The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing

Jun, Joon-Il; Kim, Ki-Hyun; Lau, Lester F.

doi:10.1038/ncomms8386

Article
Published: 16 June 2015

The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing

Joon-Il Jun¹,
Ki-Hyun Kim¹ &
Lester F. Lau¹

Nature Communications volume 6, Article number: 7386 (2015) Cite this article

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Abstract

Neutrophil infiltration constitutes the first step in wound healing, although their timely clearance by macrophage engulfment, or efferocytosis, is critical for efficient tissue repair. However, the specific mechanism for neutrophil clearance in wound healing remains undefined. Here we uncover a key role for CCN1 in neutrophil efferocytosis by acting as a bridging molecule that binds phosphatidylserine, the ‘eat-me’ signal on apoptotic cells and integrins α_vβ₃/α_vβ₅ in macrophages to trigger efferocytosis. Both knockin mice expressing a mutant CCN1 that is unable to bind α_vβ₃/α_vβ₅ and mice with Ccn1 knockdown are defective in neutrophil efferocytosis, resulting in exuberant neutrophil accumulation and delayed healing. Treatment of wounds with CCN1 accelerates neutrophil clearance in both Ccn1 knockin mice and diabetic Lepr^db/db mice, which suffer from neutrophil persistence and impaired healing. These findings establish CCN1 as a critical opsonin in skin injury and suggest a therapeutic potential for CCN1 in certain types of non-healing wounds.

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Introduction

Wound healing encompasses a highly coordinated series of events requiring the interaction of various cell types to achieve tissue integrity and homeostasis after injury. To this end, wound repair proceeds in three overlapping but functionally distinct phases—initiating with an inflammatory phase marked by infiltration of neutrophils and macrophages, followed by a proliferative phase that includes tissue formation and extracellular matrix deposition and concluding with a maturation phase that brings about matrix remodelling and resolution of the granulation tissue^1,2. Inflammation begins immediately after wounding, when blood clotting with entrapped platelets and neutrophils release a host of factors that amplify the coagulation cascade and stimulate further recruitment of inflammatory cells. Neutrophils are the first immune cells to extravasate and infiltrate the wound, functioning to initiate debridement of devitalized tissues and to combat invading microbes by releasing reactive oxygen species, antimicrobial peptides, eicosanoids, proteases and neutrophil extracellular traps^3,4. However, neutrophils must be eliminated in due course for wound healing to progress, as excessive or persistent neutrophil activity can cause further tissue damage and impede healing^5,6. Indeed, chronic non-healing or slow-healing wounds, including diabetic wounds and pressure or venous ulcers, are often associated with prolonged inflammation and neutrophil persistence^7,8,9.

Unless their recruitment is sustained at the wound site, the short-lived, expended neutrophils undergo apoptosis and are largely removed by macrophages in situ within days^10,11. Efferocytosis, or engulfment of apoptotic cells, stimulates macrophages to exhibit M2 phenotypes, which include the downregulation of pro-inflammatory cytokines and upregulation of anti-inflammatory and reparative cytokines such as transforming growth factor-β1 and interleukin-10 (ref. 12). Failure to eliminate apoptotic neutrophils often leads to secondary necrosis, resulting in tissue damage and sustained inflammation through the release of cytotoxic, pro-inflammatory and immunogenic molecules by the lysing cells¹³. Impaired clearance of apoptotic neutrophils is also associated with a variety of inflammatory diseases, including chronic obstructive pulmonary disease (COPD), asthma, pulmonary fibrosis, cystic fibrosis and atherosclerosis, underscoring the importance of this process^{14,15,16,17,18,19}.

Macrophages may recognize apoptotic cells for efferocytosis through receptors that bind phosphatidylserine (PS), the ‘eat-me’ signal that is normally localized in the inner leaflet of the plasma membrane and becomes exposed on the outer leaflet of the lipid bilayer on apoptosis. Among known efferocytosis receptors that recognize PS directly are brain-specific angiogenesis inhibitor-1 (BAI-1), Stabilin 1 and 2, and members of the T-cell immunoglobulin mucin domain protein family^11,12. Alternatively, specific factors in serum or produced by macrophages can serve as bridging molecules that bind apoptotic cells via recognition of PS and couple them to macrophages through interaction with various efferocytosis receptors. Known bridging molecules include milk-fat-globule-epidermal growth factor 8 (MFG-E8/Lactaherin)²⁰, thrombospondin-1 (ref. 21) and protein S/growth arrest-specific gene 6 (Gas6)^22,23, and each recognizes specific efferocytosis receptors on phagocytes including integrins α_vβ₃/α_vβ₅, CD36 or Tyro3/Axl/Mer (TAM), respectively^12,24. Although the existence of these diverse mechanisms of PS recognition may provide functional redundancy, specific PS recognition molecules or mechanisms may play preferential roles in various organs or contexts, possibly due to their cell-type specificity and time course of the expression^11,25. Despite the importance of neutrophil clearance, no specific bridging molecule or efferocytosis receptor has been identified as key mediators of neutrophil efferocytosis in cutaneous wound healing.

