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. 2020 Oct 22;23(11):101699. doi: 10.1016/j.isci.2020.101699

Distinct Tissue Damage and Microbial Cues Drive Neutrophil and Macrophage Recruitment to Thermal Injury

Francisco Barros-Becker 1,2,8, Jayne M Squirrell 3,8, Russell Burke 1, Julia Chini 1,6, Julie Rindy 1,4, Aos Karim 5, Kevin W Eliceiri 3,7, Angela Gibson 5, Anna Huttenlocher 1,4,9,∗
PMCID: PMC7644964  PMID: 33196024

Summary

Tissue damage triggers a rapid innate immune response that mediates host defense. Previously we reported that thermal damage of the larval zebrafish fin disrupts collagen organization and induces a robust and potentially damaging innate immune response. The mechanisms that drive damaging versus protective neutrophil inflammation in interstitial tissues remain unclear. Here we identify distinct cues in the tissue microenvironment that differentially drive neutrophil and macrophage responses to sterile injury. Using live imaging, we found a motile zone for neutrophils, but not macrophages, in collagen-free regions and identified a specific role for interleukin-6 (IL-6) receptor signaling in neutrophil responses to thermal damage. IL-6 receptor mutants show impaired neutrophil recruitment to sterile thermal injury that was not present in tissues infected with Pseudomonas aeruginosa. These findings identify distinct signaling networks during neutrophil recruitment to sterile and microbial damage cues and provide a framework to limit potentially damaging neutrophil inflammation.

Subject Areas: Microbiology and Immunology

Graphical Abstract

graphic file with name fx1.jpg

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Highlights

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    Neutrophils, but not macrophages, rapidly migrate into a burn collagen-free zone

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    Lack of ATP or ROS affects neutrophil and macrophage recruitment distinctively

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    Absence of Il-6r strongly affects neutrophil recruitment to a sterile burn wound

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    Il-6r neutrophil recruitment defect can be rescued during P. aeruginosa infection

Microbiology and Immunology

Introduction

Disruption of tissue homeostasis by damage triggers intricate and highly regulated tissue repair programs. Innate immune cells, including neutrophils and macrophages, are among the first responders to tissue damage and infection (de Oliveira et al., 2016). Macrophages clear damaged tissue and initiate tissue repair, whereas neutrophils, although necessary for host defense, can mediate damaging inflammation that delays wound healing and regeneration (Schofield et al., 2013). Innate immune cells rapidly mobilize to both sterile and infected tissues by sensing both soluble and insoluble cues that orchestrate the inflammatory response (Lämmermann et al., 2013; Niethammer et al., 2009; de Oliveira et al., 2014; Yoo et al., 2011). Complex injuries, such as burns, generate an abrupt remodeling of the tissue, accompanied by a rapid and long-lasting immune response (Miskolci et al., 2019). However, few studies have addressed the mechanisms of leukocyte recruitment to thermal injury and the cues that mediate potentially damaging inflammation (Karim et al., 2019a; Lateef et al., 2019).

Damage caused by thermal injury generates a high number of apoptotic and necrotic cells (Gravante et al., 2006a). This necrotic tissue, as well as the affected surrounding tissue, mediates the release of pro-inflammatory factors. Cell disruption releases a variety of Danger Associated Molecular Patterns (DAMPs), such as exposed DNA, reactive oxygen species (ROS), adenosine triphosphate (ATP), and N-formyl peptides (Bayliss et al., 2014; Rani et al., 2017; Shupp et al., 2010; Zhang et al., 2010). These factors, in turn, induce production of pro-inflammatory cytokines like tumor necrosis factor alpha (TNF-α), interleukin (IL)-8, IL-1β, and IL-6, which can sustain the inflammatory response (Rani et al., 2017; Rodriguez et al., 1993). Immediately following a burn injury, organized collagen is lost, innate immune cells migrate toward the damaged tissue, and tissue remodeling precedes tissue repair and regeneration (Lateef et al., 2019; LeBert et al., 2018; Miskolci et al., 2019).

Here, we take advantage of zebrafish larvae to examine the spatiotemporal events occurring during the innate immune response to burn wounds. We show that neutrophils infiltrate rapidly into a collagen-free zone after thermal injury, whereas macrophages infiltrate more slowly, in a two-step process. Furthermore, we identified distinct mechanisms of neutrophil and macrophage responses. Although IL-6 is known to mediate tissue inflammation, few studies have addressed its role in neutrophil recruitment. We identify a specific role for the IL-6 receptor in neutrophil, but not macrophage, motility and recruitment to sterile burn injury. Furthermore, we demonstrate that IL-6 receptor signaling is not necessary for neutrophil responses to thermal injury in the presence of a microbial cue.

Results

Thermal Injury Triggers Distinct Neutrophil and Macrophage Responses

Our recent studies show that thermal injury causes more complex tissue damage and generates a differential immune response compared with the well-studied transection wound (Miskolci et al., 2019). In this current study, to better understand this more complex damage and immunological response, we imaged the tissue response to caudal fin thermal injury in live larvae. During the initial burn response, there were large-scale tissue morphology changes and some of the injured fins generated a tissue mass that detached from the fin within the first 24 h post burn (hpb) (Figure 1A). Additionally, within the first 6 h after burn injury, there was robust recruitment of both neutrophils and macrophages into the damaged fin. To further characterize the dynamics of the leukocyte response, we imaged live larvae from a double transgenic line Tg(mpeg1:H2B-GFP/lyzc:H2B-mCherry) that labels macrophage and neutrophil nuclei, permitting automated tracking (von Dassow et al., 2009; Miskolci et al., 2019; Yoo et al., 2012). After thermal injury, both neutrophils and macrophages migrated into the damaged tissue, with neutrophils infiltrating out to the wound edge (Figure 1A). However, macrophages did not migrate as far as neutrophils into the distal tissue during the early wound response (by 6 hpb), as assessed by measuring the distance that macrophages and neutrophils traveled beyond the distal end of the notochord (Figures 1B and 1C). These data suggest that neutrophils and macrophages exhibit different migration dynamics in burned tissue.

Figure 1.

Figure 1

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Leukocytes Exhibit Different Recruitment Kinetics to Thermal Injury

(A) Maximum intensity projection of confocal microscopy time-lapse stills of neutrophil (magenta) and macrophage (green) migration to burn wounds (DIC). Brackets indicate general burn region and arrows show the presence of the detaching tissue mass. t = 0 represents start of the recording, approximately 5 min following thermal injury. Scale bar, 200 μm.

(B) Schematic illustrating quantification of neutrophil and macrophage distance into the burn tissue. In the last frame of time-lapse recordings, the distance from the proximal edge of a cell nucleus to a line perpendicular to the posterior tip of the notochord was measured. Only cells posterior to the perpendicular line were quantified.

(C) Comparison of distance of single leukocytes from distal end of the notochord at 6 hpb. Symbols indicate individual cells.

(D) Tracks from 6 h time-lapse recordings were analyzed using automated tracking (Imaris). Diagram showing analysis method for assessing individual cell speed prior to (t < 0) or after (t > 0) entering into the burn wound zone (t = 0), delineated by red dashed line perpendicular to the distal tip of the notochord. Colored lines illustrate a single neutrophil track (magenta) and a single macrophage track (green).

(E) Instantaneous speeds, determined in Imaris, of individual neutrophils, with red dashed line indicating time (t = 0) when cell crossed into wound zone, as shown in (D).

(F) Instantaneous speeds, determined in Imaris, of individual macrophages, with red dashed line indicating time (t = 0) when cell crossed into wound zone, as shown in (D). Dark lines indicate arithmetic mean.

(G) Comparison of the ratio of the average speed during the 20 min following t = 0 to the 20 min preceding t = 0 of individual neutrophils (magenta) or macrophages (green). Symbols indicate individual cells.

(H) Tail fins were divided into trunk (blue) and wound (red) zones, delineated by the distal end of the notochord. Images of the cumulative automated neutrophil (magenta) and macrophage (green) tracks for 2 h time intervals for each cell type were converted to binary (lower images) and the percentage area occupied by those tracks was determined. Scale bar, 100 μm.

(I) Comparison of the ratio of the percentage of area occupied by tracks in the wound zone to the percentage of area occupied by tracks in the trunk zone at three time intervals. Symbols and corresponding dark lines represent arithmetic mean with SE. Pale lines represent individual larvae. Graphs (E)–(G) and (I) represent two independent experiments. Graphs (C) and (G) columns are arithmetic mean with SE. Additional statistical values in Table S1. See also Video S1.

