STING coordinates resolution of inflammation during wound repair by modulating macrophage trafficking through STAT3

doi:10.21203/rs.3.rs-3609946/v1

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STING coordinates resolution of inflammation during wound repair by modulating macrophage trafficking through STAT3

https://doi.org/10.21203/rs.3.rs-3609946/v1

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Efficient cutaneous wound healing requires a coordinated transition between inflammatory phases mediated by dynamic changes in leukocyte subset populations. Here, we identify STING as a key innate immune mediator governing timely resolution of inflammation by regulating macrophage dynamics during skin repair. Using a mouse model, we show STING deficiency caused delayed wound closure associated with abnormal persistence of TNFα⁺ leukocytes. This resulted from the failed contraction of neutrophils coupled with impaired macrophage recruitment. STING controlled the trafficking of bone marrow myeloid cells into blood and wounds, intrinsically enhancing macrophage migratory capacity through STAT3 activation. Specifically, STING modulated the production of monocyte chemokines and their receptors CCR2/CCR5 to enable efficient egress and wound infiltration. Consequently, disrupted systemic and local STING-STAT3-chemokine signaling combine to delay macrophage influx and prevent timely neutrophil-to-macrophage inflammatory phase transition. This study elucidates STING as a critical rheostat tuning macrophage responses through STAT3 to orchestrate inflammatory resolution necessary for efficient wound healing. Our findings have broad implications for targeting STING therapeutically in both regenerative medicine and inflammatory disease contexts.

Biological sciences/Immunology/Inflammation

Health sciences/Diseases/Immunological disorders

Wound healing

Inflammation

Macrophages

STING

STAT3

Wound healing is a highly coordinated process involving complex interactions between keratinocytes, fibroblasts, endothelial cells, nerves and immune cells to restore the integrity and function of damaged tissues [1]. This process can be conceptually divided into overlapping inflammatory, proliferative and remodeling phases. Dysregulation at any step can lead to chronic non-healing wounds, which represent a major healthcare burden affecting millions worldwide [2]. Therefore, understanding the molecular mechanisms governing efficient tissue repair is crucial for identifying therapeutic targets.

The inflammatory phase initiating immediately after injury is critical for clearing debris and pathogens and orchestrating downstream events through cytokine and chemokine production [3]. Neutrophils infiltrate first and predominate during early inflammation. After debris clearance, neutrophil levels subside as macrophages become the primary immune subsets [4]. This timely switch enables transition from pro-inflammatory to pro-resolving phenotypes regulating granulation tissue formation, angiogenesis and collagen deposition [5]. Consequently, delayed contraction of inflammation due to aberrant leukocyte dynamics often underlies poor healing outcomes.

Pattern recognition receptors (PRRs) are key innate immune sensors regulating inflammation and cytokine production during wound healing [6]. In particular, the cytosolic DNA sensor STING (stimulator of interferon genes) represents an emerging player. STING signaling canonically leads to type I interferon (IFN) induction downstream of DNA pathogen detection [7]. However, STING also regulates IFN-independent immune processes, including production of numerous cytokines and chemokines relevant to tissue repair [8, 9]. Consistently, recent evidence indicates STING promotes efficient healing in multiple injury models through regulating inflammation. In corneal wounds, STING activation improved re-epithelialization and stromal remodeling, effects attributed to enhanced neutrophil recruitment early post-injury followed by accelerated transition to a macrophage-predominant state [10, 11]. STING signaling has also been shown to limit excessive inflammation in chemical burns while promoting angiogenesis [9, 12]. These findings highlight the importance of STING in timely orchestrating leukocyte responses during tissue repair. However, the mechanisms underlying STING-mediated regulation of myeloid cell dynamics and the functional relevance to healing outcomes remain incompletely understood. Notably, STING signaling appears highly context-dependent, capable of either augmenting or suppressing inflammation depending on cell type and activation state [13]. Potentially detrimental effects of unrestrained STING activity are highlighted in inflammatory disease models, wherein genetic deletion or pharmaceutical inhibition of STING protected against tissue damage [8, 14]. This contrasts with the largely beneficial impacts observed in injury models. Therefore, extensive further research is required to elucidate the complex and nuanced mechanisms governing STING functionality during wound healing.

Myeloid cells including neutrophils and macrophages have emerged as major targets and mediators of STING signaling during tissue repair processes. In lung injury, STING was shown to suppress neutrophil-driven inflammation while promoting protective macrophage polarization and fibrosis resolution [15]. STING also limited macrophage inflammation in models of sterile liver damage [16]. Consistently, STING deficiency caused excessive leukocyte accumulation and chemokine induction in chemical skin wounds. However, the healing relevance of these STING-regulated myeloid responses remains unclear, as macroscopic repair outcomes were not investigated. Further evidence suggests STING controls myeloid trafficking and infiltration indirectly by regulating chemokine production from other cell types like T-lymphocytes [17, 18]. However, potential cell-intrinsic STING signaling in myeloid cells themselves has not been defined. Dissecting both extrinsic and intrinsic STING functionality in myeloid subsets is key for comprehensively understanding its coordinated regulation of wound inflammation. Efforts are also needed to delineate the upstream signaling pathways through which STING controls myeloid chemokine induction relevant to healing.