One of the proteins whose expression is associated with wound healing is CCN1, a matricellular protein that regulates diverse cellular functions primarily through interaction with distinct integrins in a cell type-specific manner^26,27. CCN1 is organized into four conserved domains with sequence similarities to insulin-like growth factor binding proteins (IGFBP), von Willebrand factor type C repeat (vWC), thrombospondin type 1 repeat (TSR) and a cysteine-knot in the carboxyl terminus (CT). Specific integrin-binding sites have been identified in the vWC, TSR and CT domains^26,27. We have previously shown that CCN1 functions to dampen and resolve fibrosis in wound healing by triggering cellular senescence in myofibroblasts through the engagement of integrin α₆β₁ via its CT domain during the tissue maturation phase^28,29. Here we show that surprisingly, CCN1 is also indispensable for the clearance of neutrophils, thus serving a distinct function in the early inflammatory phase of wound healing. Mechanistically, it acts as a bridging molecule by binding PS on apoptotic neutrophils through its TSR domain and to integrins α_vβ₃/α_vβ₅ on macrophages through its vWC domain, thereby activating Rac1 in macrophages to trigger efferocytosis. Application of CCN1 protein on slow-healing wounds with persistent neutrophil accumulation, including wounds of diabetic mice, accelerates neutrophil clearance. These findings reveal CCN1 as the key opsonin for neutrophil efferocytosis in cutaneous wound healing, and suggest a potential therapeutic role for CCN1 in certain types of slow-healing wounds.

Results

Ccn1 knockdown impedes cutaneous wound healing

Our investigation on the role of Ccn1 in cutaneous wound healing uncovered a biphasic pattern of expression in mRNA and protein, with an early peak in the inflammatory phase (day 3) and a late peak in the maturation phase (days 7–9), suggesting distinct functions in these phases (Fig. 1a,b). To evaluate this possibility, we applied antisense oligonucleotides (AS-ODN) to excisional cutaneous wounds to knockdown Ccn1 expression (Fig. 1c). When wounds were treated with daily administration of Ccn1 AS-ODN from days 3–7 to target the inflammation and tissue formation phases of wound healing, wound closure was delayed with associated robust inflammatory cell accumulation and poor granulation tissue formation (Fig. 1d,e). Accumulation of inflammatory cells and impaired granulation tissue formation can be seen when AS-ODN was applied from day 3 to day 5 to target early Ccn1 expression specifically, but not when applied from day 7 to day 8 to target late expression in the maturation phase (Supplementary Fig. S1), during which CCN1 was previously shown to trigger myofibroblast senescence to restrict fibrosis²⁸. These findings indicate that CCN1 may play distinct roles in the early and late phases of wound healing, corresponding to its biphasic expression pattern.

**Figure 1: *Ccn1* knockdown impedes healing and neutrophil clearance in cutaneous wounds.**

In sense-ODN-treated control wounds, resolution of inflammation, formation of granulation tissue and re-epithelialization were observed within a 9-day period (Fig. 1d–f,i). By contrast, immunohistochemical and double immunofluorescence staining of AS-ODN-treated wounds for neutrophils and macrophages with anti-Ly6G and CD68 antibodies, respectively, revealed an aberrant and exuberant accumulation of neutrophils (Fig. 1f and Supplementary Fig. S2). Myeloperoxidase (MPO) activity was >4-fold higher in AS-ODN-treated wounds than sense-ODN-treated controls, confirming a large accumulation of neutrophils (Fig. 1g). Consistently, the pro-inflammatory cytokines Il-1β and Tnfα were expressed at 7- and 2.3-fold higher levels in AS-ODN-treated wounds, respectively (Fig. 1h). Neutrophils and macrophages are major cell types expressing CCN1 in the wounds during the inflammatory phase as judged by the co-localization of CCN1 with these immune cells, and AS-ODN effectively eliminated CCN1 protein expression in these cells (Supplementary Fig. S3). Consistent with the accumulation of neutrophils and the short lifespan of these cells, enhanced accumulation of apoptotic cells was observed in AS-ODN-treated wounds (Supplementary Fig. S4). Moreover, AS-ODN-treated wounds exhibited impaired re-epithelialization as shown by immunostaining of the stratified epithelial cell marker cytokeratin 14 (CK14; Fig. 1i). However, CCN1 does not have a direct effect on keratinocyte migration (Supplementary Fig. S5), suggesting that impaired re-epithelialization is secondary to other defects in these wounds. Together these results indicate that Ccn1 knockdown leads to neutrophil persistence and delayed wound healing, suggesting that wounds deficient in CCN1 function are stalled at the inflammatory phase.

Impaired skin wound healing in Ccn1^D125A/D125A knockin mice

CCN1 exerts diverse effects on cellular functions primarily through direct binding to distinct integrins in a cell type-specific manner²⁶. To dissect CCN1 functions through distinct integrin receptor pathways, we examined knockin mice expressing mutant CCN1 unable to bind specific integrins. For example, Ccn1^DM/DM mice carry mutations in the overlapping binding sites for integrin α₆β₁, the major CCN1 receptor in fibroblastic cells³⁰, and integrin α_Mβ₂, the adhesion receptor for monocytes³¹. However, Ccn1^DM/DM knockin mice did not exhibit any observable inflammatory phenotype, but showed exacerbated fibrosis due to the inability to activate α₆β₁ in fibroblasts²⁸. Although lacking an RGD sequence, CCN1 binds integrins α_vβ₃ and α_vβ₅ through a non-canonical site in its vWC domain³². To assess the role of CCN1 mediated through α_v integrins, we constructed knockin mice in which the Ccn1 genomic locus was replaced by Ccn1^D125A (Fig. 2a), a mutant allele encoding a single amino-acid substitution (Asp-125 to Ala) that abolished CCN1 binding to α_v integrins³². Knockin of the mutant allele was confirmed by standard molecular methods including direct sequencing, and illustrated by the presence of the predicted PCR products (Fig. 2b). Whereas Ccn1-null mice are embryonic lethal due to cardiovascular defects^33,34, Ccn1^D125A/D125A mice are viable, fertile and exhibit no apparent abnormalities, indicating the CCN1-D125A mutant protein is biologically active and does not impair essential functions during development.