To better understand the recruitment of neutrophils and macrophages to thermal injury, we imaged Tg(mpeg1:H2B-GFP/lyzc:H2B-mCherry) larvae at a high temporal resolution (<2 min between time points) for the first 6 hpb (Video S1). Individual neutrophils and macrophages were tracked and migration speed was quantified as the immune cells migrated from the trunk of the larva into the wound area—defined as crossing the plane of the distal end of the notochord, with the time of crossing designated as t = 0 (Figures 1D–1F). Interestingly, although both neutrophils and macrophages reduced their average speed rapidly after crossing into the wounded area, macrophages showed a greater reduction in speed than neutrophils after entering the wound area (Figure 1G). Additionally, we quantified the residency of neutrophils and macrophages in the designated burn wound region over time using the cell track foot prints (Figures 1H and 1I). Cumulative tracks for both neutrophils and macrophages were quantified for three time periods during early burn response: 0–2, 2–4, and 4–6 hpb. The percentage area of the wound region occupied by the tracks was compared with a similar region of the trunk adjacent to the wound, as delineated by the end of the notochord (Figure 1H). Over time, there was an increase in wound residency of both neutrophils and macrophages relative to that in the trunk. However, the increased occupation of neutrophils in the wound area relative to the trunk during early burn wound responses is greater than that of the macrophages (Figure 1I). Overall, these data indicate that there is a strong recruitment of both neutrophils and macrophages but, in contrast to tail transection wounds, there appears to be a limitation in the migration of macrophages into the burn wound area that does not affect the migration of neutrophils. Taken together, these data support the idea that macrophages and neutrophils interact differently with the thermally damaged tissue in vivo.

Video S1. Neutrophil and Macrophage Migration into Burn Tissue, Related to Figure 1

The caudal fin of a larva, expressing Tg(mpeg1:H2B-GFP;lyzc:H2B-mCherry) to label neutrophil (magenta) and macrophage (green) nuclei, was burned. Confocal imaging began within 5 min of wounding and continued for 7 h with 2 min cycles (left panel). Automated tracking (right panel) shows the nuclei (sphere) and tracks (lines) of neutrophils and macrophages (magenta and green, respectively) to the burn tissue cumulatively during the burn injury response. Neutrophils demonstrate a rapid migration to the burn, and an earlier infiltration into the burn tissue, compared with macrophages. Red lines show the burn area at the beginning and end of the video. Scale bar, 100 μm. Time shown as hh:mm:ss:msec.

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Differential Neutrophil and Macrophage Residency in the Collagen Free Zone

One possible contributor to the differential movement of leukocytes into the burn wound is changes in tissue architecture. Therefore, we further characterized neutrophil and macrophage migration to thermal injury by imaging leukocyte responses in conjunction with Second Harmonic Generation (SHG) imaging to visualize collagen organization (LeBert et al., 2018; Miskolci et al., 2019). SHG combined with multiphoton based fluorescence imaging was performed on live larvae following thermal injury using the Tg(mpeg1:EGFP) and Tg(mpx:EGFP) zebrafish lines, both in casper backgrounds to avoid pigmentation. As we previously reported, organized collagen fibers are not detected in the burn wound area (Figures 2A and 2C) (LeBert et al., 2018; Miskolci et al., 2019). We defined three zones within the burn wound based on SHG imaging; collagen zone (undamaged fibers), edge of collagen (end of the collagen fibers), and collagen-free zone (no detectable collagen fibers) (Figures 2A and 2C, Video S2). Neutrophils and macrophages were followed after thermal injury within each zone over time. To determine whether there was a difference between the ability of neutrophils and macrophages to transit between the different tissue microenvironments, we measured the total leukocyte volume as a tool to quantify changes in cell density over time at the collagen edge and the collagen free zone (Figures 2B and 2D). Additionally, these location-dependent differences in leukocyte residency between neutrophils and macrophages were further delineated by quantifying the change in residency during different time windows following thermal injury (Figure 2E). Neutrophils readily migrated into the collagen-free zone, reaching a peak of recruitment around 4.5 hpb (Figures 2B and 2E). After 6.5 hpb, neutrophils began leaving the collagen-free zone. The volume of neutrophils at the edge of the collagen zone remained relatively constant over time, suggesting that there was no major impediment to their movement into or out of the collagen-free tissue region (Figures 2B and 2E). However, macrophage recruitment to the collagen-free zone was reduced compared with neutrophils (Figures 2D and 2E), with peak recruitment after 15 hpb. Furthermore, macrophages accumulated at the edge of the collagen, compared with the collagen-free zone, suggesting that macrophages paused at the edge of the collagen before infiltrating the collagen-free area (Figure 2D). Additionally, following entry into the collagen free zone neutrophil morphology did not seem to be altered while macrophage morphology changed from elongated to more round (Figures S1A and S1B). These data suggest that changes in the tissue microenvironment induced by specific types of damage differentially influence the behavior of neutrophils and macrophages.

Figure 2.

Figure 2

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Macrophages and Neutrophils Differ in Their Residency in the Collagen-Free Region of the Burn Wound

(A and C) Images generated from multiphoton microscopy images of (A) neutrophils or (C) macrophages and collagen fibers during the first 18.5 hpb in a live larva. Top row: Brightfield images show overall tissue changes. Middle row: Surface rendered leukocytes (Imaris) were categorized by location: in the collagen zone (white), at the edge of the collagen (blue) or in the collagen-free zone (red), with SHG fibers in magenta. Bottom row: Side view of the surface rendered, 3D reconstruction demonstrating the 3D nature of these zones. Scale bars, 50 μm.

(B) Total volume of the surface rendered, categorized neutrophils in edge and collagen-free zone over time. Gray boxes indicate time windows used for graph in (E) Symbols indicate arithmetic mean with SE.

(D) Total volume of the surface rendered, categorized macrophages in each zone over time. Gray boxes indicate time windows used for graph in (E) Symbols indicate arithmetic mean with SE.

(E) Comparison of the change (slope) in total volume of neutrophils (magenta) and macrophages (green) for each larva in either edge (blue columns with round symbols) or collagen-free (peach columns with triangle symbols) wound zones in three different time windows, identified by the gray boxes in (B) and (D). Symbols represent individual larva; columns are arithmetic mean with SE. Additional statistical values in Table S1. See also Figure S1 and Video S2.

Video S2. Animated 3D Reconstruction of Neutrophils, Macrophages and SHG Fibers after Thermal Injury, Related to Figure 2

Concatenated 3D reconstruction video of thermally injured caudal fin, at 4.5 hpb, of a larva expressing GFP-tagged neutrophils followed by a caudal fin of a larvae expressing GFP-tagged macrophages. Each segment highlights the following features: slice view of the brightfield image showing the burn damaged tissue, a surface rendering (gray) of tissue autofluorescence to provide tissue context, with added SHG (magenta) and leukocyte (green) images that are surface rendered. The leukocyte surface rendering was color coded by location relative to the collagen fibers, as detected by SHG (see Methods for description of categorization). Note that categorization was assessed on the original SHG data, not on the surface rendering, which under-represents fine fibers. The surface rendered reconstruction is rotated through 360°. Anterior is to the left. Similar data are presented as still images in Figure 2.

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Neutrophil and Macrophage Recruitment to Thermal Injury Depends on Different Chemotactic Cues

Compared with excision wounds, burn wounds elicit a stronger and more prolonged immune response (Miskolci et al., 2019; Valvis et al., 2015). Mammalian burn injuries have zones of high tissue damage containing large numbers of apoptotic and necrotic cells (Jackson, 1969; Shupp et al., 2010). Using acridine orange (Abrams et al., 1993), we found that thermal injury led to an increase in apoptotic cells compared with uninjured larvae at 6 hpb (Figure S2A). To address whether the presence of damaged tissue contributes to leukocyte recruitment, we excised the burn tissue at 24 hpb and quantified leukocyte recruitment at 48 hpb (Figure 3A). Larvae that received double transection wounds (first at 0 h and again at 24 h) exhibited an increase in neutrophils and macrophages at the wound at 48 hpw compared with a single transection wound at 0 h (Figures 3B and 3C). In contrast, excision of the burn tissue at 24 hpb trended toward a reduction in neutrophils and a significant reduction in macrophages at the wound, compared with a non-excised burn (Figures 3B and 3C). These findings indicate that the damaged tissue in burn wounds serves as a key signal source for sustained leukocyte recruitment and that removing the damaged tissue may reduce tissue inflammation.

Figure 3.

Figure 3

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Neutrophil Recruitment to Thermal Injury Depends on Both ATP and ROS, whereas Macrophage Recruitment Depends Only on ROS

(A) Schematic illustrating the experiment design. Burn or tail transection was performed on 3-dpf larvae. After 24 h of the initial insult, burn tissue was excised or a second transection was performed.

(B) Neutrophil recruitment quantified 24 h after the second insult (48 h after initial injury). Neutrophil numbers were normalized to fin area for each larva. Symbols indicate individual larvae.

(C) Macrophage recruitment quantified 24 h after the second insult (48 h after initial injury). Macrophage numbers were normalized to fin area for each larva. Symbols indicate individual larvae.

(D) Representative images of 3-dpf larvae burned in the presence of apyrase then macrophage (green) and neutrophil (magenta) recruitment were quantified at 2 and 6 hpb. Scale bars, 200 μm.

(E and F) Recruitment of (E) neutrophils and (F) macrophages to the burn wound with apyrase treatment. Symbols indicate individual larvae.

(G) Representative images of 3-dpf larvae burned in the presence of DPI (1 h pre- and post-burn treatment) and macrophage (green) and neutrophil (magenta) recruitment was quantified at 2 and 6 hpb. Scale bars, 200 μm.

