-20 min before cell death and loss of fluorophore integrity. Time is shown as hours:minutes. Scale bar: 50 μm. Number of observable apoptotic events was increased in isopimpinellin larvae (unpaired t-test; ***P<0.001; n=18, performed as three independent experiments). (G) TUNEL assay in mpx:GFP larvae treated with DMSO or 25 μM isopimpinellin from 6 hpi and fixed at 12 hpi. Numbers of TSA-positive neutrophils and TSA/TUNEL double-positive apoptotic neutrophils at the site of injury were measured to calculate percentage neutrophil apoptosis, which was increased in isopimpinellin-treated larvae (unpaired t-test; ***P<0.001; n=115, performed as two independent experiments). (H) Larvae were treated with DMSO, 100 μM Z-VAD-FMK (zVAD), 25 μM isopimpinellin (Iso) or in combination (Iso+zVAD) from 4 hpi and imaged from 6 hpi. Number of observable apoptotic events was increased with isopimpinellin alone but the effect was lost with the addition of Z-VAD-FMK (one-way ANOVA with Bonferroni's multiple-comparison post-test to compare selected columns; *P<0.05; ns, non-significant; n=14, performed as three independent experiments). (I) Larvae were treated with DMSO, 20 μM roscovitine or 50 μM pyocyanin from 4 hpi and imaged from 6 hpi. Number of observable apoptotic events was increased with pyocyanin (one-way ANOVA with Bonferroni's multiple-comparison post-test to compare selected columns; *P<0.05; n=18, performed as three independent experiments).

Mentions: We originally identified isopimpinellin as a new pro-resolution compound in our screen for accelerators of inflammation resolution (Robertson et al., 2014). On further investigation, we found that when zebrafish larvae were exposed to isopimpinellin once inflammation was already established at 6 hpi, there was a concentration-dependent reduction in neutrophil numbers at the wound at 12 hpi (Fig. 3A). Isopimpinellin did not affect total neutrophil number in whole larvae (Fig. 3B). In our previous study, we showed that we could pharmacologically drive inflammation resolution by promoting neutrophil reverse migration (Robertson et al., 2014). To investigate whether isopimpinellin could also act via this mechanism, we photoconverted neutrophils specifically at the wound region at 6 hpi in Tg(mpx:Gal4);Tg(UAS:Kaede)i222 larvae, as described (Elks et al., 2011; Holmes et al., 2012). However, we found that fewer photoconverted neutrophils migrated away from the wound over time in isopimpinellin-treated larvae compared to the vehicle controls (Fig. 3C).Fig. 3.


Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen
Isopimpinellin accelerates inflammation resolution in vivo by inducing neutrophil apoptosis. (A) Inflammation resolution assay in mpx:GFP larvae treated with varying doses of isopimpinellin at 6 hpi. Isopimpinellin significantly reduces neutrophil numbers at the wound at 12 hpi in a dose-dependent manner (one-way ANOVA with Dunnett's multiple-comparison post-test; **P<0.01, ***P<0.001; n=18, performed as three independent experiments). Dotted line at y=18.5 indicates mean neutrophil number at wound in DMSO control larvae. (B) Total neutrophil number measured in mpx:GFP larvae treated with DMSO or 25 μM isopimpinellin for 24 h. Isopimpinellin did not affect total neutrophil number (unpaired t-test; P=0.8696; n=18, performed as three independent experiments). (C) Reverse-migration assay in mpx/Kaede larvae treated with DMSO or 25 μM isopimpinellin from 4 hpi. Neutrophils at the site of injury were photoconverted at 6 hpi and the numbers of photoconverted cells that moved away from the wound were quantified over 5 h. Neutrophils migrated away from the wound at a slower rate in isopimpinellin-treated larvae compared to DMSO control larvae. (D) Representative image of isopimpinellin-treated mpx/Kaede larvae at 8 hpi (scale bar: 70 μm). Solid white line in the left panel indicates the outline of the tail-fin, and the boxed area is magnified in the right-hand panel. White arrows in magnified view indicate neutrophils that appear apoptotic. (E,F) FRET assay in Tg(mpx:FRET)sh237 larvae treated with DMSO or 25 μM isopimpinellin from 4 hpi and imaged from 6 hpi. Cleavage of the caspase-3 target site results in separation of the fluorophores and loss of the FRET signal (red, F). Acceptor (neutrophil) fluorescence (green, F) persists for a further 10-20 min before cell death and loss of fluorophore integrity. Time is shown as hours:minutes. Scale bar: 50 μm. Number of observable apoptotic events was increased in isopimpinellin larvae (unpaired t-test; ***P<0.001; n=18, performed as three independent experiments). (G) TUNEL assay in mpx:GFP larvae treated with DMSO or 25 μM isopimpinellin from 6 hpi and fixed at 12 hpi. Numbers of TSA-positive neutrophils and TSA/TUNEL double-positive apoptotic neutrophils at the site of injury were measured to calculate percentage neutrophil apoptosis, which was increased in isopimpinellin-treated larvae (unpaired t-test; ***P<0.001; n=115, performed as two independent experiments). (H) Larvae were treated with DMSO, 100 μM Z-VAD-FMK (zVAD), 25 μM isopimpinellin (Iso) or in combination (Iso+zVAD) from 4 hpi and imaged from 6 hpi. Number of observable apoptotic events was increased with isopimpinellin alone but the effect was lost with the addition of Z-VAD-FMK (one-way ANOVA with Bonferroni's multiple-comparison post-test to compare selected columns; *P<0.05; ns, non-significant; n=14, performed as three independent experiments). (I) Larvae were treated with DMSO, 20 μM roscovitine or 50 μM pyocyanin from 4 hpi and imaged from 6 hpi. Number of observable apoptotic events was increased with pyocyanin (one-way ANOVA with Bonferroni's multiple-comparison post-test to compare selected columns; *P<0.05; n=18, performed as three independent experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920152&req=5