Macrophage recruitment to wound sites relies heavily on CCR2-CCL2 chemokine receptor-ligand interactions [19, 20]. CCL2 production from epithelial cells and fibroblasts stimulates mobilization of Ly6C^highCCR2⁺ monocytes from bone marrow which give rise to wound macrophages. Accordingly, deficiencies in CCL2 or CCR2 severely impair monocyte trafficking and cause healing impairments in multiple models [21, 22]. Whether STING signaling intersects with this critical chemokine axis to control timely macrophage influx during repair has not been explored. The transcription factor STAT3 is emerging as a key node governing macrophage migration and chemokine production downstream of various growth factors and cytokines [23]. In tumors, STAT3 drives CCL2 expression promoting macrophage infiltration and growth [24]. During myocardial infarction, STAT3 deletion suppressed Ly6C^high monocyte recruitment to wounds [25]. STAT3 also directly increased macrophage motility via upregulating traction force pathways [26]. Intriguingly, STING was shown to activate STAT3 signaling in dendritic cells. However, it remains unknown whether STING similarly engages STAT3 to regulate macrophage functions in the context of tissue repair.

In summary, efficient healing requires coordinated resolution of inflammation mediated by well-orchestrated changes in leukocyte subset dynamics. STING has emerged as a central innate immune mediator regulating myeloid cell responses during tissue repair. However, the mechanisms by which STING controls key macrophage phenotypes and trafficking pathways relevant to efficient healing remain poorly defined. Elucidating these processes is crucial to better understand STING functionality in context-dependent promotion versus inhibition of inflammation during wound healing. In this study, we investigate the role of STING in mediating timely resolution of inflammation during cutaneous wound repair. Using a mouse model deficient in STING, we demonstrate aberrant persistence of inflammatory macrophages causes delayed wound healing. Mechanistically, we reveal STING regulates both systemic macrophage mobilization and cell-intrinsic migratory capacity through modulating monocyte chemokine receptor/ligand axes via STAT3 signaling. Collectively, our findings elucidate STING as a critical rheostat controlling myeloid cell dynamics through STAT3 activation to enable efficient transition between inflammatory phases necessary for productive tissue repair.

1.Impaired inflammation kinetics underlie delayed repair in STING-deficient wounds

Utilizing a mouse model of impaired wound contraction, we uncovered a critical role for STING in timely resolution of inflammation during skin repair. Macroscopically, the rate of wound closure was substantially delayed in Sting^−/− mice compared to wildtype controls at days 2, 4, 6 and 8 post-wounding (Fig. 1a). Histological analyses further confirmed that the length of the neo-epithelium tongue was markedly shorter in wounds of Sting^−/− mice versus wildtype mice at days 2, 4 and 6 post-injury. Additionally, granulation tissue formation was inferior in Sting^−/− wounds relative to wildtype wounds at these same time points (Fig. S1a-b).

To elucidate the mechanisms contributing to aberrant wound healing in STING-deficient mice, we performed a detailed characterization of inflammation kinetics. First, we analyzed the wound recruitment dynamics of pro-inflammatory TNF-α⁺ and IL-6⁺ leukocytes, which modulate debris clearance and tissue remodeling. In wildtype wounds, TNF-α⁺ cell numbers rapidly declined with healing progression, consistent with timely inflammation resolution. However, TNF-α⁺ cells displayed abnormal persistence in Sting^−/− wounds, with significantly fewer cells at early timepoints (days 1–2) but elevated numbers at later timepoints (days 4–6) compared to wildtype controls. This reveals failed contraction of TNF-α⁺ cell accumulation in Sting^−/− wounds, indicative of delayed inflammation termination (Fig. 1b).

Interestingly, IL-6⁺ cell numbers in Sting^−/− wounds showed opposite trend with TNFα⁺ cell in WT wounds at early stages. Sting^−/− wounds showed increased IL-6⁺ cells compared with WT wounds. However, no significant differences were evident at later timepoints (days 4–6) (Fig. 1c).

Intriguingly, expression of the anti-inflammatory cytokine IL-10 showed an opposite pattern compared to pro-inflammatory mediators. Both immunohistochemistry and immunoblotting revealed significantly higher IL-10 levels in Sting^−/− versus wildtype wounds at day 2 post-injury. However, no genotype-dependent differences were detected at later timepoints (days 4–6) (Fig. 1d).

