Myeloid-derived growth factor regulates neutrophil motility in interstitial tissue damage

Abstract

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.

Figure S1.

mydgf expression in sterile injuries and infection. (A) Expression of mydgf, measured by RNA sequencing (fragments per kilobase of transcript per million mapped reads [fpkm]), in three cell types is displayed 3 h following multiple wounding along the tail fin tissue. Each dot represents one independent replicate. Sample preparation and RNA-sequencing dataset was published previously (Houseright et al., 2020). (B and C) RT-qPCR measurement of mydgf expression in WT zebrafish tails 3 h following tail transection (Tt) or tail burn wound (burn; B) and P. aeruginosa (Pa; C) otic infection at 2 h after infection. Data comprise three (burn and infection) to five (Tt) independent experiments performed in technical triplicates and are normalized to mydgf expression in unwounded (Unwnd) tails and to ef1α. n = 50 tails per condition per independent replicate. *, P < 0.05. Fold changes in gene expression were compared with the normalized value of 1 using one-sample t tests. Data are displayed as mean with 95% CI.

Figure 1.

MYDGF regulates neutrophil response to tissue damage, but not infection. (A) Ribbon and surface representation of the human MYDGF NMR solution structure (PDB accession no. 6O6W). Residues identical in zebrafish MYDGF are shown in red. (B) Schematic of zebrafish mydgf gene, with exon 5 gRNA sequences highlighted for CRISPR-Cas9 mutagenesis. gRNA sequence is shown in gray and protospacer adjacent motif is shown in magenta; scale bar = 100 bp. (C) Exon 5 DNA sequence of WT (top) and mydgf^−/− (bottom) zebrafish mutants showing a 12-bp deletion. (D) Representative Western blot for zebrafish MYDGF and β-tubulin from pooled 3 dpf zebrafish larvae. Data are representative of three independent replicates. Size marker (mol wt [MW]) in left lane. (E) InstantBlue stain of 12% SDS-PAGE of zebrafish MYDGF in CCM by HEK293 cells at 72 h after transfection by empty or zebrafish MYDGF-expressing pCS2 constructs. (F and G) Representative images (F) and quantification (G) of mCherry-labeled neutrophils at the otic vesicle of WT or mydgf^−/− larvae, following microinjection with CCM ± zebrafish MYDGF protein at 2 h after injection; three independent replicates with n = 29 +/+ control, 32 +/+ zebrafish MYDGF, 54 −/− control and 52 −/− zebrafish MYDGF; scale bar = 50 µm. (H and I) Otic vesicle of 3 dpf WT or mydgf^−/− zebrafish larvae were microinjected with P. aeruginosa (Pa; 5000 CFU), followed by fixation at 2 h after injection. Neutrophils are visualized by Sudan Black staining. In H and I, representative brightfield images (H) and quantification (I) of the number of neutrophils at the otic vesicle are shown; three independent replicates with n = 43 +/+ and 57 −/−; scale bar = 100 µm. In G and I, data are expressed as mean with 95% CI; each symbol represents one larva, and different colors represent independent replicates. ****, P < 0.0001. P values were calculated by ANOVA with Tukey’s multiple comparisons. Ctrl, control; hpi, h postinjection; zMYDGF, zebrafish MYDGF.

Figure 2.

MYDGF depletion leads to a neutrophil-dependent defect in wound healing. (A) Caudal fins of 3 dpf WT or mydgf^−/− zebrafish larvae with mCherry-labeled neutrophils were wounded by thermal injury and fixed at 3 and 24 hpb. Neutrophils were counted in the burn area. (A and B) Representative images (A) and quantification (B) of neutrophils in the burn are shown; three independent replicates with n = 24 +/+ and 24 −/− at 3 hpb and 21 +/+ and 16 −/− at 24 hpb; scale bar = 100 µm. (C–F) Caudal fins of 2 dpf (for morpholinos) or 3 dpf (for all other experiments) larvae were wounded by thermal injury and fixed at 24, 48, and 72 hpb. Wound healing was determined by quantifying the area of tail fin regrowth, measured from the caudal arteriovenous loop to the wound edge. (C and D) Representative brightfield images (C) and quantification (D) of tail fin regrowth area in WT and mydgf^−/− larvae are shown; two independent replicates with n = 45 +/+ and 51 −/− at 24 hpb, 46 +/+ and 48 −/− at 48 hpb, and 41 +/+ and 47 −/− at 72 hpb; scale bar = 100 µm. (E and F) Representative brightfield images (E) and quantification (F) of tail fin regrowth area in WT (WT Rac2) or neutrophil motility–impaired (Rac2D57N) larvae, with or without mydgf-targeting morpholino #1, at 24 hpb; three independent replicates with n = 50 WT/control mo, 32 WT/mydgf mo, 48 D57N/control mo, and 32 D57N/mydgf mo; scale bar = 100 µm. In B, D, and F, data are expressed as mean with 95% CI; each symbol represents one larva, and different colors represent independent replicates. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. P values were calculated by ANOVA with Tukey’s multiple comparisons.