DMM024935F3: Isopimpinellin accelerates inflammation resolution in vivo by inducing neutrophil apoptosis. (A) Inflammation resolution assay in mpx:GFP larvae treated with varying doses of isopimpinellin at 6 hpi. Isopimpinellin significantly reduces neutrophil numbers at the wound at 12 hpi in a dose-dependent manner (one-way ANOVA with Dunnett's multiple-comparison post-test; **P<0.01, ***P<0.001; n=18, performed as three independent experiments). Dotted line at y=18.5 indicates mean neutrophil number at wound in DMSO control larvae. (B) Total neutrophil number measured in mpx:GFP larvae treated with DMSO or 25 μM isopimpinellin for 24 h. Isopimpinellin did not affect total neutrophil number (unpaired t-test; P=0.8696; n=18, performed as three independent experiments). (C) Reverse-migration assay in mpx/Kaede larvae treated with DMSO or 25 μM isopimpinellin from 4 hpi. Neutrophils at the site of injury were photoconverted at 6 hpi and the numbers of photoconverted cells that moved away from the wound were quantified over 5 h. Neutrophils migrated away from the wound at a slower rate in isopimpinellin-treated larvae compared to DMSO control larvae. (D) Representative image of isopimpinellin-treated mpx/Kaede larvae at 8 hpi (scale bar: 70 μm). Solid white line in the left panel indicates the outline of the tail-fin, and the boxed area is magnified in the right-hand panel. White arrows in magnified view indicate neutrophils that appear apoptotic. (E,F) FRET assay in Tg(mpx:FRET)sh237 larvae treated with DMSO or 25 μM isopimpinellin from 4 hpi and imaged from 6 hpi. Cleavage of the caspase-3 target site results in separation of the fluorophores and loss of the FRET signal (red, F). Acceptor (neutrophil) fluorescence (green, F) persists for a further 10-20 min before cell death and loss of fluorophore integrity. Time is shown as hours:minutes. Scale bar: 50 μm. Number of observable apoptotic events was increased in isopimpinellin larvae (unpaired t-test; ***P<0.001; n=18, performed as three independent experiments). (G) TUNEL assay in mpx:GFP larvae treated with DMSO or 25 μM isopimpinellin from 6 hpi and fixed at 12 hpi. Numbers of TSA-positive neutrophils and TSA/TUNEL double-positive apoptotic neutrophils at the site of injury were measured to calculate percentage neutrophil apoptosis, which was increased in isopimpinellin-treated larvae (unpaired t-test; ***P<0.001; n=115, performed as two independent experiments). (H) Larvae were treated with DMSO, 100 μM Z-VAD-FMK (zVAD), 25 μM isopimpinellin (Iso) or in combination (Iso+zVAD) from 4 hpi and imaged from 6 hpi. Number of observable apoptotic events was increased with isopimpinellin alone but the effect was lost with the addition of Z-VAD-FMK (one-way ANOVA with Bonferroni's multiple-comparison post-test to compare selected columns; *P<0.05; ns, non-significant; n=14, performed as three independent experiments). (I) Larvae were treated with DMSO, 20 μM roscovitine or 50 μM pyocyanin from 4 hpi and imaged from 6 hpi. Number of observable apoptotic events was increased with pyocyanin (one-way ANOVA with Bonferroni's multiple-comparison post-test to compare selected columns; *P<0.05; n=18, performed as three independent experiments).
Mentions: We originally identified isopimpinellin as a new pro-resolution compound in our screen for accelerators of inflammation resolution (Robertson et al., 2014). On further investigation, we found that when zebrafish larvae were exposed to isopimpinellin once inflammation was already established at 6 hpi, there was a concentration-dependent reduction in neutrophil numbers at the wound at 12 hpi (Fig. 3A). Isopimpinellin did not affect total neutrophil number in whole larvae (Fig. 3B). In our previous study, we showed that we could pharmacologically drive inflammation resolution by promoting neutrophil reverse migration (Robertson et al., 2014). To investigate whether isopimpinellin could also act via this mechanism, we photoconverted neutrophils specifically at the wound region at 6 hpi in Tg(mpx:Gal4);Tg(UAS:Kaede)i222 larvae, as described (Elks et al., 2011; Holmes et al., 2012). However, we found that fewer photoconverted neutrophils migrated away from the wound over time in isopimpinellin-treated larvae compared to the vehicle controls (Fig. 3C).Fig. 3.

View Article: PubMed Central - PubMed

ABSTRACT

Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available &lsquo;cromones&rsquo; are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo. These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease.

No MeSH data available.


Related in: MedlinePlus