Collectively, these findings demonstrate that timely inflammation resolution is disrupted in STING-deficient wounds, with attenuated early TNF-α⁺ and IL-6⁺ leukocyte responses. However, anti-inflammatory IL-10 induction appears to occur prematurely.

2. Reduced macrophage infiltration causes delayed inflammation resolution in STING-deficient wounds

Based on the aberrant inflammatory phenotypes observed in Sting^−/− wounds, we hypothesized that altered myeloid cell dynamics may underlie failed inflammation resolution. We first quantified total CD11b⁺ myeloid cell infiltration into wounds by flow cytometry and immunohistochemistry. Compared to wildtype controls, Sting^−/− wounds contained significantly fewer CD11b⁺ myeloid cells at day 1 and day 2 post-injury. However, by day 6, CD11b⁺ cell numbers were conversely higher in Sting^−/− wounds (Fig. 2a-b).

To determine the specific myeloid subsets impacted by STING deficiency, we performed further flow cytometry analyzing CD11b⁺ subpopulations. We found that the early myeloid response in wildtype wounds was dominated by Ly6G⁺ neutrophils, which constituted ~ 70% of CD11b⁺ cells and peaked in number on day 1 post-injury. Following their role in debris clearance, Ly6G⁺ neutrophils rapidly declined by day 4 in concordance with resolving inflammation. Concurrently, the Ly6G⁻ monocyte/macrophage subset dynamically expanded from 20–60% of CD11b⁺ cells over this period as they transitioned to tissue remodeling roles. In contrast, maximal neutrophil infiltration was substantially delayed until day 4 in Sting^−/− wounds. While Ly6G⁺ neutrophil numbers similarly declined by day 6, the Ly6G⁻ monocyte/macrophage population strikingly failed to expand. Instead, this subset remained static at the basal uninjured proportion of about 35%. These data reveal a specific defect in timely conversion from neutrophil to monocyte/macrophage driven myeloid influx in STING-deficient wounds (Fig. 2c). We further confirmed impaired macrophage recruitment by quantifying the macrophage marker F4/80 via immunohistochemistry and immunoblotting. Both assays showed significantly reduced F4/80⁺ cell numbers and protein levels in Sting^−/− versus wildtype wounds at 2, 4, and 6 days post-injury (Fig. 2d-e).

In summary, these findings demonstrate that loss of STING signaling decouples timely switching between neutrophil and macrophage phases of the wound myeloid response. Consequently, reduced macrophage recruitment likely contributes directly to delayed inflammation resolution and healing impairment in STING-deficient mice.

3. STING orchestrates systemic myeloid cell mobilization during wound healing

Thus far, our findings indicate defective regulation of local myeloid cell responses in Sting^−/− wounds. We next investigated whether similar disruption of macrophage dynamics occurs systemically following injury. First, we analyzed CD11b⁺ populations in peripheral blood mononuclear cells (PBMCs) over the wound healing time course. Mirroring the local response, PBMCs from Sting^−/− mice contained reduced CD11b⁺ cells compared to wildtype controls at days 1–2 post-wounding. However, this difference inverted by day 6, with elevated CD11b⁺ cells in Sting^−/− PBMCs (Fig. 3a).

To determine the specific circulating subsets impacted by STING deficiency, we examined PBMCs in unchallenged and wounded mice. In wildtype PBMCs, both Ly6G⁺ neutrophils and Ly6G⁻ monocytes/macrophages concurrently peaked at day 1 before declining. This reveals coordinated mobilization into circulation following injury. In contrast, Sting^−/− mice displayed delayed expansion of Ly6G⁺ neutrophils, which instead peaked at day 6 rather than day 1. Monocyte/macrophage numbers were also reduced in Sting^−/− PBMCs and failed to peak over the time course examined (Fig. 3a).

We next investigated bone marrow myeloid cells to identify the origins of these disrupted blood trafficking dynamics. In wildtype marrow, total CD11b⁺ cells decreased substantially by day 2 post-wounding as myeloid cells mobilized into circulation. CD11b⁺ populations then recovered by day 4 and day 6 as myelopoiesis re-established homeostatic levels. However, CD11b⁺ cells in Sting^−/− marrow remained static or even expanded during the course analyzed (Fig. 3b).

Analysis of wildtype marrow subsets revealed synchronized nadirs in both neutrophils and monocytes/macrophages at day 2, reflective of simultaneous trafficking into blood. In contrast, Sting^−/− marrow displayed delayed and disjointed neutrophil and monocyte dynamics. Specifically, maximal neutrophil depletion was delayed until day 6, while monocyte egress failed to occur (Fig. 3b).

