An anti-inflammatory activation sequence governs macrophage transcriptional dynamics during tissue injury in zebrafish

Macrophages are essential for tissue repair and regeneration. Yet, the molecular programs, as well as the timing of their activation during and after tissue injury are poorly defined. Using a high spatio-temporal resolution single cell analysis of macrophages coupled with live imaging after sensory hair cell death in zebrafish, we find that the same population of macrophages transitions through a sequence of three major anti-inflammatory activation states. Macrophages first show a signature of glucocorticoid activation, then IL-10 signaling and finally the induction of oxidative phosphorylation by IL-4/Polyamine signaling. Importantly, loss-of-function of glucocorticoid and IL-10 signaling shows that each step of the sequence is independently activated. Lastly, we show that IL-10 and IL-4 signaling act synergistically to promote synaptogenesis between hair cells and efferent neurons during regeneration. Our results show that macrophages, in addition to a switch from M1 to M2, sequentially and independently transition though three anti-inflammatory pathways in vivo during tissue injury in a regenerating organ.

timing of the transitions as well as the genetic programs triggered by each activation state during tissue regeneration will be unvaluable to design targeted immunomodulatory therapies.
Here we took advantage of the rapid sensory hair cell (HC) regeneration that occurs in the 60 zebrafish lateral line (20,21) to uncover the molecular "What" (signals and genetic program) and "When" (sequence of activation) that control macrophage activity and dynamics during injury of a regenerating organ in zebrafish. This regenerating lateral line sensory system is powerful for several reasons: (i) antibiotic/neomycin treatment induces rapid HC death within minutes (21); (ii) HC regeneration occurs within hours and the first new pair of regenerated HCs is detected five 65 hours after neomycin treatment (22); and (iii) the optical clarity of the larvae allows us to follow the behavioral dynamics of macrophages using confocal microscopy at high spatio-temporal resolution. Together, these characteristics enabled us to interrogate the transcriptional and behavioral dynamics of macrophage activation at an unprecedented spatio-temporal resolution.

The same population of effector macrophages resolves inflammation within five hours in neuromasts
Tissue injury triggers an inflammatory response that involves the recruitment of macrophages to the injury site. These macrophages will be responsible for clearing cellular debris, remodeling the extracellular matrix and in some cases, provide signals to the tissue stem cells to start the repair 75 process (23). We designated this population as 'effector macrophages' (24). It is not clear if effectors represent a single, or several macrophage populations. A recent study of muscle regeneration in zebrafish showed that two populations of macrophages were dynamically regulated after injury (25). One population reacted rapidly to tissue injury and left the damaged area within twenty-four hours, while a second population stayed in contact with the muscle stem cells for a 80 longer period. Thus, the first step in understanding how effectors are molecularly regulated is to describe their dynamic after HC death. To follow both macrophages and HCs over time we study Tg(mpeg:GFP) and Tg(she:lckmScarletI) transgenic larvae that label macrophages and neuromast cells, respectively (Fig1A). Neuromast HCs are superficially located all along the larval body (Fig1A, movieS1). Treatment with neomycin for 30 min rapidly kills HCs by caspase-independent 85 cell death (movieS2) (26). Macrophages start phagocytosing dead HCs as early as 15 min after the first cells die (movieS2-S3) (27). To describe the dynamics of effector macrophages from the time they enter the neuromast to when they leave during HC regeneration, we performed a macrophage recruitment assay. We treated the larvae with neomycin for 30 min and imaged both macrophages and neuromasts 1H, 3H, 5H and 7H after treatment (Fig1B, movieS4). Quantification of the 90 number of macrophages surrounding and inside the neuromasts shows a rapid recruitment of effectors with a peak at 1H after neomycin (Fig1C-D). Subsequently, cells progressively return to homeostatic levels which they reach at 7H after neomycin treatment. Thus, there is a window of five hours when effector macrophages interact with neuromasts, which coincides with the appearance of the first regenerated HCs (22).

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To decipher if effector macrophages represent a specific population, we performed time-lapse recordings of macrophages and neuromasts during neomycin treatment and homeostasis in a large area of the larval trunk to identify where the effectors reside prior to HC death (movieS5).
Quantification of the location of effector and non-effector macrophages before HC death shows that effectors are located closer to the neuromast than non-effectors (71µm+/-14µm vs 175µm+/-100 15µm, respectively) (Fig1E). In contrast to non-effectors, effectors show a rapid, directional migration toward the neuromasts in response to HC death (Fig1F). Interestingly, non-effector cells show an increase in non-directed cell velocity after neomycin treatment suggesting that HC death initially triggers a global injury response (Fig1G).
To assess if a single or multiple populations of effectors are recruited to neuromasts during the 105 five hour window, we specifically photoconverted effector cells in neuromasts 1H after neomycin and quantified the ratio of photoconverted vs non-photoconverted macrophages 3H and 5H after HC death (Fig1H). Photoconverted cells represent an average of 98%+/-2% and 100% of the macrophages inside neuromasts at 3H and 5H after HC death, respectively, demonstrating that a single population of effectors is recruited during the five hour window (Fig1I).

