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. 2023 Oct 1;16(10):dmm050056.
doi: 10.1242/dmm.050056. Epub 2023 Oct 13.

Identification of histone deacetylase inhibitors as neutrophil recruitment modulators in zebrafish using a chemical library screen

Sijia Fan  1 , Jinlong Jiang  1 , Huan Zhang  1 , Cuihong Wang  1 , Shang Kong  2 , Tingting Zhao  2 , Ling Meng  1 , Yang Liu  1 , Jingjing Qin  1 , Xiuqin Rong  1 , Zhenting He  1 , Qinke He  1 , Ke He  1 , Ketong Chen  1 , Ling Lei  1 , Xinyu Hai  1 , Hong Nie  2 , Chunguang Ren  1
Affiliations

Identification of histone deacetylase inhibitors as neutrophil recruitment modulators in zebrafish using a chemical library screen

Sijia Fan et al. Dis Model Mech. .

Abstract

Tissue injury-induced neutrophil recruitment is a prerequisite for the initiation and amplification of inflammatory responses. Although multiple proteases and enzymes involved in post-translational modification (PTM) of proteins regulate leukocyte recruitment, an unbiased functional screen of enzymes regulating inflammatory leukocyte recruitment has yet to be undertaken. Here, using a zebrafish tail fin amputation (TFA) model to screen a chemical library consisting of 295 compounds that target proteases and PTM enzymes, we identified multiple histone deacetylase (HDAC) inhibitors that modulate inflammatory neutrophil recruitment. AR-42, a pan-HDAC inhibitor, was shown to inhibit neutrophil recruitment in three different zebrafish sterile tissue injury models: a TFA model, a copper-induced neuromast damage and mechanical otic vesicle injury (MOVI) model, and a sterile murine peritonitis model. RNA sequencing analysis of AR-42-treated fish embryos revealed downregulation of neutrophil-associated cytokines/chemokines, and exogenous supplementation with recombinant human IL-1β and CXCL8 partially restored the defective neutrophil recruitment in AR-42-treated MOVI model fish embryos. We thus demonstrate that AR-42 non-cell-autonomously modulates neutrophil recruitment by suppressing transcriptional expression of cytokines/chemokines, thereby identifying AR-42 as a promising anti-inflammatory drug for treating sterile tissue injury-associated diseases.

Keywords: AR-42; Cell migration; HDAC; Neutrophil; Zebrafish.

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Conflict of interest statement

Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Fig. 1.
Fig. 1.
HDACIs suppress tissue injury-induced neutrophil recruitment. (A) Schematic diagram of the 3-day-post-fertilization (dpf) Tg(mpx:GFP)i114 zebrafish embryonic tail fin amputation (TFA) model with imaging at 6 h post amputation. The diagram was created with BioRender.com. (B) Classification of 41 of 295 inhibitors targeting proteases and post-translational modification (PTM) enzymes that significantly inhibited neutrophil recruitment in zebrafish TFA model. (C) Representative fluorescence images of 3-dpf Tg(mpx:GFP)i114 embryos treated with ten HDACs inhibitors from the library that suppressed zebrafish neutrophil recruitment in the TFA model. The red dashed rectangles denote the areas used for neutrophil counting. Scale bar: 100 µm. (D) Quantitative analysis of neutrophil recruitment in C (n=30) (see Materials and Methods for details). Each data point represents an individual embryo. Each HDACI group was individually compared with the DMSO group. Error bars represent mean±s.d. ****P<0.0001 (one-way ANOVA with Dunnett's test). The experiments in C,D were repeated four times.
Fig. 2.
Fig. 2.
Pan-HDACI AR-42 inhibits tissue injury-induced neutrophil migration in the zebrafish TFA model in a dose-dependent manner. (A,B) Representative fluorescence images (A) and quantitative analysis (B) of neutrophil recruitment to the wound area of 3-dpf Tg(mpx:GFP)i114 embryos treated with DMSO or AR-42 in the TFA model (n=30). Scale bar: 100 µm. The red dashed rectangles denote the areas used for neutrophil counting. Each data point represents an individual embryo (one-way ANOVA with Dunnett's test). The AR-42-treated group was individually compared with the DMSO group. (C) Representative migration trajectories of neutrophil chemotaxis toward the wound in DMSO- or 10 µM AR-42-treated 3-dpf Tg(mpx:GFP)i114 embryos in the TFA model. Scale bar: 100 µm. ‘N’ denotes the number of GFP+ neutrophils that were tracked in one embryo. (D,E) Quantitative analysis of neutrophil migratory velocity (D) and deflexion angle (E) in C. Each data point represents an individual GFP+ neutrophil (two-tailed unpaired Student's t-test). Error bars represent mean±s.d. **P<0.01; ****P<0.0001. The experiments in A,B and C-E were repeated four and three times, respectively.
Fig. 3.
Fig. 3.
AR-42 suppresses the zebrafish neutrophil recruitment in CIND and MOVI models. (A) Schematic diagram of the copper-induced neuromast damage (CIND) model. (B) Representative fluorescence images of DMSO- or AR-42-treated 3-dpf Tg(mpx:GFP)i114 embryos in the CIND model. Scale bar: 200 µm. The red dashed ovals denote the neuromast regions used for neutrophil counting. (C) Quantification of neutrophils recruited to zebrafish neuromasts in B (n=30). (D) Schematic diagram of the mechanical otic vesicle injury (MOVI) model. (E) Representative fluorescence images of DMSO- or AR-42-treated 3-dpf Tg(mpx:GFP)i114 embryos in the MOVI model. Scale bar: 100 µm. The white dashed lines denote the contours of the embryos. The red dashed circles denote the otic vesicle contours. (F) Quantification of neutrophils recruited to the zebrafish otic vesicle in E (n=30). Each data point represents an individual embryo (one-way ANOVA with Dunnett's test) in C,F. Error bars represent mean±s.d. ****P<0.0001. The experiments in B,C,E,F were repeated four times.
Fig. 4.
Fig. 4.
AR-42 inhibits neutrophil inflammatory recruitment in the mouse peritonitis model. (A) Representative dot plots of neutrophil percentages that were analyzed by flow cytometry. (B) Quantitative analysis of the percentage of neutrophils in CD45+ cells in peritoneal lavage fluid. (C) Quantitative analysis of total cell number in peritoneal lavage fluid. (D) Quantitative analysis of the number of neutrophils in peritoneal lavage fluid. Each data point represents an individual mouse (two-tailed unpaired Student's t-test). Error bars represent mean±s.d. ***P<0.001. The experiments in A-D were repeated three times.
Fig. 5.
Fig. 5.
AR-42 treatment downregulates neutrophil migration-associated cytokine/chemokine expression. (A) Volcano plot of RNA-seq data with differentially expressed genes between the AR-42_TFA and DMSO_TFA groups (see Materials and Methods for details). The red dashed oval highlights il11a, which is among the top ten downregulated genes. (B) Differentially expressed related pathways between the AR-42_TFA and DMSO_TFA groups were analyzed by GO pathway enrichment. (C) Differentially expressed related pathways between the AR-42_TFA and DMSO_TFA groups were analyzed by KEGG pathway enrichment. (D,E) Fold change of neutrophil migration associated-cytokines/chemokines in the DMSO_TFA group compared to the DMSO group (D) and in the AR-42_TFA group compared to the DMSO_TFA group (E). The fold change was normalized to tuba1a expression. Each data point represents a biological replicate (one-way ANOVA with Dunnett's test). (F) FPKM values of neutrophil migration associated-cytokines/chemokines in the DMSO_TFA and AR-42_TFA groups. Each data point represents a biological replicate (two-way ANOVA with Šídák's test). Data show the mean±s.d. ns, not statistically significant; *P<0.05; ***P<0.001; ****P<0.0001. The experiments in A-F were repeated four times.
Fig. 6.
Fig. 6.
AR-42 suppresses the expression of zebrafish il1b, cxcl8a, cxcl8b.1 and cxcl18b. (A-D) qRT-PCR analysis of the expression levels of il1b (A), cxcl8a (B), cxcl8b.1 (C) and cxcl18b (D) in 3-dpf wild-type zebrafish embryos treated with DMSO or 10 µM AR-42 for 1 h with or without TFA modeling. Eighty larvae were used for each treatment. The relative expression was quantified by the 2-ΔΔct method. tuba1a was used as an internal control. Each data point represents a biological replicate (one-way ANOVA with Dunnett's test). Error bars represent mean±s.d. ****P<0.0001. The DMSO and AR-42_TFA groups were compared to the DMSO_TFA condition. The experiments in A-D were repeated three times.
Fig. 7.
Fig. 7.
Exogenous supplementation of recombinant human IL-1β and CXCL8 partially rescues the defective neutrophil recruitment upon AR-42 treatment. (A-C) Representative fluorescence images (A) and quantitative analysis (B,C) of neutrophil recruitment to the otic vesicle in DMSO- or AR-42-treated 3-dpf Tg(mpx:GFP)i114 embryos with or without recombinant human IL-1β/CXCL8 proteins injection in a MOVI model (n=30). Scale bar: 100 µm. The red dashed circles denote the otic vesicle contours. Each data point represents an individual embryo (one-way ANOVA with Tukey's test). Error bars represent mean±s.d. ****P<0.0001. The experiments in A-C were repeated three times.
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