In summary, these findings reveal STING coordinates timely trafficking of myeloid cells from bone marrow into circulation following wounding. Disruption of this process provides a likely upstream source of the defective circulating and wound-infiltrating myeloid responses in STING-deficient mice.

4. STING regulates macrophage migratory capacity through cell-intrinsic signaling

Thus far, our data indicate STING orchestrates macrophage dynamics through systemic control of mobilization from bone marrow reservoirs. We next investigated whether STING also directly regulates macrophage migratory capacity through cell-intrinsic signaling.

First, we performed in vitro Transwell migration assays using bone marrow-derived macrophages (BMDMs). Stimulation with the STING agonist DMXAA significantly enhanced the migration of wildtype but not Sting^−/− BMDMs (Fig. 4a). This demonstrates that STING activation can directly increase macrophage motility independent of systemic factors. We next developed an in vivo system to track macrophage recruitment following adoptive transfer into wound-bearing mice (Fig. 4b). Briefly, BMDMs from wildtype or Sting^−/− mice were pre-stained with the fluorescent membrane dye DiD and injected intravenously into recipient mice. Immunofluorescence and flow cytometry performed 3 days after wounding revealed significantly fewer recruited Sting^−/− BMDMs in wounds compared to wildtype controls (Fig. 4c-d).

Together, these in vitro and in vivo findings indicate STING signaling intrinsically promotes macrophage migration capacity downstream of wound-induced activation. Augmentation of mobility provides a complementary cellular mechanism, along with coordinated mobilization, by which STING signaling optimizes timely macrophage infiltration to mediate efficient inflammation resolution.

5. STING signaling regulates chemokine pathways essential for monocyte recruitment

After identifying decreased macrophage infiltration as a cause of delayed wound healing in Sting^−/− mice, we sought to define the underlying molecular mechanisms governing their trafficking. Monocyte egress from bone marrow and recruitment to tissues relies on chemokine receptor/ligand axes, particularly CCR2/CCL2.

We first quantified Ly6C^highCCR2⁺ inflammatory monocytes, which predominantly give rise to wound macrophages. Flow cytometry revealed significantly fewer Ly6C^highCCR2⁺ cells in Sting^−/− versus wildtype wounds at days 1, 2, and 4 post-injury (Fig. 5a). A similar deficit was observed in circulation, with reduced Ly6C^highCCR2⁺ monocyte levels in naive Sting^−/− mice. After wounding, Ly6C^highCCR2⁺ monocytes substantially decreased in both genotypes by day 2 coinciding with recruitment to wounds before recovering by day 6. However, the kinetics were blunted in Sting^−/− mice (Fig. 5b).

Extending our analyses to bone marrow, we found Ly6C^highCCR2⁺ monocytes displayed coordinated regulation in wildtype mice. Bone marrow Ly6C^highCCR2⁺ numbers decreased by day 2 post-injury, reflecting efficient egress. They remained depleted until day 4 before recovering by day 6 once mobilization was complete. In contrast, Ly6C^highCCR2⁺ populations in Sting^−/− marrow failed to decline after wounding and instead expanded. This reveals retention of Ly6C^highCCR2⁺ monocytes in Sting^−/− marrow rather than timely release into circulation (Fig. 5c).

Based on these findings, we hypothesized STING regulates monocyte dynamics by controlling chemokine production. Indeed, stimulation of wildtype BMDMs with the STING agonist DMXAA induced robust upregulation of the key monocyte chemokines CCL2 and CCL5 (Fig. 5d-f), along with their respective receptors CCR2 and CCR5 (Fig. 5g-h). However, chemokine induction was completely abrogated in Sting^−/− BMDMs.

In summary, these data demonstrate STING orchestrates efficient monocyte mobilization during wound healing through timely stimulation of chemokine cues essential for promoting their egress and recruitment. Dysregulation of this STING-chemokine signaling axis likely causes impaired monocyte trafficking and subsequent macrophage deficits observed in Sting^−/− mice.

6. STING promotes macrophage chemokine production and migration via STAT3 activation

After determining STING regulates myeloid cell dynamics during wound healing through chemokine pathway modulation, we next investigated the underlying signaling mechanisms. We focused on STAT3, a transcription factor controlling immune cell trafficking. Stimulation of wildtype BMDMs with DMXAA led to rapid and sustained STAT3 phosphorylation, indicative of activation. As expected, STAT3 activation was completely absent in Sting^−/− BMDMs (Fig. 6a). We also found that CCL2 also activated STAT3 specifically in wildtype BMDMs (Fig. 6b).

To directly assess the role of STAT3 in STING-mediated chemokine induction relevant to wound healing, we used the small molecule STAT3 inhibitor WP1066. Indeed, WP1066 prevented DMXAA-stimulated upregulation of CCR2 (Fig. 6c), CCR5 (Fig. 6d), and CCL2 (Fig. 6e) expression in wildtype BMDMs. WP1066 also abrogated the promigratory effects of DMXAA observed in Transwell assays (Fig. 6f).