**Figure 2: *Ccn1*^D125A/D125A knockin mice phenocopy mice with *Ccn1* knockdown in wound healing.**

Expression of mutant Ccn1 is elevated in cutaneous wounds in Ccn1^D125A/D125A mice, similar to wild-type (WT) mice (Fig. 2c). Ccn1^D125A/D125A mice experienced significantly slower cutaneous wound closure than WT mice, with poor granulation tissue formation and aberrant accumulation of inflammatory cells similar to the effects of Ccn1 knockdown (Fig. 2d,e). Immunostaining for Ly6G and CD68 and double immunofluorescence showed that neutrophils were accumulated in wounds of Ccn1^D125A/D125A mice (Fig. 2f), and this result was confirmed by MPO assay (Fig. 2g). Moreover, immunostaining for CK14 and vimentin indicated that re-epithelialization and granulation tissue formation, respectively, were impaired (Fig. 2h,i).

As CCN1 is known to induce angiogenesis through α_vβ₃ (refs 35, 36), we examined angiogenesis in wounds of Ccn1^D125A/D125A mice. Indeed, these mice exhibited reduced numbers of CD31-positive endothelial cells and blood vessels, fewer vessel-associated mural cells or pericytes as judged by staining for NG2 and desmin, as well as diminished expression of angiogenic growth factors (Supplementary Fig. S6). Since the α₆β₁-binding site required for CCN1 induction of myofibroblast senescence was not affected by the D125A mutation²⁸, senescence occurred successfully in wounds of Ccn1^D125A/D125A mice (Supplementary Fig. S7a). However, collagen deposition and tissue maturation was delayed, peaking at day 11 instead of day 9 in WT, consistent with delayed healing in these mice (Supplementary Fig. S7b). The phenotypes of Ccn1^D125A/D125A mice closely resemble the effects of Ccn1 knockdown, suggesting that critical CCN1 functions in the inflammatory phase of wound healing are mediated through α_v integrins.

Neutrophil depletion accelerates repair in Ccn1 knockin mice

Consistent with increased neutrophil accumulation in Ccn1^D125A/D125A wounds, elevated expression of the pro-inflammatory cytokines Tnfα and Il-1β was observed (Fig. 3a,b). Expression of the neutrophil chemoattractants Cxcl1 (KC, GROα) and Cxcl5 (LIX), as well as their receptor Cxcr2, was elevated and more sustained in Ccn1^D125A/D125A wounds (Fig. 3c–e), suggesting that there may be enhanced recruitment of neutrophils to the wound site. Since macrophages in wounds are mostly derived from monocytes recruited from circulation, the elevated and sustained expression of Ccl2 encoding the monocyte chemotactic protein-1 was also consistent with increases in macrophages (Fig. 3f). These cytokine expression patterns may be due to aberrant recruitment of, or gene expression by, inflammatory cells, or alternatively, impaired neutrophil apoptosis or clearance⁶. However, there was no difference in the number of neutrophils in the peripheral blood of WT and Ccn1^D125A/D125A mice (Fig. 4a), suggesting no disparity in the systemic level of neutrophil production and mobilization. The expression of granulocyte colony-stimulating factor (Gcsf) in the wounds was also similar (Supplementary Fig. S8). Moreover, CCN1 protein had no effect on apoptosis of bone marrow-derived neutrophils (Supplementary Fig. S9), and there was no defect in Ccn1^D125A/D125A macrophage gene expression when exposed to M1- or M2-polarizing agents (Supplementary Fig. S10). Together, these results indicate that neutrophil accumulation in Ccn1^D125A/D125A mice is not likely due to aberrant production, mobilization or apoptosis of neutrophils, nor deranged gene expression in macrophages.

