A high-throughput chemically induced inflammation assay in zebrafish

Abstract

Background: Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening.

Results: Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction.

Conclusions: This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

Figure 1

Leukocytes migrate specifically to damaged lateral line neuromasts in zebrafish larvae. (a-j) 56-hours postfertilization (56-hpf) BACmpx::GFP or lysC::DsRED2 transgenic zebrafish larvae exhibit green or red fluorescent leukocytes, respectively. (a and b) Untreated fish show the normal distribution of labeled cells, mostly localized in the ventral trunk and tail. (c and d) In copper-treated siblings, leukocytes become localized preferentially to a few clusters along the horizontal midline of the trunk and tail. (e-j) A detailed view of this region in copper-treated animals shows that while many cells disperse throughout the body, other cells congregate in discrete clusters (arrows); no overt tissue damage to the larvae is observed in bright-field images. (k-r) A mating cross of cldnB::GFP and lysC::DsRED2 transgenic fish labels neuromasts in green and leukocytes in red. Posterior trunk neuromasts were imaged immediately after adding copper (k-n) or 20 minutes after copper treatment (o-r) using bright-field red or green fluorescence illumination. Few, if any, leukocytes are seen near neuromasts at the beginning of treatment. (m and n) Here a case where a single leukocyte is present is shown. (q and r) In contrast, copper-treated fish have numerous red fluorescent leukocytes interspersed within the neuromast cells. Note the extent of damage induced by copper in the neuromast cells (compare Figures 1l and 1p).

Figure 2

Quantification of infiltrating leukocytes in the lateral line after diverse treatments: the chemically induced inflammation (ChIn) assay. (a) Schematic view of a 3 days postfertilization (dpf) larva. The boxed area corresponds to the horizontal myoseptum (line). An area of approximately 10 cell diameters is delimited around the myoseptum (dotted red lines) and corresponds to the area where leukocytes were counted in all manual quantification experiments. (b) Significant induction of leukocyte recruitment to the lateral line by copper treatment. The graph shows average leukocyte numbers in the lateral line in negative controls (untreated fish or cadmium chloride-treated fish) and in copper-treated fish. (c) The effect of copper on leukocyte recruitment to the lateral line is concentration dependent. (d) Effectiveness of other metals in the ChIn assay. (e) Neomycin, at concentrations that eliminate hair cells, also induces leukocyte recruitment, but less effectively than copper. (f) Larvae of different ages, from 56 to 128 hpf, were exposed to 10 μM CuSO₄ and were monitored for leukocytes present at the myoseptum every 20 minutes thereafter until 120 minutes. Fish at all stages analyzed showed similar behaviors and exhibited increased presence of leukocytes at the lateral line by 20 minutes after initiating exposure to copper. For all experiments, at least 15 larvae were used for each condition. ***P < 0.001.

Figure 3

Proof of principle of the ChIn assay using known anti-inflammatory drugs. The ChIn assays were carried out by manually counting leukocytes recruited to the lateral line after copper treatment (10 μM) as described in Results. In this experiment, dimethyl sulfoxide (DMSO) was included in all samples, and drugs were added 1 hour prior to copper treatment at the indicated concentrations. As before, experiments were carried out with 15 larvae per condition. ***P < 0.001. **0.001 <P < 0.01. *0.01 <P < 0.05.

Figure 4

Detection of H₂O₂ inhibition and genetic mutations using ChIn assays. (a) Reactive oxygen species signaling is critical for copper-induced inflammation. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, impairs wounds to leukocyte signaling, resulting in reduced leukocyte numbers at damaged neuromasts. Larvae at age 56 hpf were treated with 100 μM DPI for 1 hour prior to copper treatment (10 μM CuSO₄ for 40 minutes). DPI treatment results in significantly lower leukocyte numbers within the myoseptum compared to untreated control larvae. (b) The ChIn assay enables detection of genetic mutations affecting an inflammatory response. Clutches from matings between homozygous Wiskott-Aldrich syndrome (was) gene males and heterozygous was females were treated with 10 μM CuSO₄, fixed and scored by Sudan Black staining, and individual larvae were subsequently genotyped. Both homozygous and heterozygous was mutants recruited significantly lower numbers of leukocytes to the myoseptum than wild-type larvae upon copper-induced neuromast damage. In addition, the inflammatory response of homozygous mutants is significantly lower than that of heterozygous mutants. **P = 0.0045. ***P < 0.001.

Figure 5

Automated ChIn assay. (a-d) Image acquisition method using compound transgenic larvae cldnB::GFP and lysC::DsRED2. Images show control (DMSO) (a and b) and treated (CuSO₄) (c and d) fish revealing neuromasts (green, arrows) and leukocytes (red). Shown are the raw images (a and c) and the number and identity of the neuromasts that were automatically detected by the software (b and d) (white squares). The image analysis software determines the average red fluorescence intensity per square (neuromast area) and calculates the data averaged for all squares detected within one larva. Note that the program is able to detect most, but not all, of the visible neuromasts. The variable neuromast detection success is compensated by using more larvae than in the manual method: 24 per plate, in triplicate, averaging around 50 data-producing larvae per condition. (e) A control experiment using the automated ChIn assay. Untreated or metal-exposed double-transgenic fish were imaged, and red fluorescence was averaged from three experiments. Results are comparable to manual ChIn assays. (f and g) Comparison of ChIn assay results between the manual quantification method (f) and automated detection (g) of anti-inflammatory drug activity.