Collectively, these results mechanistically link STING to enhanced macrophage chemokine production and migration through STAT3 signaling pathway activation.

Inflammation is a crucial initial phase of wound healing, but timely resolution is equally important to prevent detrimental effects of sustained leukocyte activity and enable transition to later reparative stages [27–29]. Using a mouse model of impaired contraction, we identify STING as a critical regulator of inflammation kinetics during skin repair. STING deficiency caused delayed closure associated with aberrant persistence of TNF-α⁺ leukocytes and reduced early IL-6⁺ cells. Mechanistically, these phenotypes resulted from disrupted macrophage dynamics, including impaired trafficking from bone marrow reservoirs and decreased wound recruitment. STING intrinsically promoted macrophage chemokine production and migration through STAT3 activation. Together, these defects in systemic and local STING-mediated control of macrophage responses provide a cellular basis for failed inflammation termination and subsequent healing impairment.

Neutrophils and macrophages have dichotomous roles during wound healing [30, 31]. Neutrophils predominate the early inflammatory phase and clear debris via phagocytosis and protease release [32]. As their numbers decline, macrophages expand and transition towards angiogenesis promotion, extracellular matrix remodeling and granulation tissue formation [33, 34]. This timely switch in myeloid populations enables productive progression between inflammatory and proliferative phases. Consequently, dysregulation of leukocyte subset dynamics often critically impacts repair outcomes. For example, delayed neutrophil apoptosis caused sustained inflammation and poor healing in diabetic wounds. Conversely, accelerating macrophage turnover using CSF-1 blockade improved collagen deposition [35–37]. Our findings now identify STING as a key innate immune regulator coupling dynamic changes in wound neutrophil and macrophage populations.

We demonstrate peak neutrophil infiltration was substantially delayed in STING-deficient wounds. This was associated with reduced early TNF-α⁺ leukocytes, consistent with the key role of neutrophil-derived TNF-α in initiating inflammation. However, TNF-α⁺ cells then displayed aberrant persistence at later timepoints in STING-deficient wounds, indicative of sustained inflammatory phenotypes. IL-6 induction occurred independently of STING signaling during the inflammatory phase. While IL-6 synergizes with TNF-α to amplify leukocyte recruitment, it transitions to an anti-inflammatory role in remodeling by restricting neutrophil influx. The relative preservation of early IL-6⁺ cells despite reduced TNF-α⁺ numbers may represent attempted compensatory acceleration of inflammation resolution in STING-deficient wounds. However, this was insufficient to rescue timely closure.

In contrast to the pro-inflammatory defects, we found anti-inflammatory IL-10 upregulation occurred prematurely in STING-deficient wounds. This likely also represents compensation for reduced TNFα⁺ and IL-6⁺ leukocytes to limit excessive inflammation due to failed STING-mediated contraction. IL-10 induction is a hallmark of leukocyte phenotypic switching towards resolution. Accelerated IL-10 production has been associated with improved healing. Sustained TNF-α⁺ cells despite heightened IL-10 levels in STING-deficient wounds indicates IL-10 upregulation alone was insufficient to overcome intrinsic defects in inflammation termination.

Extending our analyses to macrophage dynamics, we found reduced early CD11b⁺ myeloid infiltration in STING-deficient wounds associated with the TNFα⁺ cell defects. However, CD11b⁺ cells conversely accumulated by late stages, consistent with failed resolution. Further dissection of specific subsets revealed macrophages failed to expand and replace neutrophils in the absence of STING. Consequently, delayed neutrophil apoptosis coupled with impaired macrophage recruitment provides a likely mechanistic basis for sustained inflammation and healing impairment.

Neutrophil clearance relies in part on macrophage efferocytosis and phagocytosis. Reduced apoptotic cell uptake by macrophages can delay inflammation resolution [38]. As STING promotes macrophage phagocytic capacity, impairment of this process may have additionally contributed to neutrophil persistence. Reciprocally, failed macrophage influx may further augment neutrophil survival by preventing phagocytic removal. This reveals ongoing bidirectional crosstalk between disrupted myeloid populations combining to inhibit timely inflammation termination in STING-deficient wounds.

In addition to regulating local wound leukocyte responses, we found STING controlled systemic myeloid cell trafficking. Following injury, neutrophils and Ly6C^high inflammatory monocytes concurrently expanded in circulation to supply wound infiltration [21, 39]. However, this coordinated response was decoupled in STING-deficient mice. Circulating monocytes critically give rise to wound macrophages, highlighting upstream dysregulation of mobilization as a likely contributor to impaired recruitment.