**Figure 3: Sustained expression of chemokines and cytokines in *Ccn1*^D125A/D125A wounds.**

**Figure 4: Neutrophil depletion accelerates wound healing in *Ccn1*^D125A/D125A mice.**

It was observed that the deletion of macrophages in the inflammatory phase of wound healing led to vascularization defect, reduced granulation tissue formation and impaired re-epithelialization, similar to aspects of the Ccn1^D125A/D125A phenotype³⁷. Since deletion of macrophages may lead to impaired neutrophil clearance, we tested the hypothesis that neutrophil persistence may be the cause of wound-healing defects in Ccn1^D125A/D125A mice by depleting neutrophils using a well-characterized monoclonal antibody (mAb) against Ly6G (RB6-8C5)³⁸. Previous studies showed that neutrophil depletion during wound healing either had no effect or accelerated re-epithelialization^39,40. A single intraperitoneal (i.p.) injection of anti-Ly6G mAb 1 day before wounding decreased neutrophils from ∼15–20% of blood cells to <4% by day 2 post wounding in both WT and Ccn1^D125A/D125A mice; thereafter, neutrophils recovered to the normal level by day 8 (Fig. 4a)⁴⁰. In WT mice, wound closure was unaffected by anti-Ly6G mAb treatment up to day 7 but was slightly retarded by day 9, although the difference did not reach statistical significance (P>0.05; Fig. 4b). By contrast, neutrophil depletion accelerated wound closure in Ccn1^D125A/D125A mice to essentially the same rate as similarly treated WT mice (*P<0.001; **P<0.0001; Fig. 4b). Anti-Ly6G mAb-treated Ccn1^D125A/D125A wounds showed much reduced neutrophil accumulation and improved granulation tissue formation as judged by staining with haematoxylin and eosin (H&E) and immunostaining for Ly6G and CD68 (Fig. 4c,d). MPO assay also confirmed reduction of neutrophils in day 7 wounds (Fig. 4e). Moreover, neutrophil depletion in Ccn1^D125A/D125A mice resulted in greatly improved re-epithelialization, as evidenced by CK14 staining (Fig. 4f). Concomitantly, substantial reduction in the expression of the monocyte chemokine Ccl2 as well as the neutrophil chemokines Cxcl1 and Cxcl5 and their receptor Cxcr2 was also observed (Fig. 4g). Thus, neutrophil depletion accelerated wound closure and rectified many of the wound healing-associated defects in Ccn1^D125A/D125A mice, indicating that neutrophil persistence is the underlying cause of elevated chemokine expression, impaired granulation tissue formation, and delayed wound healing in Ccn1^D125A/D125A mice.

CCN1 mediates neutrophil efferocytosis through α_v integrins

Since integrin α_vβ₃ is known to function as an efferocytosis receptor on macrophages²¹, we hypothesized that CCN1 might regulate phagocytosis of apoptotic neutrophils by macrophages through α_v integrins. To evaluate this possibility, we harvested neutrophils and macrophages from bone marrow of WT and Ccn1^D125A/D125A mice (Supplementary Figs S9 and S10). When WT macrophages (labelled with CellTracker Green CMFDA) were incubated with aged WT neutrophils (labelled blue with 4′,6-diamidino-2-phenylindole (DAPI)), efferocytosis was evident with multiple neutrophils detected inside macrophages (Fig. 5a). However, WT neutrophils were inefficiently engulfed by macrophages from Ccn1^D125A/D125A mice, indicating a role for CCN1 in efferocytosis (Fig. 5a). Efferocytosis was substantially diminished when either neutrophils or macrophages were from Ccn1^D125A/D125A mice, and further reduced when both neutrophils and macrophages were from these mice. These results indicate that CCN1 functions to regulate both cell types in efferocytosis in a non-cell-autonomous manner (Fig. 5b).

**Figure 5: CCN1 mediates efferocytosis through integrins α_vβ₃/α_vβ₅.**

Next, we examined the effect of exogenously added CCN1 protein on efferocytosis (Fig. 5c). WT CCN1 protein was able to restore efferocytosis in Ccn1^D125A/D125A macrophages to WT levels, showing that CCN1 can directly promote efferocytosis. However, the CCN1-D125A mutant protein inhibited efferocytosis in WT macrophages (Fig. 5c), suggesting that it might be able to act in a dominant-negative manner. Indeed, addition of an increasing amount of CCN1-D125A protein progressively inhibited the stimulatory effect of WT CCN1 on efferocytosis, showing dominant-negative action (Fig. 5d). This observation may help to explain the reduced efferocytsis observed when either neutrophils or macrophages were isolated from Ccn1^D125A/D125A mice (Fig. 5b), since the mutant CCN1 protein inhibits WT CCN1 function.

Blocking α_v integrins with known antagonists such as echistatin or RGD peptide inhibited efferocytosis, whereas the RGE control peptide had no effect (Fig. 5e). Inhibition by RGD peptide was observed even in the presence of exogenously added CCN1 protein, consistent with α_v integrins acting downstream of CCN1 (Fig. 5f). Furthermore, macrophages pretreated with function-blocking mAbs against integrins α_v, β₃, β₅ and α_vβ₃ abrogated CCN1-stimulated efferocytosis, whereas IgG control or anti-β₁ mAb had no effect (Fig. 5g). Together with the loss of efferocytosis activity in the D125A mutant, these results establish that CCN1 acts through integrins α_vβ₃ and α_vβ₅.

Phagocytosis requires cytoskeletal reorganization and movement to form phagosomes, a process dependent on the activation of the small GTPase Rac1 (ref. 24). Activation of α_vβ₅ is thought to trigger the recruitment of the p130^Cas/CrkII/Dock180 complex, which activates Rac1 and promotes efferocytosis⁴¹. We found that CCN1 stimulated the formation of the p130^Cas/CrkII complex in macrophages, as shown by reciprocal co-immunoprecipitation (Fig. 6a,b). Tyrosine phosphorylation of the focal adhesion kinase (FAK) was also dependent on the formation this p130^Cas/CrkII signalling complex⁴², which occurred in a CCN1-dependent manner (Fig. 6c). Furthermore, we demonstrate that CCN1 induced the activation of Rac1 in bone marrow-derived macrophages (Fig. 6d). CCN1-induced formation of the p130^Cas/CrkII signalling complex, FAK phosphorylation and activation of Rac1 are each dependent on CCN1 interaction with α_vβ₃/α_vβ₅, as they were abolished in the D125A mutant (Fig. 6).