(H and I) Recruitment of neutrophils and macrophages, (H) and (I) respectively, to the burn wound with DPI treatment. Symbols indicate individual larvae. For (B), (C), (E), (F), (H), and (I) columns are mean with SE. Additional statistical values in Table S1. See also Figure S2.

To address the question of what signals from the damaged tissue contribute to the sustained inflammatory response, we considered soluble chemotactic signals released by damaged tissue, such as adenosine triphosphate (ATP) and hydrogen peroxide (H2O2), that regulate early leukocyte recruitment to a wound (Harada et al., 2018; Niethammer et al., 2009; de Oliveira et al., 2014; Yoo et al., 2011). ATP is a well-described damage signal with pleiotropic downstream inflammatory effects (Cauwels et al., 2014). To test the impact of ATP signaling on leukocyte recruitment to the thermal injury, larval caudal fins were burned in the presence of apyrase (Figure 3D), an ATP-diphosphohydrolase that leads to the degradation of ATP (Gault et al., 2014). Apyrase treatment with thermal injury decreased the number of neutrophils at 6 hpb (Figure 3E). However, macrophage recruitment was unaffected by apyrase treatment during these timepoints, suggesting that macrophage recruitment is not dependent on ATP during early time points (Figure 3F).

Hydrogen peroxide is also an important leukocyte chemoattractant in vivo (Chang et al., 2013; Klyubin et al., 1996; Niethammer et al., 2009; Yoo et al., 2011). Blocking H2O2 release reduces the number of macrophages and neutrophils at a tail transection wound in zebrafish (Niethammer et al., 2009; de Oliveira et al., 2014; Tauzin et al., 2014; Yoo et al., 2011). To determine the role of H2O2 following thermal injury, larvae were incubated with diphenyleneiodonium chloride (DPI), which prevents H2O2 production during wounding (Figure 3G) (Yoo et al., 2011). Consistent with the previous observations of leukocyte recruitment to tail transection (Niethammer et al., 2009; de Oliveira et al., 2014; Yoo et al., 2011), H2O2 production inhibition with DPI impaired both neutrophil and macrophage recruitment to a burn wound at 2 and 6 hpb (Figures 3H and 3I). Of note, DPI treatment had some direct damaging effects on fin morphology compared with control vehicle but, despite this damage, leukocyte recruitment was still impaired. These results suggest that, although some common cues, such as H2O2, mediate both neutrophil and macrophage recruitment, distinct cues, like ATP, may differentially mediate leukocyte responses to thermal injury.

Neutrophils Are Present in Human Burns and Impair Zebrafish Burn Wound Healing

To ascertain whether a neutrophil response observed in zebrafish was present in human burn wounds we examined the RNA expression of several sets of genes in human burn tissue samples. Utilizing previously published expression data from biopsies of burn patients with both partial and full thickness burns (Karim et al., 2019b), we re-examined these expression data to determine the levels of neutrophil-related genes. In both deep partial-thickness (DPT) and full-thickness (FT) burns, neutrophil markers increased compared with the control (Figure 4A, Data S1), establishing that neutrophil response to thermal injury in humans is increased, similar to that observed in zebrafish larvae. This is consistent with published histology showing neutrophil infiltration in burns. Furthermore, the timing of recruitment seen in our work follows similar trends as seen by others in different burn models (Van De Groot et al., 2009; Lateef et al., 2019). We next sought to determine whether the presence of neutrophils impacts burn wound resolution. To accomplish this, we assessed burn wound regrowth in Tg(mpx:Rac2D57N-mCherry) transgenic larvae. This dominant negative Rac2 prevents neutrophil migration out of the caudal hematopoietic tissue and subsequent recruitment to a wound (Deng et al., 2011). Tissue regrowth at 72 h after burn injury was significantly improved in Rac2D57N larvae as compared with wild-type (Figure 4B). This suggests that the presence of neutrophils is inhibitory to burn wound healing in this model, consistent with studies in mouse models (Dovi et al., 2003).

Figure 4.

Figure 4

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Neutrophil Recruitment to a Burn Wound Is IL-6 Dependent

(A) RNA expression of neutrophil-related genes (source: geneontology.org) of normal, deep partial-thickness (DPT) and full-thickness (FT) burn samples from human biopsies. Symbols indicate patient biopsies.

(B) Fin regeneration area at 72 hpb in wild-type and Rac2D57N larvae. Symbols indicate individual larvae.

(C) Expression of IL-6-related pathway genes (Source: Biocarta) in human burn patient biopsies. Data for (A) and (C) from single sample Gene set Enrichment Analysis (ssGSEA). Symbols indicate patient biopsies.

(D) Representative images showing neutrophils (magenta) and macrophages (green) in 3-dpf larvae, carrying WT (+/+), heterozygous (+/−), or mutant alleles (−/−) for il-6r, at 2, 6, and 24 h following thermal injury. Scale bar, 200 μm.

(E) Number of neutrophils present in the burn wound in the different il-6r allele backgrounds. Symbols indicate individual larvae.

(F) Number of macrophages present in the burn wound in the different il-6r allele backgrounds. Symbols indicate individual larvae.

(G) Quantification of neutrophil recruitment to the burn wound at 6 hpb in larvae with and without injection of il-6r mRNA (isoform #1). Symbols indicate individual larvae.

(H) Quantification of neutrophil number at 6 hpb when IL-6R binding to gp130 was inhibited with SC144 treatment. Symbols indicate individual larvae.

(I) Representative images of cumulative automated tracks of neutrophils (magenta) and macrophages (green) overlayed on DIC images of il-6r+/+ or il-6r−/− thermally injured caudal fins at 6 hpb. Scale bar, 100 μm.

(J) Quantification of mean neutrophil speed in il-6r+/+ or il-6r−/− over 6 h following thermal injury. Symbols indicate individual larvae.

(K) Quantification of mean macrophage speed in il-6r+/+ or il-6r−/− over 6 h following thermal injury. Symbols indicate individual larvae. For (A)–(C), (E)–(H), (J), and (K) columns are arithmetic mean with SE. Additional statistical values in Table S1. See also Figure S3 and Videos S3 and S4.

Neutrophil, but Not Macrophage, Recruitment Depends on IL-6

Owing to the importance of neutrophil infiltration into burn tissue, we next aimed to investigate signaling pathways that could influence this behavior. Our recent studies implicate IL-6 signaling in neutrophil motility in 3D in vitro (Hind et al., 2018). Furthermore, IL-6 is upregulated in burn patient serum and circulating levels of IL-6 correlate with reduced patient survival (Gauglitz et al., 2008; Yeh et al., 1999). We identified a significant increase in expression of IL-6-related pathway genes in full thickness burns compared with control (Figure 4C, Data S1). Although IL-6 blockade is used to treat chronic inflammatory disease (Noack and Miossec, 2017), the specific role of IL-6 signaling on inflammation in response to burn injury remains unclear. To investigate the role of IL-6 signaling in response to thermal injury in zebrafish, we characterized an IL-6 receptor (il-6r) mutant zebrafish line (Sa42709; ZIRC). Protein sequence alignment predicts several key conserved domains between human and zebrafish il-6r (Figure S3A; ENSEMBL: ENSDART00000186042.1). These conserved domains include the IL-6 binding domain, signal transduction domains and IL-6 receptor family cysteine residues (Yawata et al., 1993). The il-6r mutant zebrafish line (il-6r −/−) is predicted to have a premature stop codon within exon 1 (L42 > Stop; Figure S3A) and, indeed, RT-qPCR showed that mRNA transcript abundance was decreased in il-6r−/− larvae, suggesting that the mutation affects mRNA stability (Figure S3B). il-6r−/− zebrafish were outcrossed into the Tg(mpeg1:H2B-GFP/lyzc:H2B-mCherry) line, permitting quantification of leukocyte responses to thermal injury in the absence of the il-6r (Figure 4D). Neutrophil recruitment to the burn wound was significantly decreased in the il-6r −/− larvae compared with both il-6r+/+ and il-6r+/- matched siblings at 2, 6, and 24 hpb (Figure 4E). Additionally, heterozygous il-6r+/- larvae exhibited an intermediate decrease in neutrophil recruitment compared with il-6r+/+ siblings at 6 hpb (Figure 4E), suggesting that the concentration of the IL-6R regulates neutrophil recruitment to burn injury. In contrast, macrophage recruitment to burn injury was generally unaffected by loss of il-6r (Figure 4F). At 6 h after burn il-6 expression exhibited increased levels in the tail compared with control, although this trend was not statistically significant (Figure S3C). However, we also observed reduced recruitment of neutrophils to a tail transection wound in IL-6R-deficient larvae (Figure S3D), supporting previous reports that IL-6 also plays a role in other types of injuries (Biffl et al., 1996; Tompkins, 2015). Depletion of IL-6R also did not affect the total number of either neutrophils or macrophages in larval zebrafish (Figure S3E), indicating that lower neutrophil recruitment to the thermal injury is not due to developmental defects in leukocyte production.