Analyses of bone marrow populations provided key insights into the origins of these disrupted circulation dynamics. In wildtype marrow, both neutrophils and monocytes displayed synchronized nadirs as they egressed after wounding before recovering once mobilization was complete. In contrast, STING deficiency caused delayed and exaggerated neutrophil depletion uncoupled from monocyte egress. This reveals neutrophil mobilization became independent of efficient macrophage release. Consequently, failed coordinated myeloid trafficking provides a basis for impaired macrophage influx and sustained neutrophil presence combining to delay inflammation resolution.

Intriguingly, STING regulation of macrophage dynamics showed both extrinsic and intrinsic components. Along with disrupting systemic trafficking pathways, we found STING intrinsically promoted macrophage migration in response to wound signals. STING-deficient macrophages displayed cell-autonomous defects in chemokine-induced migration and wound recruitment. This identifies direct STING signaling as a amplifier of macrophage motility and influx. Coordinated enhancement of both mobilization and intrinsic migratory capacity enables STING to exert tight control over macrophage trafficking and subsequent inflammation resolution.

To elucidate the molecular mechanisms underlying STING-mediated regulation of macrophage dynamics, we identified chemokine pathway modulation as a key process. Leukocyte migration relies heavily on chemokine receptor-ligand interactions [40, 41]. The CCR2-CCL2 axis is particularly important for mediating Ly6C^high monocyte egress and macrophage recruitment during inflammation and wound healing [20, 42, 43]. We found STING deficiency impaired induction of multiple monocyte-relevant chemokines in macrophages. STING-deficient mice also showed reduced wound and circulating CCR2⁺ monocytes associated with failed bone marrow egress. This mechanistically links STING to regulation of the CCR2-CCL2 chemokine axis essential for timely monocyte trafficking and macrophage wound infiltration.

Finally, we identified STAT3 activation as the key signaling mechanism through which STING controls macrophage chemokine production and migration. STAT3 is emerging as a crucial regulator of myeloid cell dynamics through transcriptional regulation of trafficking-associated genes. We demonstrated STING signaling induced rapid STAT3 phosphorylation indicative of activation. Furthermore, pharmacological STAT3 inhibition prevented STING-induced chemokine expression and macrophage migration. This causatively links STAT3 to translation of STING signals into enhanced motility and chemokine production phenotypes regulating efficient inflammation resolution.

Our findings elucidate a STING-STAT3-chemokine signaling axis governing timely macrophage trafficking to enable coordinated inflammatory phase transitions necessary for productive wound healing. These results have broader implications for understanding context-dependent roles of STING in promoting versus resolving inflammation during tissue injury responses. Importantly, we identify macrophage recruitment and chemokine production as key processes underlying beneficial impacts of STING signaling on repair outcomes.

In contrast, unrestrained STING activity elicits pathologic hyper-inflammation in sterile injury models through amplifying dendritic cell-mediated cytokine production. Our data now highlight the importance of balanced STING signals tuned to enhance macrophage trafficking and chemokine induction while avoiding systemic hyperactivation and leukocyte over-accumulation. Defining these nuanced signaling thresholds sensitive to specific cell types and cytokine milieus remains an important goal for optimizing therapeutic targeting of STING in a context-specific manner.

An intriguing implication of our findings is that the beneficial impacts of STING signaling on wound healing may conversely worsen outcomes in inflammatory disease settings by augmenting macrophage recruitment and chemokine production. Indeed, STING deficiency protected against tissue damage in models of inflammatory bowel disease and arthritis. Our results now provide cellular and molecular mechanisms to explain exacerbation of inflammation by STING signaling in these contexts. This highlights the need to restrict therapeutic STING targeting specifically to damaged tissues while avoiding off-target effects promoting inflammation in nearby diseased sites.

In summary, we identify STING as a critical innate immune rheostat coordinating changes between myeloid cell subsets necessary for timely inflammation resolution enabling productive wound healing. Mechanistically, STING signaling regulates macrophage dynamics through STAT3-dependent modulation of chemokine pathways both systemically and intrinsically in myeloid cells. Our findings provide key insights into STING as a context-dependent mediator of inflammation during tissue injury responses, with important implications for therapeutic targeting in both regenerative medicine and chronic inflammatory disease settings.

Animal experiment

Wildtype (WT) C57BL/6 mice (6–8 weeks old) were obtained from the Animal Institutes of the Army Medical University (Chongqing, China). Sting global knockout (Sting-/-) mice (C57BL/6 N background) were generously provided by Zhenyu Ju (Guangzhou, China). The Animal Ethics Committee of the Army Medical University approved the mice experiments, and the animal license is SCXK (J) 2007-017. Mice were anesthetized with pentobarbital sodium and the dorsal surface was shaved with an electric clipper followed by a depilatory agent to remove any remaining hair. Full-thickness wounds (6-mm biopsy punch) were made on the back skin of the mice. Silicone ring (10mm outer diameter and 8mm inner diagram rim) was used to splint the wound to avoid skin contraction. Then the wounds were photographed on 0, 2,4,6 and 8 days after punch and measured by the Image J software. The formula used for calculating the wound area percentage was as follows: wound area % = SWn/SW0I´100%, where SW0 is the size of the initial wound, and SWn is the size of the wound on the nth day after surgery.