**Figure 6: CCN1 activates Rac1 and p130^Cas-CrkII complex formation in murine macrophages.**

CCN1 functions as a bridging molecule

As phagocytic receptors on macrophages, integrins α_vβ₃ and α_vβ₅ do not directly recognize the so called ‘eat-me’ signals, such as PS, on apoptotic cells. Instead, a molecule that can bind both PS and the integrins is required to bridge the apoptotic cells to α_vβ₃/α_vβ₅ on macrophages¹⁰. To test whether CCN1 may act as a bridging molecule, we first examined its ability to bind PS directly using solid-phase enzyme-linked immunosorbent assay. We found that PS efficiently bound to CCN1-WT protein as well as to the CCN1-D125A³² (unable to bind α_vβ₃/α_vβ₅) and CCN1-DM³⁰ (unable to bind α₆β₁/α_Mβ₂) with half maximal binding occurring at ∼20–25 nM CCN1, but not to laminin (Fig. 7a). Although laminin was previously shown to bind to glycolipids and phospholipids, it required a much higher concentration (>10 μg) of lipids for binding⁴³. To confirm that CCN1 binding to PS mediates its function in efferocytosis, PS-containing liposomes were used as competitive inhibitors (Fig. 7b). Although exogenously added CCN1 protein enhanced efferocytosis, its effect was strongly inhibited when CCN1 was preloaded with PS-liposomes, indicating that CCN1 binding to PS is critical for its bridging function.

CCN1 contains a thrombospondin type I repeat (TSR) in its third domain (Fig. 7c). As the TSR motif in BAI-1 can bind PS⁴⁴, we tested whether CCN1 binding to PS is mediated through its TSR. For this purpose, several deletion mutants were constructed (Fig. 7c) and the binding of purified deletion mutant proteins to PS was tested using membranes on which various phospholipids were spotted (Fig. 7d). Bound CCN1 was detected using anti-CCN1 vWC domain antibodies, which recognized all deletion mutants equally (Fig. 7c). Consistent with the solid-phase binding assay above, CCN1 showed strong binding to PS, but not to other negatively charged lipids. It also bound to the mitochondrial inner membrane lipid cardiolipin and sulfatide, as observed for the TSR of BAI-1 (ref. 44). Both CCN1-ΔCT and D125A-ΔCT proteins were also capable of binding to PS, cardiolipin and sulfatide, indicating that the C-terminal domain (CT) of CCN1 is dispensable for PS binding. These deletion mutants also bound phosphatidic acid (PA), which was not recognized by full-length CCN1, suggesting that removal of the CT domain may reveal a cryptic binding site for PA in TSR. By contrast, CCN1-I.II, which is lacking both the TSR and CT domains, did not bind to any lipid on the strip, indicating that CCN1 binding to PS requires the TSR domain (Fig. 7d). Since the mutant protein CCN1-I.II does not bind PS and the D125A-ΔCT does not bind integrins α_vβ₃/α_vβ₅, these mutant proteins did not stimulate neutrophil efferocytosis (Fig. 7e). By contrast, CCN1-WT and CCN1-ΔCT, being able to bind both PS and integrins α_vβ₃/α_vβ₅, enhanced neutrophils efferocytosis by macrophages (Fig. 7e). Together, these results show that CCN1 is a novel opsonin molecule for efferocytosis by bridging apoptotic neutrophils to macrophages through direct interaction with PS and integrins α_vβ₃/α_vβ₅, leading to the activation of Rac1 and phagocytosis.

CCN1 accelerates neutrophil clearance in slow-healing wounds

In view of the activity of CCN1 in efferocytosis, we tested its ability to accelerate neutrophil clearance in slow-healing wounds. A single dose of recombinant WT CCN1 protein or vehicle (PBS) was applied to wounds of Ccn1^D125A/D125A mice at day 6 post wounding and wounds were analysed 1 day later (Fig. 8a). H&E staining showed high neutrophil accumulation in PBS-treated Ccn1^D125A/D125A mice, whereas the number of neutrophils significantly decreased in CCN1-treated mice with proportionately more associated macrophages (Fig. 8a). MPO assays confirmed that the treatment of wounds with WT CCN1 significantly reduced neutrophils in Ccn1^D125A/D125A mice, whereas treatment with CCN1-D125A protein had no effect (Fig. 8b). Moreover, expression of the pro-inflammatory cytokine Il-1β was reduced on treatment with CCN1 protein in Ccn1^D125A/D125A mice, consistent with curtailed inflammation (Fig. 8c).

