A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide

<div><p>Tissue injury and infection trigger innate immune responses. However, dysregulation may result in chronic inflammation and is commonly treated with corticosteroids and non-steroidal anti-inflammatory drugs. Unfortunately, long-term administration of both therapeutic classes can cause unwanted side effects. To identify alternative immune-modulatory compounds we have previously established a novel screening method using zebrafish larvae. Using this method we here present results of an <i>in vivo</i> high-content drug-repurposing screen, identifying 63 potent anti-inflammatory drugs that are in clinical use for other indications. Our approach reveals a novel pro-inflammatory role of nitric oxide. Nitric oxide affects leukocyte recruitment upon peripheral sensory nervous system or epithelial injury in zebrafish larvae both via soluble guanylate cyclase and in a soluble guanylate cyclase -independent manner through protein S-nitrosylation. Together, we show that our screening method can help to identify novel immune-modulatory activities and provide new mechanistic insights into the regulation of inflammatory processes.</p></div

Experimental setup and compound categorization of the automated ChIn screen.

<p><b>(a)</b> Schematic diagram illustrating the screening strategy for compounds modulating inflammation. Individual larvae are distributed in 384-well plates. Drugs are added 1 h prior to treatment with CuSO₄ for 1h at 28°C in the dark followed by an exchange of medium and a 6h time-lapse analysis. Finally, screening data are automatically processed and analyzed. <b>(b)</b> Pie chart displaying the identified compound categories upon screening 1120 compounds of an FDA-approved library and the ICCB library of known bioactives. Numbers indicate percentages.</p

Inflammation induces S-nitrosylation in zebrafish larvae.

<p>Changes in total protein S-nitrosylation during inflammation was assessed with the modified biotin switch technique and detected by Western Blot. S-nitrosylated proteins in samples of control (lane 1) larvae and larvae with chemically induced inflammation (lane 2 and 3) were detected by anti-TMT immunoblotting. Chemically induced inflammation significantly induces S-nitrosylation, marked by additional strong anti-TMT immunoblotting signals (arrowheads), in protein extracts of CuSO₄ treated larvae. Inhibition of Nos with 250 μM L-NAME prior to CuSO₄ treatment prevents changes in protein S-nitrosylation. EF–1α was used as loading control.</p

Functional classification and validation of selected anti-inflammatory hits.

<p>Anti-inflammatory candidates were classified in functional or therapeutic groups. Selected anti-inflammatory hit compounds exert dose-dependent anti-inflammatory effect on chemically induced inflammation. <b>(a)</b> Pie chart dividing the 70 anti-inflammatory hit compounds in functional or therapeutic groups containing at least 2 representatives (numbers). Groups with only one representative are summarized in “other”. (<b>b—e)</b> Dose-response behavior of selected anti-inflammatory hit compounds Tenatoprazole <b>(b)</b>, NECA <b>(c)</b>, Candesartan <b>(d)</b>, and 3-Bromo-7-nitroindazole <b>(e)</b>. Bar charts indicate means ± SEM of the initial inflammatory index (%) 90 minutes after CuSO₄ treatment (t = 0). Three individual experiments were performed with 15 replicate larvae for each condition, respectively. Statistics were evaluated with unpaired one-sided t-tests. ns–not significant: P > 0.05, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001.</p

Genetic knockdown of zebrafish <i>nos2b</i> and sGC (<i>gucy</i>) but not <i>nos1</i> and <i>nos2a</i> impair the inflammatory response.

<p>Assessment of the role of the 3 zebrafish Nos isoforms and sGC on peripheral sensory nervous system <b>(a)</b> and epithelial <b>(b)</b> inflammation upon genetic knockdown. <b>(a)</b><i>nos2b</i> and <i>gucy</i> knockdown significantly reduce initial inflammation at injured neuromasts as compared to wildtype (WT) larvae. <b>(b)</b> Knockdown of <i>nos2b</i> and <i>gucy</i> significantly reduce the number of neutrophils recruited to ventral fin wounds. Bar charts represent means of the initial inflammatory index (%) ± SEM 90 minutes after CuSO₄ treatment <b>(a)</b> and 1 h after mechanical injury of the ventral fin <b>(b)</b>. Three individual experiments were performed with 15 replicate morphant larvae for each condition, respectively. Statistics were evaluated with unpaired one-sided t-tests. ns–not significant: P > 0.05, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001.</p

Overview of compound concentration-dependent pronephric phenotypes.

<p>Illustrative examples of pronephroi of a (A) non-treated embryo, and after treatment with (B) 20 mM penicillin, (C) 40 mM ampicillin, (D) 40 mM gentamicin, (E) 40 mM kanamycin, (F) 40 mM acetaminophen, (G) 40 mM captopril, (H) 10 mM losartan. For examples of phenotypes after Indomethacin treatment see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082137#pone-0082137-g004" target="_blank">Figure <b>4A-E</b></a>. Arrow and arrowheads in (A) indicate the different morphological parameters of the pronephros scored to evaluate compound effect on the developing kidney. Arrow: fused glomeruli; Arrowhead: angle between the neck segment and the proximal convoluted tubule. (I-M) Heatmaps showing (I) lethality rates, (J) edema rates and (K-M) changes in morphological parameters of the pronephros. In detail, (K) incomplete glomerular fusion, (L) glomerular malformation and (M) tubular angle. For further details see Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082137#pone.0082137.s003" target="_blank">Tables <b>S1</b>-<b>S3</b></a>. Colour codes indicate the percentage of embryos (I-L) with particular phenotype, or the angle between neck segment and proximal convoluted tubule (M) as indicated by the colour coded legend. Grey squares indicate missing data points. Concentration ranges used are indicated above the heatmaps, or below for Indomethacin, respectively. Abbreviations: penicillin (Pen), ampicillin (Amp), gentamicin (Gen), kanamycin (Kan), acetaminophen (Ace), captopril (Cap), losartan (Los) and indomethacin (Ind). *p<0.05, **p<0.001.</p

Standardized orientation of zebrafish embryos.

<p>(A,B) Photographs of the brass tool for the simultaneous generation of agarose grooves within 96 well microtiter plates: (A) top view and (B) tilted view. For dimensions of the plate see Materials and Methods section. (C) Schematic depiction of a single well with a ventrally oriented embryo within an agarose cavity. Drawing is not to scale. (D) Illustrative example of aligned and oriented embryos. Shown are dorsal views of 48 hpf embryos acquired using a 2.5x objective on an inverted wide field screening microscope.</p

Overview of workflow for the automated imaging of the developing zebrafish pronephros.

<p>(A) Overview of the workflow for screening larval kidneys. The flowchart illustrates the different steps carried out to obtain overview images of kidneys. (B) Initial compound treatment or microinjection of embryos prior to sample preparation and imaging. (C) Schematic illustrating the transfer of embryos into agarose coated microtiter plates, and alignment and orientation of embryos. (D-G) Acquisition and processing of image Data. D to F show data of the same embryo. (D) Automated acquisition of z-stacks (33 z-slices, dZ=15µm) on an inverted widefield screening microscope. (E) Deblurring of images using deconvolution. Shown are maximum projections of z-stacks of raw data (left panel) and deconvolved data (right panel). (F) Automated detection and cropping of the kidney region. The red square indicates the position and dimensions of the cropped region. (G) Automated generation of overview images for quick assessment of overall morphological changes. Indomethacin skeletal formula in (A) taken from (<a href="http://en.wikipedia.org/wiki/file:indometacin_skeletal.svg" target="_blank"><u>http://en.wikipedia.org/wiki/File:Indometacin_skeletal.svg</u></a>).</p