To confirm specificity of the il-6r mutation, zebrafish il-6r mRNA was amplified from wild-type cDNA to rescue the mutant line. Two alternatively spliced isoforms of il-6r were identified (Figure S3A, GenBank: MW067024; MW067025). Isoform #2 is generally similar to the zebrafish il-6r consensus sequence, whereas isoform #1 lacks exon 8, generating an early stop codon (Figure S3A). Consensus Constrained TOPology prediction (CCTOP) and Phobius in silico analysis (Dobson et al., 2015; Käll et al., 2004) predicted that isoform #1 lacks the transmembrane and cytoplasmic domain (Figure S3A), suggesting this is a soluble isoform of IL-6R. This is consistent with previously described soluble human isoforms of IL-6R lacking the transmembrane domain due to alternative splicing of the last exon (Baran et al., 2018; Heaney and Golde, 1996; Horiuchi et al., 1994). Importantly, the human soluble form of IL-6R is still capable of binding IL-6 and signaling through the coreceptor, gp130 (Hurst et al., 2001; Jones et al., 2001). We found that the reduction in neutrophil recruitment to thermal injury in il-6r −/− larvae was partially rescued by injection of mRNA of the identified soluble form of the il-6r (Isoform #1) (Figure 4G). Furthermore, il-6r +/+ larvae injected with the same il-6r isoform mRNA exhibited an increase in neutrophil recruitment to a burn, further suggesting that il-6r works in a dose-dependent manner for neutrophil recruitment following thermal injury (Figure 4G). Additionally, neutrophil recruitment was decreased during a thermal injury response when the binding of the IL-6R to its co-receptor gp130 was inhibited using the small molecule SC144 (Figure 4H) (Xu et al., 2013). Together, these findings support a critical role for IL-6 signaling in neutrophil recruitment to thermal damage. These findings are consistent with previous work reporting that IL-6 is important for neutrophil migration toward IL-8, fMLP, and LPS-induced pulmonary inflammation and infection (Fielding et al., 2008; Hind et al., 2018; Wright et al., 2014; Yan et al., 2013).

To determine the impact of the IL-6 receptor on leukocyte migration, we used the il-6r−/−Tg(mpeg1:H2B-GFP/lyzc:H2B-mCherry) line described above and tracked leukocyte movement during early burn responses. In mutant larvae, neutrophils, but not macrophages, showed a striking migratory defect compared with WT larvae (Figures 4I and Videos S3 and S4). Quantification of migratory parameters showed that the mean neutrophil speed was significantly reduced in il-6r−/− compared with il-6r+/+ larvae (Figure 4J), whereas the macrophage speed was unaffected (Figure 4K). These results suggest that neutrophil, but not macrophage, motility in response to burn wounds is dependent on the IL-6 receptor.

Video S3. il-6r+/+ Neutrophils Respond Normally to a Burn Injury, Related to Figure 4

Larva, 3 dpf, expressing Tg(mpeg1:H2B-GFP;lyzc:H2B-mCherry) to label macrophage (green) and neutrophil (magenta) nuclei, and carrying homozygous wild-type (+/+) of il-6r were burned, followed by 7 h time-lapse confocal imaging with 4 min cycles (left movie panel). Automated tracking (right movie panel) shows the nuclei (sphere) and cumulative tracks (lines) of neutrophils and macrophages to the burn tissue, in magenta and green, respectively. il-6r+/+ neutrophils and macrophages have a normal response to the burn with early infiltration of neutrophils into burned tissue. Scale bar, 100 μm. Time is show as hh:mm:ss:msec.

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Video S4. il-6R−/− Neutrophils Fail to Respond Normally to a Burn Injury, Related to Figure 4

Larva, 3 dpf, expressing Tg(mpeg1:H2B-GFP;lyzc:H2B-mCherry) to label macrophage (green) and neutrophil (magenta) nuclei and carrying homozygous mutant (−/−) alleles of il-6r were burned, followed by 7 h time-lapse imaging with 4 min cycles (left movie panel). Automated tracking (right movie panel) shows the nuclei (sphere) and cumulative tracks (lines) of neutrophils and macrophages to the burn tissue, in magenta and green, respectively. il-6r−/− larvae present an impaired neutrophil migration phenotype, whereas macrophage migration is unchanged. By 7 hpb only a few neutrophils are in close proximity to the burn. Scale bar, 100 μm. Time is show as hh:mm:ss:msec.

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The IL-6 Receptor Is Not Necessary for Neutrophil Recruitment to Pseudomonas aeruginosa-Infected Burn Wounds

To further investigate the role of il-6r during thermal injury, we imaged the response of leukocytes to infected wounds. Pseudomonas aeruginosa is one of the most common pathogens found in burn patients and leads to higher morbidity and mortality (Estahbanati et al., 2002). Therefore, wound responses were investigated in the presence of P. aeruginosa (PA14 strain) infection after thermal injury (Figures 5A, 5B,and S4A). We found that tissue re-growth was impaired with P. aeruginosa-infected wounds (Figure 5C). Although P. aeruginosa infection was mostly cleared by 96 hbp (Figure S4A), infection of burn wounds resulted in an increased and sustained recruitment of both neutrophils and macrophages at the injured site from 2 to 96 hpb (Figures S4B and S4C). In both control and IL-6 receptor-depleted larvae neutrophil recruitment was increased with infection compared with uninfected wounds at 6 and 24 hpb (Figure S4D). However, the fold change in neutrophil recruitment to P. aeruginosa-infected wounds in il-6r−/− mutant larvae was not decreased compared with the infected il-6r+/+ at 6 hpb. This is in contrast to the dramatic decrease in neutrophil recruitment observed in uninfected il-6r−/− compared with wild-type larvae (Figures 5D and S4D). Additionally, the fold change in neutrophil recruitment in il-6r−/− mutant larvae was greater in the presence of P. aeruginosa infection at both 6 and 24 hpb. Together, these data suggest that il-6r is not required for neutrophil recruitment to infected wounds. Macrophage recruitment to infected burn wounds was slightly decreased in il-6r−/− larvae at 6 hpb, but there was no difference in recruitment observed at 24 hpb (Figures 5E and S4E). In all, these findings suggest that the IL-6 receptor is required for neutrophil recruitment to tissue damage but not to infected wounds.

Figure 5.

Figure 5

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IL-6 Receptor Is Not Required for Neutrophil Recruitment to Infected Burn Wounds

(A) Diagram describing the protocol for infection of burn wounds with P. aeruginosa (PA14 strain). Larvae, 3 dpf, were burned, followed immediately by a 1-h exposure to bacteria or PBS. Fin regeneration and leukocyte recruitment were quantified and larvae were subsequently genotyped.

(B) Fluorescent micrographs showing the presence of PA14 bacteria in thermally injured caudal fin at 24 hpb. Arrows indicate bacteria in infected tails; asterisk denotes non-specific background fluorescence. Scale bar, 200 μm.

(C) Area of tissue regrowth in burned caudal fins with and without PA14 infection, measured as the fin tissue caudal to the posterior end of the notochord. Symbols indicate individual larvae.

(D) Comparison of neutrophil recruitment to the burn wound site with bacteria (PA14 - beige columns) or without bacteria (PBS - blue columns) in il-6r+/+ or il-6r−/− larvae depicted as the fold change in neutrophil number relative to the mean of the corresponding wild-type. Symbols indicate individual larvae.

(E) Comparison of macrophage recruitment to the burn wound site, with bacteria (PA14, beige columns) or without bacteria (PBS, blue columns) in il-6r+/+ or il-6r−/− larvae, depicted as the fold change in macrophage number relative to the mean of the corresponding wild-type. Symbols indicate individual larvae. Graphs with neutrophil counts and heterozygote data are presented in Figure S4. For (C)–(E) columns are arithmetic mean with SE. Additional statistical values in Table S1. See also Figure S4.

Discussion

Thermal injury induces complex tissue damage that has significant morbidity and mortality worldwide. Both damage and microbial cues recruit innate phagocytes, and the balance of this inflammation can determine clinical outcome (Church et al., 2006; Ipaktchi et al., 2006). We identified IL-6 as a key signal that controls neutrophil responses to sterile thermal injury but is not necessary for neutrophil recruitment to wounds infected with P. aeruginosa, a common pathogen found in human burn injuries (Gonzalez et al., 2018). Our findings highlight context-dependent signals that differentially regulate neutrophil and macrophage responses that impact tissue repair.

Leukocyte behavior is regulated by the tissue microenvironment; however, the ability to image leukocyte movement within interstitial tissues with high resolution has limited the field. Few models allow for the direct visualization of leukocyte-ECM dynamics in real time. Zebrafish caudal fins contain radially organized collagen fibers (LeBert et al., 2015), which are lost after burn injury (LeBert et al., 2018; Miskolci et al., 2019), similar to the loss of ECM organization observed in murine skin after thermal injury (Lateef et al., 2019). Here we show distinct motile behaviors of neutrophils and macrophages in interstitial tissues after thermal injury, with striking differences in neutrophil and macrophage recruitment into the “collagen-free” zone. Macrophages exhibit pausing and slower migration at the interface between collagen-containing and the wound-induced collagen-free zones. By contrast, neutrophils exhibit rapid, swarming migration into the collagen-free zone in a fashion similar to other migratory amoeboid cells in vivo (Lämmermann et al., 2013). For example, neutrophils move into other collagen-free zones including the luminal parts of the intestine and vasculature (Massena et al., 2010; Sumagin et al., 2014). Our previous studies demonstrated that macrophages exhibit a mesenchymal-like mode of migration in interstitial tissues in zebrafish that requires proteolytic activity, whereas neutrophils demonstrate amoeboid migration that is not protease sensitive (Barros-Becker et al., 2017). Taken together, these findings show that, in contrast to macrophages, neutrophils are less sensitive to changing collagen ECM structure and can rapidly migrate in areas devoid of organized collagen in vivo.