Figure S2.

Morpholino-mediated depletion of MYDGF phenocopies neutrophil accumulation at sterile injury observed in mydgf homozygous mutant. (A) Quantification of the tail length of unwounded WT and mydgf^−/− larvae at 6 dpf; two independent replicates with n = 13 +/+ and 10 −/−. (B) Schematic of zebrafish mydgf gene, with sequences targeted by splice-blocking morpholinos #1 and #2 highlighted at the junction of exon 3 and intron 3 and the junction of exon 5 and intron 5, respectively; scale bar = 100 bp. (C) Representative Western blot for zebrafish MYDGF and β-actin from pooled, 2 dpf larvae treated with either mismatch control mo or mo targeting mydgf. (D) Quantification of Western blot in C; representative of three independent replicates. (E–G) Representative brightfield images of Sudan Black staining (E) and quantification of the number of neutrophils in the wound microenvironment of larvae treated with either mismatch control mo and mydgf-targeting mo #1 (F) or mo #2 (G) at 1 and 6 hpw following tail transection of the caudal fin; three independent replicates with n = 52 control and 52 mo #1 at 1 hpw and 53 control and 34 mo #1 at 6 hpw (F), and three independent replicates with n = 47 control and 51 mo #2 at 1 hpw and 44 control and 58 mo #2 at 6 hpw (G); scale bar = 100 µm. (H) Quantification of the total number of neutrophils in 3 dpf whole larvae treated with mismatch control or mo #1 targeting mgdyf; three independent replicates with n = 59 control and 57 mo #1. For F–H, data are expressed as mean with 95% CI; each symbol represents one larva, and different colors represent the results of three independent replicates. ***, P < 0.001; ****, P < 0.0001. P values were calculated by ANOVA with Tukey’s multiple comparisons.

Figure 3.

MYDGF depletion alters neutrophil motility in the wound microenvironment. (A) Caudal fins of 3 dpf WT or mydgf^−/− zebrafish larvae with mCherry-labeled neutrophils were wounded by tail transection, and fixed at 1 and 6 hpw. Neutrophils were counted at the wound, distal to the tip of the notochord. (A and B) Representative images (A) and quantification (B) of neutrophils in the wound are shown; three independent replicates with n = 27 +/+ and 20 −/− at 1 hpw and 42 +/+ and 37 −/− at 6 hpw; scale bar = 100 µm. (C) Quantification of total number of mCherry-labeled neutrophils in 3 dpf WT and mydgf^−/− whole larvae; three independent replicates with n = 55 WT and 58 −/−. In B and C, data are expressed as mean with 95% CI; each symbol represents one larva, and different colors represent independent replicates. *, P < 0.05; ***, P < 0.001. P values were calculated by ANOVA with Tukey’s multiple comparisons. (D) Representative serial images from time-lapse imaging 0–6 h following tail transection of the caudal fin of WT and mydgf^−/− larvae; see Video 1. Lines represent neutrophil tracks over time, with warmer colors indicating a longer time since track start; four independent replicates with n = 7 +/+ and 11 −/− larvae; scale bar = 100 µm. (E) Quantification of the number of neutrophils at the wound microenvironment over the course of time-lapse imaging. (F) Quantification of the instantaneous speed during the later phase (3–6 hpw) of neutrophil recruitment; 0 min represents the time at which each neutrophil enters the wound microenvironment.

Figure 4.