Chronic non-healing or slow-healing wounds, including diabetic wounds, are often associated with prolonged or exacerbated inflammation, and excessive neutrophils have been reported in chronic pressure and venous ulcers^7,9,45. Delayed wound healing with prolonged inflammation and neutrophil persistence is also reported in wounds of Lepr^db/db mice, which carry an inactivating mutation in the leptin receptor gene and develop obesity and diabetes⁸. To investigate the activity of CCN1 in a diabetic context, excisional wounds of Lepr^db/db mice were treated with daily administration of CCN1 protein from days 5 to 8 and analysed on day 9 (Fig. 8d,e). H&E staining showed inflammatory cell accumulation in PBS-treated control wounds, but not in CCN1-treated wounds (Fig. 8d). Immunostaining for Ly6G confirmed that the robust neutrophil accumulation was eliminated by CCN1 treatment in Lepr^db/db wounds (Fig. 8d). Correspondingly, CCN1 treatment results in a dramatic reduction (>5-fold) in MPO activity in Lepr^db/db wounds to the level similar to that of unwounded skin (Fig. 8e). Next, we assessed whether defects in neutrophil clearance in Lepr^db/db wounds might be related to impaired Ccn1 expression. In contrast to significant increases in Ccn1 mRNA and protein in WT wounds (Fig. 1a,b), Ccn1 mRNA in Lepr^db/db wounds showed only marginal increases on wounding that did not reach statistical significance (Fig. 8f). Moreover, the total CCN1 protein actually decreased on wounding in Lepr^db/db mice, possibly due to a protease-rich environment in which neutrophils are aberrantly accumulated (Fig. 8g). Thus, there is a deficiency in Ccn1 expression in Lepr^db/db wounds, leading to the accumulation of neutrophils. These results underscore the importance of CCN1 in promoting neutrophil efferocytosis in wound healing, suggesting a therapeutic potential for CCN1 in the treatment certain types of slow-healing wounds including diabetic wounds.

Discussion

Restoration of tissue integrity and homeostasis following injury to the skin is of vital importance, as the integument provides the first barrier against invading microbes and pathogens. Acute inflammation occurring immediately after injury plays a critical role for host defense and debridement of necrotic tissues, although deregulated inflammation or its persistence can cause further tissue damage and lead to chronic non-healing wounds. Impaired wound healing causes considerable morbidity in afflicted patients, and is a leading cause of diabetes-related amputations⁴⁶. Thus, effective management of inflammation resolution is critical to wound care, and targeting excessive neutrophil activity has been proposed for treating chronic wounds such as venous ulcers⁴⁵. Here we have uncovered a critical role for CCN1 in triggering efferocytosis of neutrophils by macrophages to promote the resolution of inflammation in skin wounds. CCN1 appears to be the first bridging molecule identified to play this role in cutaneous wound healing. Moreover, application of CCN1 accelerates neutrophil clearance in diabetic mouse wounds, suggesting a therapeutic potential for CCN1 in the treatment of certain types of impaired wound healing.

To dissect the functions of CCN1 through distinct integrins, we have constructed an allelic series of knockin mice in which Ccn1 is replaced by alleles expressing CCN1 mutants unable to bind specific integrins. This approach circumvents the embryonic lethality of Ccn1-null mice due to cardiovascular defects³⁴, and avoids potential limitations in cell type-specific deletions as CCN1 may be secreted by diverse cell types in the tissue microenvironment. In skin wound healing, analysis of Ccn1^DM/DM knockin mice expressing CCN1 unable to bind α₆β₁/α_Mβ₂ led to the discovery that CCN1 controls fibrosis through triggering myofibroblast senescence by engagement of α₆β₁ (ref. 28), and studies on Ccn1^D125A/D125A mice reported herein revealed a critical function in promoting the resolution of inflammation through efferocytosis. Interestingly, fibroblast-specific deletion of Ccn1, even in combination with deletion of the homologous family member Ccn2, did not result in any change in wound closure rate⁴⁷, suggesting that CCN1 produced from various cell types in the wound microenvironment adequately compensates for the loss of fibroblast-derived CCN1. Indeed, CCN1 is expressed in macrophages⁴⁸, neutrophils (Supplementary Fig. S2), endothelial cells³³ and fibroblasts²⁸. No apparent defect in macrophage function was noted in Ccn1^DM/DM knockin mice, even though CCN1 can induce gene expression in macrophages through α_Mβ₂ in culture⁴⁹, suggesting that inflammatory cytokines or regulators other than CCN1 may prevail in regulating macrophage gene expression in the cutaneous wound.

Multiple efferocytosis receptors and bridging molecules have been described, and they may play differential roles in various developmental and pathological contexts depending on their cell type specificity and time course of expression. For example, both Gas6 and Protein S contribute to the phagocytosis of photoreceptor outer segments by retinal pigment epithelial cells, but no other efferocytosis defect was observed among a myriad of phenotypes in Gas6, Protein S and TAM receptor mutant mice^25,50. Mice lacking TSP-1, a bridging molecule, exhibit impaired angiogenesis in cutaneous wound healing but efferocytosis defect has not been reported⁵¹. Likewise, MFG-E8-null mice show reduced angiogenesis in skin wound healing without notable efferocytosis defect⁵². Indeed, whereas MFG-E8 is critical for the elimination of apoptotic B cells in the germinal centres⁵³ and mammary epithelial cells during mammary gland involution⁵⁴, MFG-E8 is downregulated in virtually all inflammatory conditions and inflammation-related pathologies⁵⁵, suggesting that it may not participate in neutrophil elimination.

CCN1 binds to α_vβ₃/α_vβ₅ on macrophages, PS on apoptotic neutrophils and α₆β₁ on myofibroblasts through its vWC, TSR and CT domains, respectively, to exert distinct functions during wound healing (Fig. 9). Since CCN family proteins emerged through evolution by exon shuffling^26,27, it appears likely that this process recombined various structural domains from extracellular matrix proteins to confer unique functions governing multiple aspects of wound healing. CCN1 is known to dampen and resolve fibrosis in the maturation phase of wound healing by inducing myofibroblast senescence²⁸. Our current findings suggest the possibility that CCN1 might further contribute to the restriction of fibrosis by promoting efferocytosis of apoptotic myofibroblasts. In support of this hypothesis, we found that CCN1 can promote macrophage engulfment of apoptotic fibroblastic cells (Supplementary Fig. S10c), and apoptotic myofibroblasts have been associated with restriction of fibrosis⁵⁶.