Pro-inflammatory signals, such as DAMPs, are released from damaged tissues and mediate leukocyte recruitment in response to apoptotic and necrotic cells (Gravante et al, 2006a, 2006b; Shupp et al., 2010; Singer et al., 2008). Damage signals that modulate early leukocyte recruitment include adenosine triphosphate (ATP) and reactive oxygen species (ROS). ATP released during injuries rapidly induces leukocyte migration (Boucher et al., 2010; de Oliveira et al., 2014). Both neutrophils and macrophages strongly respond to ATP gradients (Chen et al., 2006; Kronlage et al., 2010; Wang and Chen, 2018; Wang and Kubes, 2016) and this effect can be reduced by treatment with apyrase (Kronlage et al., 2010; de Oliveira et al., 2014). Interestingly, we found that neutrophil, but not macrophage, recruitment to burn injury was reduced by apyrase, suggesting that the cues mediating the recruitment of neutrophils and macrophages to thermal injury are separable. It is not clear if the neutrophil effect was a direct result of ATP reduction or indirect through effects of ATP on other signaling pathways, including ROS-mediated production of MIP-2 in macrophages, which then could induce neutrophil recruitment, as documented in mice (Kawamura et al., 2012). In contrast to ATP, we found that ROS signaling was important for both early and late neutrophil and macrophage recruitment to thermal injury. ROS is widely recognized as an important neutrophil chemoattractant to damage, resulting in recruitment early after tail transection (Niethammer et al., 2009; Yoo et al., 2011). Although it is not clear if these effects are direct, neutrophils can sense H2O2 through the oxidation of downstream signaling components, like Lyn, as an early signaling mechanism (Yoo et al., 2011). Alternatively, H2O2 could be acting through general tissue activation of the NF-kB/AP1 signaling pathway and contribute to later recruitment via the production of other pro-inflammatory mediators, such as IL-8 and IL-6 (Chang et al., 2013; de Oliveira et al., 2014; Wittmann et al., 2012). Interestingly, removal of the damaged tissue after burn reduces recruitment of both neutrophils and macrophages to the wound. Early excision of burns in humans with a large total body surface area injury decreases mortality, an effect thought to be due to the reduction of the inflammatory burden of the necrotic tissue (Ong et al., 2006). Further study is needed to determine if removal of the damaged tissue reduces DAMP-mediated recruitment or whether this removal is impacting other wound-specific signals.

Our results, along with previous reports, show that IL-6 is upregulated after burn injury in human patients and in murine burn models (Abdullahi et al., 2014; Drost et al., 1993; Hager et al., 2018). We detected a similar trend in il-6 expression through our zebrafish RT-qPCR, although these results are not statistically significant. This cytokine regulates neutrophil migration toward IL-8 and fMLP, thereby impacting LPS-induced pulmonary inflammation, infection, and peritoneal acute inflammation (Fielding et al., 2008; Hind et al., 2018; Wright et al., 2014; Yan et al., 2013). Our findings show that depletion of the IL-6R in zebrafish decreases neutrophil recruitment to burn injury. IL-6 expression can be induced by several factors, including LTB4, ATP, INF-γ, and NF-kB activation (Faggioli et al., 1997; Ihara et al., 2005; Tanaka et al., 2014). This leads to the activation of STAT3 and several pro-inflammatory genes, including chemokine receptors such as the IL-8 receptor, CXCR2 (Nguyen-Jackson et al., 2010). IL-6 expression also generates a positive feedback loop, further activating IL-6 signaling pathways (Hendrayani et al., 2016; Lee et al., 2012). Our data suggest that IL-6 signaling could be involved in initiating and maintaining a primed state in neutrophils after burn injury, which is impaired in the il-6r−/− mutants, leaving neutrophils less sensitive to specific external inflammatory cues. Bacterial infection of the burn injury by P. aeruginosa shows that neutrophils from il-6r mutants are capable of migrating in great numbers to the damaged area in the presence of microbial cues, albeit to a slightly lesser degree than that of the infected wild-type larvae. This suggests that neutrophil deficiency after loss of IL-6R is not related to impaired cell movement. It is possible that IL-6 acts as a neutrophil motility priming factor that mediates recruitment to sterile damage cues. Interestingly, loss of IL-6R has no noticeable effect on macrophage recruitment to burn injury. IL-6 signaling has been shown to regulate macrophage polarization, favoring an M2 state, by alternatively activating macrophages (Chen et al., 2018; Fernando et al., 2014; Mauer et al., 2014). Therefore, although IL-6 signaling does not affect macrophage recruitment to burn injury in zebrafish larvae, more work is necessary to explore the impact of IL-6 on macrophage polarization within burn wounds.

Both microbes and tissue damage signaling recruit neutrophils to the site of insult. A recent report by Huang and Niethammer (2018) showed that neutrophils require tissue damage signaling to respond to microbial cues in the otic vesicle of zebrafish. Here, we show that IL-6 is a tissue damage cue that mediates neutrophil recruitment to sterile thermal injury. The presence of bacteria can override the neutrophil recruitment inhibition in il-6r mutants, suggesting that even when specific tissue damage cues are blocked, neutrophils can still respond to microbial cues. It is possible that the hierarchy of recruitment signaling is context dependent such that the extensive disruption of the matrix architecture that occurs with thermal injury may induce distinct tissue damage cues that are separable from microbial cues. This distinction potentially provides a therapeutic target to block damaging neutrophil inflammation in burns with IL-6 blockade, while still permitting neutrophil responses to pathogenic infection.

In conclusion, zebrafish provide a new model in which to study the innate immune response during early burn injury. Tissue morphology changes and loss of collagen fibers affect macrophage, but not neutrophil, recruitment to the wound area. We demonstrate that IL-6 signaling is critical for neutrophil motility and recruitment during early burn wound response but not in the presence of pathogenic microbes. Further work will be needed to determine the complete role of IL-6 signaling and its potential for therapeutic benefit in burn patients. This work provides a framework for the identification of pathways that limit damaging neutrophil inflammation in sterile injuries while not impacting necessary neutrophil responses to microbial injury.

Limitations of the Study

This study shows the complex environment under which neutrophils and macrophages need to respond during early burn injuries. The specific abundance of different signals, soluble and insoluble, can affect each cell type individually, influencing their response. However, further studies are needed to define the specifics of the relationship between changes in the matrix architecture of a burn injury and neutrophil and macrophage migration. Moreover, further studies are needed to elucidate the cellular origin of il-6 as well as the molecular downstream effects of il-6 on neutrophil activation, especially during a sterile burn injury.

Resource Availability

Lead Contact

Further information and requests for data, resources, and reagents should be directed to, and will be fulfilled, by the Lead Contact, Anna Huttenlocher (huttenlocher@wisc.edu).

Materials Availability

Plasmids and zebrafish lines generated in this study are available from the lead contact upon request.

Data and Code Availability

Original/source data for all figures published in this study are available from the lead contact upon request. The sequences generated in this study are available at GenBank (Accession number MW067024 for il-6r isoform #1 and MW067025 for il-6r isoform #2). This study did not generate any new code.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

We would like to thank all Huttenlocher lab members for useful discussion and input. We would like to thank Drs. Veronika Miskolci and Adam Horn for critical reading of the manuscript. We would like to thank Jens Eickhoff for advice on statistical analysis. This work was supported by NIH R35 GM1 18027 (A.H.).

Author Contributions

Conceptualization, F.B.-B., J.M.S., and A.H. Methodology, F.B.-B. and J.M.S. Investigation, F.B.-B., J.M.S., R.B., J.C., and J.R. Formal analysis, F.B.-B., J.M.S., R.B., and A.K. Statistical analysis, J.M.S. Human expression data analysis, A.K. Visualization, F.B.-B. and J.M.S. Writing, F.B.-B., J.M.S., and A.H., with input of all other authors. Resources, A.H. and K.W.E. Funding acquisition, A.H.

Declaration of Interests

The authors declare no competing interests.

Published: November 20, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101699.