MYDGF depletion impairs neutrophil reverse migration and resolution following injury. (A and B) Representative images (A) and quantification (B) of green (total) and red (photoconverted) dendra2-labeled neutrophils in the wound microenvironment at 6 hpw following tail transection of 3 dpf WT and mydgf^−/− larvae; three independent replicates with n = 33 +/+ and 36 −/−; scale bar = 100 µm. (C) Quantification of the percentage of photoconverted neutrophils present at the wound at 2 hpw that are no longer present at 6 hpw in the same larva. (D and E) Representative images (D) and quantification (E) of green (total) and red (photoconverted) dendra2-labeled neutrophils in the burn at 24 hpb following thermal injury of the caudal fin of 3 dpf WT and mydgf^−/− larvae; three independent replicates with n = 20 +/+ and 15 −/−; scale bar = 100 µm. (F) Quantification of the percentage of photoconverted neutrophils present in the burn microenvironment at 3 hpb that are no longer present at 24 hpb in the same larva. (G) Representative images of immunostaining for active caspase-3 (casp3) following tail transection of 3 dpf WT and mydgf^−/− larvae with mCherry-labeled neutrophils at 6 hpw; scale bar = 100 µm. (H) Quantification of total and active caspase-3–expressing neutrophils in the wound at 6 hpw; three independent replicates with n = 42 +/+ and 71 −/−. (I) Proportion of neutrophils in the wound expressing active caspase-3 at 6 hpw. In B, C, E, F, H, and I, data are expressed as mean with 95% CI; each symbol represents one larva, and different colors represent the results of three independent replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were calculated by ANOVA with Tukey’s multiple comparisons.

Figure S3.

Macrophage responses at sterile injuries in response to MYDGF depletion. (A and B) Representative brightfield images (A) and quantification (B) of tail fin regrowth area following thermal injury of the caudal fin of larvae microinjected at the posterior caudal vein with PBS or clodronate liposomes (Cld); three independent replicates with n = 48 PBS and 49 clodronate liposomes at 1 d postburn (dpb), 47 PBS and 47 clodronate liposomes at 2 dpb, 46 PBS and 47 clodronate liposomes at 3 dpb, and 44 PBS and 45 clodronate liposomes at 4 dpb; scale bar = 100 µm. P values were calculated by unpaired two-tailed t test; *, P < 0.05. (C) Quantification of the number of macrophages at the wound microenvironment at 4 and 24 hpw following tail transection of the caudal fin of 3 dpf WT and mydgf^−/− larvae; three independent replicates with n = 21 +/+ and 30 −/− at 4 hpw, 20 +/+ and 38 −/− at 24 hpw. P values were calculated by ANOVA with Tukey’s multiple comparisons. (D) Quantification of the number of physical contact between neutrophils and macrophages at the wound microenvironment over the course of time-lapse imaging following tail transection of the caudal fin of 3 dpf WT and mydgf^−/− larvae; four independent replicates with n = 7 +/+ and 11 −/− larvae; see Video 1. (E and F) Quantification of the number of macrophages (E) and the number of physical contacts between neutrophils and macrophages (F) at the burn microenvironment over the course of time-lapse imaging following thermal injury of the caudal fin of 3 dpf WT and mydgf^−/− larvae; three independent replicates with n = 10 +/+ and 10 −/− larvae; see Video 2. The number of contacts displayed in D and F are adjusted values, where the raw number of contacts was multiplied by the number of neutrophils and normalized to number of macrophages at each frame. No statistical differences were detected, as calculated by unpaired two-tailed t test. In B and C, data are expressed as mean with 95% CI; each symbol represents one larva, and different colors represent the results of three independent replicates.

Figure 5.

Neutrophil accumulation in the mydgf mutant is dependent on the HIF-1α pathway. (A) Schematic representation of HIF-1α activation in neutrophils. Pathway activation results in neutrophil persistence and survival and is characterized by increased expression of phd3. HIF-1α pathway activation can be blocked at the level of transcription factor nuclear binding using arnt-1 morpholino. Illustration was created at www.biorender.com. (B) phd3 expression in pooled tail fin tissue collect from WT larvae, either unwounded or 3 h following tail transection (Tt) or thermal injury (burn), measured by RT-qPCR. Data comprise three (burn) to five (Tt) independent replicates performed in technical triplicates and normalized to mydgf expression in unwounded tails and to ef1α. n = 50 tails per condition per independent replicate. *, P < 0.05. Fold changes in gene expression were compared with the normalized value of 1 using one-sample t tests. (C and D) Representative images (C) and quantification (D) of the number of mCherry-labeled neutrophils at the wound in WT and mydgf^−/− larvae, with control or arnt-1 morpholino, at 1 and 6 hpw after Tt; three or four independent replicates with n = 46 +/+ control mo, 33 +/+ arnt-1 mo, 50 −/− control mo, and 38 −/− arnt-1 mo at 1 hpw and 54 +/+ control mo, 38 +/+ arnt-1 mo, 68 −/− control mo, and 73 −/− arnt-1 mo at 6 hpw; scale bar = 100 µm. Data are expressed as mean with 95% CI; each symbol represents one larva, and different colors represent independent replicates. *, P < 0.05; **, P < 0.01. P values were calculated by ANOVA with Tukey’s multiple comparisons.