High temporal resolution scRNA-seq identifies a population of effector macrophages
To molecularly characterize all macrophage populations, identify the effector population and characterize their activation sequence and underlying molecular program over the course of HC death, we performed a scRNA-seq time course of Tg(mpeg:GFP) transgenic larvae. We dissociated 600 5dpf larvae each during homeostasis, and 1H, 3H and 5H after a 30min neomycin  clusters and focused our analysis on these cells as effector macrophages.

An anti-inflammatory activation sequence in macrophages during HC death
To identify the core molecular programs driving the effector macrophages activation states at each timepoint, we compared the differentially expressed genes of the three clusters and analyzed the genes that were shared (TableS2). We detected that at the fifteen minutes neomycin timepoint the Glucocorticoid (GR) pathway targets (arl5c, jdp2b, dusp1, klf9, tsc22d3, nfkbiab, sgk1, fkbp5) very few genes are found specifically upregulated at the 5H timepoint (Fig3A, S4A). grn2, a progranulin growth factor regulator of the anti-inflammatory macrophage phenotype (55) is highly upregulated. Interestingly, in grn knock-out mice, muscle injury leads to a persistence of macrophages at the injury site suggesting a role for grn in regulating macrophage dynamics (56).

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Altogether, this analysis reveals a linear sequence of macrophage anti-inflammatory activation immediately after HC death starting with Glucocorticoid signaling, followed by IL10 signaling, and lastly a combination of Polyamine and IL4 signaling, which induces oxidative phosphorylation at the transcriptional level. To our knowledge, this is the first report of an in vivo linear sequence of three major anti-inflammatory activation pathways after injury. This suggests that in addition 210 to a transition from a pro-inflammatory M1 to an anti-inflammatory M2 state, the same macrophages transition through different anti-inflammatory states to potentially regulate their dynamics/activity.

Each anti-inflammatory state is independently activated
The discovery of this linear anti-inflammatory sequence of effector macrophage activation raises 215 the interesting question if epistatic relationships between Glucocorticoid signaling and IL10 signaling and between IL10 signaling and IL4/Polyamine signaling exist.
To address this question, we first inhibited Glucocorticoid signaling during HC death using the GR inhibitor RU486. HRC-FISH for the GR target dusp1 confirmed that a 10µM treatment with RU486 was efficient in blocking GR activation (Fig4A). HCR-FISH for il10ra at the 15 min 220 timepoint, as well as for the IL10 signaling target gene fgl2a at the 1H timepoint showed no downregulation of these genes in effector macrophages after RU486 treatment (Fig4B-E). This demonstrates that GR activation is not required for IL10 activation in effector macrophages.
To test for a possible epistatic relationship between IL10 signaling and the following activation state (Polyamine + IL4 signaling), we generated a mutant for il10ra by deleting the promoter is likely not sufficient to induce proper tissue regeneration and a sequential and independent activation of the three pathways might be required.

Discussion
Macrophage molecular activation regulates the dynamic and activity of these phagocytes and modulation of their activation states and their dynamic can have dramatic effects on tissue repair and regeneration (57). Here, using a high spatio-temporal resolution analysis of macrophage activation during HC regeneration, we provide evidence that the same population of macrophages is sequentially and independently activated by three major anti-inflammatory pathways.
It is now broadly documented that macrophages adopt a pro-inflammatory activation state immediately after injury (19, 28, 58-60). The role for this pro-inflammatory phase is mainly to The anti-inflammatory role of the GR pathway has been extensively documented (39, [62][63][64]. It can both directly inhibit pro-inflammatory gene transcription by direct binding of the GR receptor to their enhancers/promoters or by tethering the pro-inflammatory activators AP-1 and Nfkb (64).

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GR also triggers a rapid and large-scale chromatin unwinding, potentially creating a permissive environment for drastic changes in gene expression required for macrophage activity (65). Therefore, the strong and transient activation of the GR pathway immediately as the first HCs start to die likely turns off the transcription of pro-inflammatory cytokines.
The short phase of GR activation is immediately followed by IL10 signaling activation and a 270 subsequent transition to a IL4/Polyamine activation state that induces oxidative phosphorylation.
A recent study in mouse Bone Marrow Derived Macrophages (BMDM) showed that part of the IL10 anti-inflammatory function is to inhibit glycolysis while promoting oxidative phosphorylation after treatment with LPS, which mimics a bacterial infection (66). Our il10ra mutant analysis demonstrates that lack of IL10 signaling after HC death does not affect the 275 induction of oxidative phosphorylation. Co-stimulation of mouse BMDM with LPS and IL10 also leads to the upregulation of the IL4 receptor (67) whereas the loss of IL10 in zebrafish does not affect the induction of il4r.1. These discrepancies suggest that IL10 and IL4 signaling are differently regulated in response to bacterial infection versus tissue injury.
The IL4/OXPHOS state is characteristic for wound healing macrophages and responsible for the 280 last step of inflammation resolution (2, [68][69][70]. In addition, the induction of the pro-repair gene manf, which is required for retina regeneration (54), suggests that at the 3H timepoint macrophages transition to a wound-healing state. Altogether, our in vivo data demonstrate the sequential and independent transition of effector macrophages through three major anti-inflammatory states. This finding has important implications for the design of targeted immunomodulatory therapies.   Tables S1 to S3