Supplemental Information

Document S1. Transparent Methods and Figures S1 and S2
mmc1.pdf (2.3MB, pdf)
Table S1. Additional Statistical Information, Related to Graphs in Figures 1 through 5

Excel file providing tables with additional statistical information related to graphs presented in all main figures.

mmc2.xlsx (30.5KB, xlsx)
Data S1. Full Gene List, Gene Analysis, and ssGSEA Scores, Related to Figure 4

Human gene data and analysis for neutrophil- and IL-6-related gene sets.

mmc3.xlsx (33.3KB, xlsx)

References

  1. Abdullahi A., Amini-Nik S., Jeschke M.G. Animal models in burn research. Cell. Mol. Life Sci. 2014;71:3241–3255. doi: 10.1007/s00018-014-1612-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abrams J.M., White K., Fessler L.I., Steller H. Programmed cell death during Drosophila embryogenesis. Development. 1993;117:29–43. doi: 10.1242/dev.117.1.29. [DOI] [PubMed] [Google Scholar]
  3. Baran P., Hansen S., Waetzig G.H., Akbarzadeh M., Lamertz L., Huber H.J., Ahmadian M.R., Moll J.M., Scheller J. The balance of interleukin (IL)-6, IL-6·soluble IL-6 receptor (sIL-6R), and IL-6·sIL-6R·sgp130 complexes allows simultaneous classic and trans-signaling. J. Biol. Chem. 2018;293:6762–6775. doi: 10.1074/jbc.RA117.001163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barros-Becker F., Lam P., Fisher R., Huttenlocher A. Live imaging reveals distinct modes of neutrophil and macrophage migration within interstitial tissues. J. Cell Sci. 2017;130:3801–3808. doi: 10.1242/jcs.206128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bayliss J., Delarosa S., Wu J., Peterson J.R., Eboda O.N., Su G.L., Hemmila M., Krebsbach P.H., Cederna P.S., Wang S.C. Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice. J. Burn Care Res. 2014;35:54–61. doi: 10.1097/BCR.0b013e31829b36d6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Biffl W.L., Moore E.E., Moore F.A., Peterson V.M. Interleukin-6 in the injured patient: marker of injury or mediator of inflammation? Ann. Surg. 1996;224:647–664. doi: 10.1097/00000658-199611000-00009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boucher I., Rich C., Lee A., Marcincin M., Trinkaus-Randall V. The P2Y2 receptor mediates the epithelial injury response and cell migration. Am. J. Physiol. Cell Physiol. 2010;299:C411–C421. doi: 10.1152/ajpcell.00100.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cauwels A., Rogge E., Vandendriessche B., Shiva S., Brouckaert P. Extracellular ATP drives systemic inflammation, tissue damage and mortality. Cell Death Dis. 2014;5:e1102. doi: 10.1038/cddis.2014.70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang S., Linderholm A., Franzi L., Kenyon N., Grasberger H., Harper R. Dual oxidase regulates neutrophil recruitment in allergic airways. Free Radic. Biol. Med. 2013;65:38–46. doi: 10.1016/j.freeradbiomed.2013.06.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen Y., Corriden R., Inoue Y., Yip L., Hashiguchi N., Zinkernagel A., Nizet V., Insel P.A., Junger W.G. ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors. Science. 2006;314:1792–1795. doi: 10.1126/science.1132559. [DOI] [PubMed] [Google Scholar]
  11. Chen L., Wang S., Wang Y., Zhang W., Ma K., Hu C., Zhu H., Liang S., Liu M., Xu N. IL-6 influences the polarization of macrophages and the formation and growth of colorectal tumor. Oncotarget. 2018;9:17443–17454. doi: 10.18632/oncotarget.24734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Church D., Elsayed S., Reid O., Winston B., Lindsay R. Burn wound infections. Clin. Microbiol. Rev. 2006;19:403–434. doi: 10.1128/CMR.19.2.403-434.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. von Dassow G., Verbrugghe K.J.C., Miller A.L., Sider J.R., Bement W.M. Action at a distance during cytokinesis. J. Cell Biol. 2009;187:831–845. doi: 10.1083/jcb.200907090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Van De Groot F., Krijnen P.A.J., Begieneman M.P.V., Ulrich M.M.W., Middelkoop E., Niessen H.W.M. Acute inflammation is persistent locally in burn wounds: a pivotal role for complement and C-reactive protein. J. Burn Care Res. 2009;30:274–280. doi: 10.1097/BCR.0b013e318198a252. [DOI] [PubMed] [Google Scholar]
  15. Deng Q., Yoo S.K., Cavnar P.J., Green J.M., Huttenlocher A. Dual roles for Rac2 in neutrophil motility and active retention in zebrafish hematopoietic tissue. Dev. Cell. 2011;21:735–745. doi: 10.1016/j.devcel.2011.07.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dobson L., Reményi I., Tusnády G.E. CCTOP: a Consensus Constrained TOPology prediction web server. Nucleic Acids Res. 2015;43:W408–W412. doi: 10.1093/nar/gkv451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Drost A.C., Burleson D.G., Cioffi W.G., Jordan B.S., Mason A.D., Pruitt B.A. Plasma cytokines following thermal injury and their relationship with patient mortality, burn size, and time postburn. J. Trauma - Inj. Infect. Crit. Care. 1993;35:335–339. doi: 10.1097/00005373-199309000-00001. [DOI] [PubMed] [Google Scholar]
  18. Estahbanati H.K., Kashani P.P., Ghanaatpisheh F. Frequency of Pseudomonas aeruginosa serotypes in burn wound infections and their resistance to antibiotics. Burns. 2002;28:340–348. doi: 10.1016/s0305-4179(02)00024-4. [DOI] [PubMed] [Google Scholar]
  19. Faggioli L., Merola M., Hiscott J., Furia A., Monese R., Tovey M., Palmieri M. Molecular mechanisms regulating induction of interleukin-6 gene transcription by interferon-gamma. Eur. J. Immunol. 1997;27:3022–3030. doi: 10.1002/eji.1830271140. [DOI] [PubMed] [Google Scholar]
  20. Fernando M.R., Reyes J.L., Iannuzzi J., Leung G., McKay D.M. The pro-inflammatory cytokine, interleukin-6, enhances the polarization of alternatively activated macrophages. PLoS One. 2014;9:e94188. doi: 10.1371/journal.pone.0094188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fielding C.A., McLoughlin R.M., McLeod L., Colmont C.S., Najdovska M., Grail D., Ernst M., Jones S.A., Topley N., Jenkins B.J. IL-6 regulates neutrophil trafficking during acute inflammation via STAT3. J. Immunol. 2008;181:2189–2195. doi: 10.4049/jimmunol.181.3.2189. [DOI] [PubMed] [Google Scholar]
  22. Gauglitz G.G., Song J., Herndon D.N., Finnerty C.C., Boehning D., Barral J.M., Jeschke M.G. Characterization of the inflammatory response during acute and post-acute phases after severe burn. Shock. 2008;30:503–507. doi: 10.1097/SHK.0b013e31816e3373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gault W.J., Enyedi B., Niethammer P. Osmotic surveillance mediates rapid wound closure through nucleotide release. J. Cell Biol. 2014;207:767–782. doi: 10.1083/jcb.201408049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gonzalez M.R., Ducret V., Leoni S., Fleuchot B., Jafari P., Raffoul W., Applegate L.A., Que Y.A., Perron K. Transcriptome analysis of Pseudomonas aeruginosa cultured in human burn wound exudates. Front. Cell. Infect. Microbiol. 2018;8:39. doi: 10.3389/fcimb.2018.00039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gravante G., Palmieri M.B., Esposito G., Delogu D., Santeusanio G., Filingeri V., Montone A. Apoptotic cells are present in ischemic zones of deep partial-thickness burns. J. Burn Care Res. 2006;27:688–693. doi: 10.1097/01.BCR.0000238101.94950.EC. [DOI] [PubMed] [Google Scholar]
  26. Gravante G., Filingeri V., Delogu D., Santeusanio G., Palmieri M.B., Esposito G., Montone A., Sconocchia G. Apoptotic cell death in deep partial thickness burns by coexpression analysis of TUNEL and Fas. Surgery. 2006;139:854–855. doi: 10.1016/j.surg.2006.01.024. [DOI] [PubMed] [Google Scholar]
  27. Hager S., Foldenauer A.C., Rennekampff H.-O., Deisz R., Kopp R., Tenenhaus M., Gernot M., Pallua N. Interleukin-6 serum levels correlate with severity of burn injury but not with gender. J. Burn Care Res. 2018;39:379–386. doi: 10.1097/BCR.0000000000000604. [DOI] [PubMed] [Google Scholar]
  28. Harada Y., Kato Y., Miyaji T., Omote H., Moriyama Y., Hiasa M. Vesicular nucleotide transporter mediates ATP release and migration in neutrophils. J. Biol. Chem. 2018;293:3770–3779. doi: 10.1074/jbc.M117.810168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Heaney M.L., Golde D.W. Soluble cytokine receptors. Blood. 1996;87:847–857. [PubMed] [Google Scholar]
  30. Hendrayani S.-F., Al-Harbi B., Al-Ansari M.M., Silva G., Aboussekhra A. The inflammatory/cancer-related IL-6/STAT3/NF-κB positive feedback loop includes AUF1 and maintains the active state of breast myofibroblasts. Oncotarget. 2016;7:41974–41985. doi: 10.18632/oncotarget.9633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hind L.E., Ingram P.N., Beebe D.J., Huttenlocher A. Interaction with an endothelial lumen increases neutrophil lifetime and motility in response to P aeruginosa. Blood. 2018;132:1818–1828. doi: 10.1182/blood-2018-05-848465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Horiuchi S., Koyanagi Y., Zhou Y., Miyamoto H., Tanaka Y., Waki M., Matsumoto A., Yamamoto M., Yamamoto N. Soluble interleukin-6 receptors released from T cell or granulocyte/macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism. Eur. J. Immunol. 1994;24:1945–1948. doi: 10.1002/eji.1830240837. [DOI] [PubMed] [Google Scholar]
  33. Huang C., Niethammer P. Tissue damage signaling is a prerequisite for protective neutrophil recruitment to microbial infection in zebrafish. Immunity. 2018;48:1006–1013.e6. doi: 10.1016/j.immuni.2018.04.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hurst S.M., Wilkinson T.S., McLoughlin R.M., Jones S., Horiuchi S., Yamamoto N., Rose-John S., Fuller G.M., Topley N., Jones S.A. Il-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation. Immunity. 2001;14:705–714. doi: 10.1016/s1074-7613(01)00151-0. [DOI] [PubMed] [Google Scholar]
  35. Ihara H., Hirukawa K., Goto S., Togari A. ATP-stimulated interleukin-6 synthesis through P2Y receptors on human osteoblasts. Biochem. Biophys. Res. Commun. 2005;326:329–334. doi: 10.1016/j.bbrc.2004.11.037. [DOI] [PubMed] [Google Scholar]
  36. Ipaktchi K., Mattar A., Niederbichler A.D., Hoesel L.M., Vollmannshauser S., Hemmila M.R., Su G.L., Remick D.G., Wang S.C., Arbabi S. Attenuating burn wound inflammatory signaling reduces systemic inflammation and acute lung injury. J. Immunol. 2006;177:8065–8071. doi: 10.4049/jimmunol.177.11.8065. [DOI] [PubMed] [Google Scholar]
  37. Jackson D.M. Second thoughts on the burn wound. J. Trauma. 1969;9:839–862. doi: 10.1097/00005373-196910000-00003. [DOI] [PubMed] [Google Scholar]
  38. Jones S.A., Horiuchi S., Topley N., Yamamoto N., Fuller G.M. The soluble interleukin 6 receptor: mechanisms of production and implications in disease. FASEB J. 2001;15:43–58. doi: 10.1096/fj.99-1003rev. [DOI] [PubMed] [Google Scholar]
  39. Käll L., Krogh A., Sonnhammer E.L.L. A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 2004;338:1027–1036. doi: 10.1016/j.jmb.2004.03.016. [DOI] [PubMed] [Google Scholar]