**Figure 9: A proposed schematic model for CCN1 actions in cutaneous wound healing.**

Since CCN1 can induce angiogenesis through interaction with integrin α_vβ₃ in endothelial cells^35,36, it is not surprising that Ccn1^D125A/D125A mice also exhibited impaired angiogenesis during wound healing. These knockin mice show reduced CD31-positive endothelial cells, fewer vessel-associated pericytes and diminished expression of angiogenesis-related genes (Supplementary Fig. S6). However, neutrophil depletion rectified many of the wound-healing defects by accelerating wound closure, improving angiogenesis and promoting granulation tissue formation (Fig. 4), suggesting that neutrophil persistence, rather than impaired angiogenesis, underlies the wound healing defects in Ccn1^D125A/D125A mice. As there are multiple angiogenic factors in the wound microenvironment, it is possible that CCN1 plays a redundant or relatively minor role in regulating angiogenesis during wound healing, and the angiogenic defect in Ccn1^D125A/D125A mice may have been exacerbated by neutrophil persistence. However, since the angiogenesis and efferocytosis functions of CCN1 are mediated through the same integrin and cannot be separated by mutation, understanding the angiogenic role of CCN1 in wound healing may require further scrutiny.

Diabetic wounds often suffer from enhanced inflammation and delayed healing, and Lepr^db/db mice exhibit these phenotypes^8,40. In contrast to WT mice in which Ccn1 expression is enhanced on wounding (Fig. 1a,b), Ccn1 mRNA is not significantly elevated in Lepr^db/db wounds and CCN1 protein actually decreased on wounding (Fig. 8f,g), potentially due to robust protease activity produced by accumulated neutrophils⁴⁵. Thus, Lepr^db/db wounds appear to suffer from impaired Ccn1 expression, leading to a deficit in CCN1 function. Consistently, CCN1 therapy of cutaneous wounds in Lepr^db/db mice efficiently eliminated excess accumulation of neutrophils (Fig. 8). Since these results are obtained in Lepr^db/db mice, the specific role of CCN1 in normal and impaired human wounds remains to be determined. It is interesting to note that the CCN1-D125A mutant protein can inhibit efferocytosis in a dominant-negative manner (Fig. 5d), most likely by sequestering neutrophils through PS binding but being unable to bridge them to macrophages. Thus, cleavage of CCN1 through its protease-sensitive central hinge region between domains II and III⁵⁷ in certain pathological contexts may potentially generate CCN1 fragments that inhibit efferocytosis in a dominant-negative manner (Fig. 7e). For example, CCN1 is cleaved in lung epithelial cells from patients with COPD⁵⁸, a disease in which impaired efferocytosis has been observed¹⁵.

Whether CCN1 plays a critical role in efferocytosis in other inflammatory or pathological contexts beyond cutaneous wound repair is currently unknown. In this regard, it is noteworthy that aberrant Ccn1 expression has been reported in myriad inflammatory conditions, including sepsis⁵⁹, inflammatory bowel disease^60,61, rheumatoid arthritis⁶², COPD⁶³, cholestasis⁶⁴ and cardiac injury repair⁶⁵. It will be important to assess whether the opsonin function of CCN1 is important in these contexts. Moreover, CCN1 controls fibrosis by inducing senescence not only in skin wounds, but also in liver injuries induced by either hepatotoxin or cholestasis²⁹, indicating that the role of CCN1 in tissue injury repair is potentially wide-ranging and not limited to cutaneous wounds.

Prolonged inflammation is the hallmark of non-healing wounds, including diabetic wounds, pressure ulcers and venous ulcers^1,66. Excessive neutrophils contribute to impaired healing, and thus wound closure in diabetic mice was accelerated by 50% on neutrophil depletion⁴⁰. Here we show that CCN1 treatment leads to effective neutrophils removal in slow-healing wounds, including wounds of Ccn1^D125A/D125A and in Lepr^db/db diabetic mice (Fig. 8). These findings suggest a potential therapeutic value in the application of CCN1 in certain types of non-healing wounds⁴⁵. Since CCN1 plays multiple and disparate roles in wound healing (Fig. 9), the possibility that application of CCN1 in wound care may both accelerate inflammation resolution and dampen fibrosis and reduce scarring clearly warrants further investigation.

Methods

CCN1 proteins and reagents

Recombinant CCN1 and mutant proteins (D125A, DM, CCN1-I.II, CCN1-ΔCT and D125A-ΔCT) were produced using a baculovirus expression system in insect cells and purified by ion-exchange or immunoaffinity chromatography^30,32. Rabbit polyclonal antibodies against CCN1 were raised against the CCN1 vWC domain⁶⁷. Rabbit and mouse IgGs, echstatin and lipopolysacharide were from Sigma. RGDSP and RGESP peptides were from Bachem. Function-blocking mAbs against mouse integrin β₁ (KM16), α_v (RMV-7), β₃ (2C9-G3), α_vβ₃ (23-C6) and β₅ (KN52) were from eBioscience. Recombinant murine interleukin-4 were from R&D Systems. Horse radish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were from Amersham Biosciences. Biotin-conjugated goat anti-rabbit IgG, biotin-conjugated goat anti-rat IgG antibody and streptavidin-conjugated HRP were from Invitrogen.