Flow cytometry

Wounds were harvested with a 2-mm margin and minced into 1-2mm pieces by sharp scissors and covered with 5mg/mL Collagenase IV (LS004189, Worthington, Lakewood, US) and 0.1mg/mL DNase I (10104159001, Roche, Basel, CH). Then the wound pieces were at 37°C for 2 hours for enzymatic digestion and filtered with 70 mm filter screens. Brefeldin A was used to inhibit the cytokine secretion for 4 hours when TNF-α and IL-6 were detected. For surface staining, the cell suspension was stained with BV605-conjugated CD11b (101257, BioLegend, California, US), FITC-conjugated Ly6G (127606, BioLegend, California, US), Percype-cy5.5-conjugated Ly6G (127616, BioLegend, California, US), eFluor450-conjugated Ly6C (48-5932-82, eBioscience, Waltham, US) and FITC-conjugated CCR2 (150607, BioLegend, California, US). Next, cells were washed and acquired by Attune Acoustic Focusing Cytometer (Life Technologies, Carlsbad, CA). For intracellular staining, cells were fixed with Cyto-Fast™ Fix Perm Solution (426803, BioLegend, California, US) for 30min and stained with PE-conjugated TNF-α (12-3697-82, eBioscience, Waltham, US) and PE-conjugated IL-6 (504504, BioLegend, California, US). The stained cells were detected by Cytometer and analyzed with FlowJo software, version 10.0.

Histologic analyses

The wound tissues were fixed with 4% paraformaldehyde and overnight before embedding in paraffin. For Hematoxylin-eosin (H&E) staining, paraffin-embedded tissues were cut into 4 µm sections, deparaffinized with xylene, and rehydrated by serials of gradient alcohol, the structures of the tissue cells were stained with hematoxylin and eosin (H&E) as before. Immunohistochemical staining, the specimens were deparaffinized, hydrated as before, and incubated with CD11b (ab133357, abcam, Cambridge, UK), F4/80 (SAF002, AiFang biological, Changsha, CHN) and IL-10 (ARC9102, Invitrogen, Waltham, US) overnight at 4°C. After washing 3 times with PBS, specimens were incubated with anti-rabbit or mouse HRP reagent (SP-9001 or SP-9002, Zhongshan Biology, Beijing, CHN) for 30 minutes and DAB for 1minutes at room temperature. Finally, the sections were rinsed with water, counterstained with hematoxylin, dehydrated, and observed under Olympus Image Viewer.

Western blot analysis

Wound tissues were harvested with a 2-mm margin and cut with sterile scissors. The wound tissues were covered with RIPA buffer with cocktail of protease/phosphatase inhibitor and PMSF (Beyotime, China) and crushed by tissue cell-destroyer (ABclonal) with 5mm steel beads. BMDMs were lysed in RIPA buffer cocktail directly. Total protein concentration of tissues or cells in the supernatant was measured by BCA Protein Concentration Assay Kit. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 0.22µm polyvinylidene fluoride (PVDF) membranes. Membranes were blocked by 5% non-fat milk at room temperature for 2 h and rinsed 3 times with tris-buffered saline with Tween 20 (TBST). Membranes were incubated with primary antibodies against F4/80 (30325, CST, USA), CCL2 ( 2029S, CST, USA), CCL5 (710001, Invitrogen, Waltham, US), p-STAT3 (9145T, CST, USA), STAT3 (1:1000, 4904T, CST, USA), ERK1/2 (1:1000, ab32537, abcam, Cambridge, UK), p-ERK1/2 (1:1000, FTE1718062, R&D Systems, USA) and β-tublin (1:5000, sungenebiotech, CHN) at 4°C overnight. After washing with TBST, membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:3000, Beyotime, CHN) for 1 h at room temperature. After rinsing with TBST, membranes were visualized by electrogenerated chemilumi-nescence (150901, Advanta, USA,) in iBright™ FL1500 Instrument (Thermo Fisher Scientific, USA) and analyzed by Image-J software.

Bone Marrow-derived Macrophage (BMDM) culture

For BMDMs isolation, bone marrow from WT and Sting^-/- mice femurs and tibias were rushed out as described previously. Then resuspended with RPMI1640 media (10% fetal bovine serum, 1% penicillin/streptomycin) and macrophage colony-stimulating factor (20 ng/ml, 315-02, PeproTech, Cranbury, NJ) for 7 days.