  40. Karim A.S., Shaum K., Gibson A.L.F. Indeterminate-depth burn injury-exploring the uncertainty. J. Surg. Res. 2019;245:183–197. doi: 10.1016/j.jss.2019.07.063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Karim A.S., Yan A., Ocotl E., Bennett D.D., Wang Z., Kendziorski C., Gibson A.L.F. Discordance between histologic and visual assessment of tissue viability in excised burn wound tissue. Wound Repair Regen. 2019;27:150–161. doi: 10.1111/wrr.12692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kawamura H., Kawamura T., Kanda Y., Kobayashi T., Abo T. Extracellular ATP-stimulated macrophages produce macrophage inflammatory protein-2 which is important for neutrophil migration. Immunology. 2012;136:448–458. doi: 10.1111/j.1365-2567.2012.03601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Klyubin I.V., Kirpichnikova K.M., Gamaley I.A. Hydrogen peroxide-induced chemotaxis of mouse peritoneal neutrophils. Eur. J. Cell Biol. 1996;70:347–351. [PubMed] [Google Scholar]
  44. Kronlage M., Song J., Sorokin L., Isfort K., Schwerdtle T., Leipziger J., Robaye B., Conley P.B., Kim H.-C., Sargin S. Autocrine purinergic receptor signaling is essential for macrophage chemotaxis. Sci. Signal. 2010;3:ra55. doi: 10.1126/scisignal.2000588. [DOI] [PubMed] [Google Scholar]
  45. Lämmermann T., Afonso P.V., Angermann B.R., Wang J.M., Kastenmüller W., Parent C.A., Germain R.N. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature. 2013;498:371–375. doi: 10.1038/nature12175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lateef Z., Stuart G., Jones N., Mercer A., Fleming S., Wise L. The cutaneous inflammatory response to thermal burn injury in a Murine model. Int. J. Mol. Sci. 2019;20:538. doi: 10.3390/ijms20030538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. LeBert D.C., Squirrell J.M., Rindy J., Broadbridge E., Lui Y., Zakrzewska A., Eliceiri K.W., Meijer A.H., Huttenlocher A. Matrix metalloproteinase 9 modulates collagen matrices and wound repair. Development. 2015;142:2136–2146. doi: 10.1242/dev.121160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. LeBert D., Squirrell J.M., Freisinger C., Rindy J., Golenberg N., Frecentese G., Gibson A., Eliceiri K.W., Huttenlocher A. Damage-induced reactive oxygen species regulate vimentin and dynamic collagen-based projections to mediate wound repair. Elife. 2018;7:e30703. doi: 10.7554/eLife.30703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Lee J., Nakagiri T., Oto T., Harada M., Morii E., Shintani Y., Inoue M., Iwakura Y., Miyoshi S., Okumura M. IL-6 amplifier, NF-κB-triggered positive feedback for IL-6 signaling, in grafts is involved in allogeneic rejection responses. J. Immunol. 2012;189:1928–1936. doi: 10.4049/jimmunol.1103613. [DOI] [PubMed] [Google Scholar]
  50. Massena S., Christoffersson G., Hjertström E., Zcharia E., Vlodavsky I., Ausmees N., Rolny C., Li J.P., Phillipson M. Achemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils. Blood. 2010;116:1924–1931. doi: 10.1182/blood-2010-01-266072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Mauer J., Chaurasia B., Goldau J., Vogt M.C., Ruud J., Nguyen K.D., Theurich S., Hausen A.C., Schmitz J., Brönneke H.S. Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin. Nat. Immunol. 2014;15:423–430. doi: 10.1038/ni.2865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Miskolci V., Squirrell J., Rindy J., Vincent W., Sauer J.D., Gibson A., Eliceiri K.W., Huttenlocher A. Distinct inflammatory and wound healing responses to complex caudal fin injuries of larval zebrafish. Elife. 2019;8:e45976. doi: 10.7554/eLife.45976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Nguyen-Jackson H., Panopoulos A.D., Zhang H., Li H.S., Watowich S.S. STAT3 controls the neutrophil migratory response to CXCR2 ligands by direct activation of G-CSF-induced CXCR2 expression and via modulation of CXCR2 signal transduction. Blood. 2010;115:3354–3363. doi: 10.1182/blood-2009-08-240317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Niethammer P., Grabher C., Look A.T., Mitchison T.J. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature. 2009;459:996–999. doi: 10.1038/nature08119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Noack M., Miossec P. Selected cytokine pathways in rheumatoid arthritis. Semin. Immunopathol. 2017;39:365–383. doi: 10.1007/s00281-017-0619-z. [DOI] [PubMed] [Google Scholar]
  56. de Oliveira S., López-Muñoz A., Candel S., Pelegrín P., Calado A., Mulero V. ATP modulates acute inflammation in vivo through dual oxidase 1-derived H2O2 production and NF-κB activation. J. Immunol. 2014;192:5710–5719. doi: 10.4049/jimmunol.1302902. [DOI] [PubMed] [Google Scholar]
  57. de Oliveira S., Rosowski E.E., Huttenlocher A. Neutrophil migration in infection and wound repair: going forward in reverse. Nat. Rev. Immunol. 2016;16:378–391. doi: 10.1038/nri.2016.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Ong Y.S., Samuel M., Song C. Meta-analysis of early excision of burns. Burns. 2006;32:145–150. doi: 10.1016/j.burns.2005.09.005. [DOI] [PubMed] [Google Scholar]
  59. Rani M., Nicholson S.E., Zhang Q., Schwacha M.G. Damage-associated molecular patterns (DAMPs) released after burn are associated with inflammation and monocyte activation. Burns. 2017;43:297–303. doi: 10.1016/j.burns.2016.10.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Rodriguez J.L., Miller C.G., Garner W.L., Till G.O., Guerrero P., Moore N.P., Corridore M., Normolle D.P., Smith D.J., Remick D.G. Correlation of the local and systemic cytokine response with clinical outcome following thermal injury. J. Trauma. 1993;34:684–685. doi: 10.1097/00005373-199305000-00011. [DOI] [PubMed] [Google Scholar]
  61. Schofield Z.V., Woodruff T.M., Halai R., Wu M.C.L., Cooper M.A. Neutrophils - a key component of ischemia-reperfusion injury. Shock. 2013;40:463–470. doi: 10.1097/SHK.0000000000000044. [DOI] [PubMed] [Google Scholar]
  62. Shupp J.W., Nasabzadeh T.J., Rosenthal D.S., Jordan M.H., Fidler P., Jeng J.C. A review of the local pathophysiologic bases of burn wound progression. J. Burn Care Res. 2010;31:849–873. doi: 10.1097/BCR.0b013e3181f93571. [DOI] [PubMed] [Google Scholar]
  63. Singer A.J., McClain S.A., Taira B.R., Guerriero J.L., Zong W. Apoptosis and necrosis in the ischemic zone adjacent to third degree burns. Acad. Emerg. Med. 2008;15:549–554. doi: 10.1111/j.1553-2712.2008.00115.x. [DOI] [PubMed] [Google Scholar]
  64. Sumagin R., Robin A.Z., Nusrat A., Parkos C.A. Transmigrated neutrophils in the intestinal lumen engage ICAM-1 to regulate the epithelial barrier and neutrophil recruitment. Mucosal Immunol. 2014;7:905–915. doi: 10.1038/mi.2013.106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Tanaka T., Narazaki M., Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb. Perspect. Biol. 2014;6:a016295. doi: 10.1101/cshperspect.a016295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Tauzin S., Starnes T.W., Becker F.B., Lam P., Huttenlocher A. Redox and Src family kinase signaling control leukocyte wound attraction and neutrophil reverse migration. J. Cell Biol. 2014;207:589–598. doi: 10.1083/jcb.201408090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Tompkins R.G. Genomics of injury: the glue grant experience. J. Trauma Acute Care Surg. 2015;78:671–686. doi: 10.1097/TA.0000000000000568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Valvis S.M., Waithman J., Wood F.M., Fear M.W., Fear V.S. The immune response to skin trauma is dependent on the etiology of injury in a mouse model of burn and excision. J. Invest. Dermatol. 2015;135:2119–2128. doi: 10.1038/jid.2015.123. [DOI] [PubMed] [Google Scholar]
  69. Wang X., Chen D. Purinergic regulation of neutrophil function. Front. Immunol. 2018;9:399. doi: 10.3389/fimmu.2018.00399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wang J., Kubes P. A reservoir of mature cavity macrophages that can rapidly invade visceral organs to affect tissue repair. Cell. 2016;165:668–678. doi: 10.1016/j.cell.2016.03.009. [DOI] [PubMed] [Google Scholar]
  71. Wittmann C., Chockley P., Singh S.K., Pase L., Lieschke G.J., Grabher C. Hydrogen peroxide in inflammation: messenger, guide, and assassin. Adv. Hematol. 2012;2012:541471. doi: 10.1155/2012/541471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Wright H.L., Cross A.L., Edwards S.W., Moots R.J. Effects of IL-6 and IL-6 blockade on neutrophil function in vitro and in vivo. Rheumatology (Oxford) 2014;53:1321–1331. doi: 10.1093/rheumatology/keu035. [DOI] [PubMed] [Google Scholar]
  73. Xu S., Grande F., Garofalo A., Neamati N. Discovery of a novel orally active small-molecule gp130 inhibitor for the treatment of ovarian cancer. Mol. Cancer Ther. 2013;12:937–949. doi: 10.1158/1535-7163.MCT-12-1082. [DOI] [PubMed] [Google Scholar]
  74. Yan B., Wei J.-J., Yuan Y., Sun R., Li D., Luo J., Liao S.-J., Zhou Y.-H., Shu Y., Wang Q. IL-6 cooperates with G-CSF to induce protumor function of neutrophils in bone marrow by enhancing STAT3 activation. J. Immunol. 2013;190:5882–5893. doi: 10.4049/jimmunol.1201881. [DOI] [PubMed] [Google Scholar]
  75. Yawata H., Yasukawa K., Natsuka S., Murakami M., Yamasaki K., Hibi M., Taga T., Kishimoto T. Structure-function analysis of human IL-6 receptor: dissociation of amino acid residues required for IL-6-binding and for IL-6 signal transduction through gp130. EMBO J. 1993;12:1705–1712. doi: 10.1002/j.1460-2075.1993.tb05815.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Yeh F.L., Lin W.L., Shen H.D., Fang R.H. Changes in circulating levels of interleukin 6 in burned patients. Burns. 1999;25:131–136. doi: 10.1016/s0305-4179(98)00150-8. [DOI] [PubMed] [Google Scholar]
  77. Yoo S.K., Starnes T.W., Deng Q., Huttenlocher A. Lyn is a redox sensor that mediates leukocyte wound attraction in vivo. Nature. 2011;480:109–112. doi: 10.1038/nature10632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Yoo S.K., Lam P., Eichelberg M.R., Zasadil L., Bement W.M., Huttenlocher A. The role of microtubules in neutrophil polarity and migration in live zebrafish. J. Cell Sci. 2012;125:5702–5710. doi: 10.1242/jcs.108324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Zhang Q., Raoof M., Chen Y., Sumi Y., Sursal T., Junger W., Brohi K., Itagaki K., Hauser C.J. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature. 2010;464:104–107. doi: 10.1038/nature08780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Dovi J.V., He L.-K., DiPietro L.A. Accelerated wound closure in neutrophil-depleted mice. J. Leukoc. Biol. 2003;73:448–455. doi: 10.1189/jlb.0802406. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Neutrophil and Macrophage Migration into Burn Tissue, Related to Figure 1

The caudal fin of a larva, expressing Tg(mpeg1:H2B-GFP;lyzc:H2B-mCherry) to label neutrophil (magenta) and macrophage (green) nuclei, was burned. Confocal imaging began within 5 min of wounding and continued for 7 h with 2 min cycles (left panel). Automated tracking (right panel) shows the nuclei (sphere) and tracks (lines) of neutrophils and macrophages (magenta and green, respectively) to the burn tissue cumulatively during the burn injury response. Neutrophils demonstrate a rapid migration to the burn, and an earlier infiltration into the burn tissue, compared with macrophages. Red lines show the burn area at the beginning and end of the video. Scale bar, 100 μm. Time shown as hh:mm:ss:msec.

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Video S2. Animated 3D Reconstruction of Neutrophils, Macrophages and SHG Fibers after Thermal Injury, Related to Figure 2

Concatenated 3D reconstruction video of thermally injured caudal fin, at 4.5 hpb, of a larva expressing GFP-tagged neutrophils followed by a caudal fin of a larvae expressing GFP-tagged macrophages. Each segment highlights the following features: slice view of the brightfield image showing the burn damaged tissue, a surface rendering (gray) of tissue autofluorescence to provide tissue context, with added SHG (magenta) and leukocyte (green) images that are surface rendered. The leukocyte surface rendering was color coded by location relative to the collagen fibers, as detected by SHG (see Methods for description of categorization). Note that categorization was assessed on the original SHG data, not on the surface rendering, which under-represents fine fibers. The surface rendered reconstruction is rotated through 360°. Anterior is to the left. Similar data are presented as still images in Figure 2.

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Video S3. il-6r+/+ Neutrophils Respond Normally to a Burn Injury, Related to Figure 4

Larva, 3 dpf, expressing Tg(mpeg1:H2B-GFP;lyzc:H2B-mCherry) to label macrophage (green) and neutrophil (magenta) nuclei, and carrying homozygous wild-type (+/+) of il-6r were burned, followed by 7 h time-lapse confocal imaging with 4 min cycles (left movie panel). Automated tracking (right movie panel) shows the nuclei (sphere) and cumulative tracks (lines) of neutrophils and macrophages to the burn tissue, in magenta and green, respectively. il-6r+/+ neutrophils and macrophages have a normal response to the burn with early infiltration of neutrophils into burned tissue. Scale bar, 100 μm. Time is show as hh:mm:ss:msec.

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Video S4. il-6R−/− Neutrophils Fail to Respond Normally to a Burn Injury, Related to Figure 4

Larva, 3 dpf, expressing Tg(mpeg1:H2B-GFP;lyzc:H2B-mCherry) to label macrophage (green) and neutrophil (magenta) nuclei and carrying homozygous mutant (−/−) alleles of il-6r were burned, followed by 7 h time-lapse imaging with 4 min cycles (left movie panel). Automated tracking (right movie panel) shows the nuclei (sphere) and cumulative tracks (lines) of neutrophils and macrophages to the burn tissue, in magenta and green, respectively. il-6r−/− larvae present an impaired neutrophil migration phenotype, whereas macrophage migration is unchanged. By 7 hpb only a few neutrophils are in close proximity to the burn. Scale bar, 100 μm. Time is show as hh:mm:ss:msec.

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Document S1. Transparent Methods and Figures S1 and S2
mmc1.pdf (2.3MB, pdf)
Table S1. Additional Statistical Information, Related to Graphs in Figures 1 through 5

Excel file providing tables with additional statistical information related to graphs presented in all main figures.

mmc2.xlsx (30.5KB, xlsx)
Data S1. Full Gene List, Gene Analysis, and ssGSEA Scores, Related to Figure 4

Human gene data and analysis for neutrophil- and IL-6-related gene sets.

mmc3.xlsx (33.3KB, xlsx)

Data Availability Statement

Original/source data for all figures published in this study are available from the lead contact upon request. The sequences generated in this study are available at GenBank (Accession number MW067024 for il-6r isoform #1 and MW067025 for il-6r isoform #2). This study did not generate any new code.

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