Transwell migration assay

WT and Sting^-/- BMDMs (2ⅹ10⁵ cells) were pre-treated with or without 10µg/mL WP1066 for 1h and 50µg/mL DMXAA in serum-free culture medium. These BMDMs were seeded in the upper chambers of PC membrane (5 µm pore size in 24-well plates, 3421, Corning, NY, USA), and 800 µl RPMI1640 with 20% serum was placed in the lower chambers. After 6 h, the cells on the upper surface of the insert were wiped off with a cotton swab, and the filters were fixed with 4% paraformaldehyde (P0099, Beyotime, CHN) and stained with crystal violet staining solution (C0121, Beyotime, CHN). Then filters were rinsed with sterilized distilled water, and photographed on a ZEISS microscope.

Did Labeling and Monocyte Migration into Skin Injury Assay

Did purple cell membrane labeling kit (C1039, Beyotime, China) was used to label BMDMs following the manufacturer’s protocol. Each WT mouse received 2×10⁶ Did -labeled BMDMs from WT mice or Sting^-/- mice via tail vein injection. After that, the injected mice underwent full-thickness wound as described above and were sacrificed on 3 days after wound. Wound tissues were acquired as described above. For flow cytometry, the digested wound cells were stained with BV421-conjugated F4/80 (123132, BioLegend, California, US) and Zombie (77143, Bio Legend, California, US). For confocal, the sections were stained with F4/80 (SAF002, AiFang biological, Changsha, CHN).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, China) was used for total RNA extraction. Reverse transcription was performed using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, China). Quantitative PCR reactions were performed using TB Green Master Mix (RR820A, Takala). Primer sequences were as follows: GAPDH-forward: 5′-GGTTGTCTCCTGCGACTTCA-3′, GAPDH-reverse: 5′-TGGTCCAGGGTTTCTTACTCC-3′, CCL2-forward, 5′-CATCTGCCC TAAGGTCTTC-3′, and CCL2-reverse, 5′GCTTGAGGTGGTTGTGGA-3′; CCL5-forward,5′-ACCACTCCCTGCTGCTTTG-3′, and CCL5-reverse, 5′-ACTTGGCG GTTCCTTCGA-3′.

Statistical analysis

The data were presented as means ± SEM. These statistical analyses were performed by unpaired T-test with Shapiro-Wilk normality test using GraphPad Prism 7.0 software. P < 0.05 was considered as statistically significant.

Acknowledgements

The authors thank the theoretical and technical guidance from all members of WH, GL and ZJ laboratories. This work was supported by the National Natural Science Foundation of China (number 82172232 to WH, number 81920108022 to GL, number 82202464 to YC, number 81772088 to JZ and number 81772088 to JZ).

Author contributions

C. C. and X. C. performed most of the experiments. Z.L. performed Western blotting assay. C.C. and X. C. performed data analysis. W. Z., J. Y., Y. C., Y. T., Y. H., W. H. and X. Z. helped with in vivo experiments; J. Z., Y. W., W. Y., R. S., Q. L. and H. S. helped with in vitro experiments. C. C. and W. H wrote the manuscript; Z. J, G. L, and W. H conceived the project.

Conflicts of interest

The authors declare no conflicts of interest.

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Supplemntaryfigure1.tif
Supplementary Figure S1. Delayed wound healing in Sting^-/- mice
(a) The re-epithelialization of the wound was examined by H&E staining. The representative images of the length (red line) and thickness (yellow line) of neoepithelial tongue (dotted black line) were shown (left panel) and were statistically analyzed (right panel). Bar = 200 μm. n = 5 biologically independent mice.
(b) The representative images of the length (length line) and area (dotted black line) of granulation tissues were shown (left panel) and statistically analyzed (right panel). Bar = 200 μm. n = 5 biologically independent mice. Data are presented as the mean ± SEM. P-value was calculated by one-way ANOVA with Sidak’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.

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STING coordinates resolution of inflammation during wound repair by modulating macrophage trafficking through STAT3

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Abstract

Figures

Introduction

Results

1.Impaired inflammation kinetics underlie delayed repair in STING-deficient wounds

2. Reduced macrophage infiltration causes delayed inflammation resolution in STING-deficient wounds

3. STING orchestrates systemic myeloid cell mobilization during wound healing

4. STING regulates macrophage migratory capacity through cell-intrinsic signaling

5. STING signaling regulates chemokine pathways essential for monocyte recruitment

6. STING promotes macrophage chemokine production and migration via STAT3 activation

Discussion

Methods

Animal experiment

Flow cytometry

Histologic analyses

Western blot analysis

Bone Marrow-derived Macrophage (BMDM) culture

Transwell migration assay

Did Labeling and Monocyte Migration into Skin Injury Assay

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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