Animals and analysis of cutaneous wound healing

Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Chicago. Two independent lines of Ccn1^D125A/D125A mice (A5 and F5) were generated in a svJ129-C57BL/6 mixed background (Fig. 2a) and backcrossed more than six times into the C57BL/6 background; results from both lines were similar. Genetically diabetic mice (BKS.Cg-m+/+ Lepr^db/J) were from Jackson Laboratories, and intercrossed to yield homozygous db/db mice. Typically, 10–12-week-old male mice with similar body weight (∼25–28 g for C57BL/6 mice) were used for cutaneous wound healing. Mice were anaesthetized with i.p. injection of ketamine/xylazine (100 mgkg⁻¹ i.p.) and the dorsum of each mouse was clipped free of hair. Full-thickness excisional wounds through panniculus carnosus were created with a 6-mm biopsy punch (Miltex) and left open during the healing process²⁸. For healing kinetic studies, digital images of wounds were taken and wound diameters were measured using Photoshop CS2 (Adobe). For histological analyses, wound tissues were snap-frozen in optimum cutting temperature compound (Tissue-Tek) and sectioned serially (7-μm thickness) using a Leica CM1950 UV cryostat. Alternatively, wound tissues were embedded in paraffin and sections were stained with H&E or probed with anti-Ly6G rat mAb (BD Pharmingen; 1A8, 1:100 dilution) or anti-CD68 rabbit polyclonal antibody (Abcam, 1:100 dilution), followed by biotin-conjugated goat anti-rat IgG secondary antibody coupled with streptavidin-HRP with 3,3′-Diaminobenzidine (Sigma) as substrate, and counterstained with haematoxylin. Apoptosis in wounds was detected using anti-activated caspase-3 polyclonal antibody (Cell Signaling) and visualized under fluorescence microscopy (Leica DM4000B). For western blot analysis, wound tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-Cl pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5% Na-deoxycholate, 1% Triton-X-100 and protease inhibitors cocktail) using polytron homogenizer, followed by sonication. Cellular senescence was evaluated by senescence-associated β-galactosidase staining of wound tissues²⁸.

Antisense oligonucleotide treatment

Antisense oligonucleotide (AS-ODN) against Ccn1 was prepared in PBS and injected intradermally (200 μg per wound) into full-thickness excisional wounds on indicated days. Sense oligonucleotides (sense-ODN) were used as control. The sequences of oligonucleotides are: antisense, 5′- GCAGGCACGGGGCAGGTGG -3′ and sense, 5′- CCACCTGCCCGCTGCCTGC -3′.

MPO assay

The samples from freshly isolated wound tissues were prepared as described⁶⁸, and MPO assay was performed in assay buffer (50 mM hexadecyl trimethyl ammonium bromide, 0.167 mg ml⁻¹ of O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide). MPO activity (absorbance) was measured using plate reader (Lab System, Multiscan MS) at 450 nm. Purified human MPO (Sigma) was used to generate a standard curve. Data were expressed as absorbance per wound weight.

RNA isolation and qRT–PCR

Skin wounds or cultured bone marrow-derived macrophages were homogenized using TRIzol reagent (Invitrogen) and total RNA was isolated using RNeasy Mini Kit (Quiagen). Total RNA (2 μg) was reverse transcribed to cDNA using MMLV-Reverse Transcriptase (Promega), and quantitative reverse transcription-PCR (qRT–PCR) was performed with the iCycler Thermal Cycler (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad). The specificity of qRT–PCR was confirmed by agarose gel electrophoresis and melting-curve analysis. A housekeeping gene (cyclophilin E) was used as an internal standard. The primers used are listed in Supplementary Table 1.

Isolation of bone marrow-derived macrophages and neutrophils

Murine neutrophils and macrophages were isolated from bone marrow of either WT or Ccn1^D125A/D125A mice as described with modifications^69,70. For neutrophil isolation, the resuspended bone marrow in Hank’s balanced salt solution (5 ml) was carefully layered over 62% Percoll (5 ml, Amersham) and centrifuged at 1,000g for 30 min. After the gradient centrifugation, a cloudy layer containing neutrophils was removed from on top of the Percoll layer and resuspended in RPMI media for culture. An aliquot of cell suspension (∼20 μl) was spread on to the slide glass to assess the purity of isolated neutrophils by Giemsa staining (Sigma). For macrophages, the bone marrow cells were resuspended in macrophage complete media (DMEM/F12 with 20% (v/v) L-929 cells conditioned media), counted, and 4 × 10⁵ cells were plated in sterile plastic petri dish (100 mm) and incubated in 37 °C in 5% CO₂ for 7 days with medium being refreshed on day 3 for full differentiation into macrophages. The macrophages differentiation was checked using F4/80 immunostaining.

Efferocytosis assay

Bone marrow-derived macrophages (10⁵ cells per well) from either WT or Ccn1^D125A/D125A mice were plated on 12-well culture dish 1 day before the assay and labelled with CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA; 5 μM, Invitrogen). Neutrophils were aged for 16 h and then incubated at 55 °C for 2 h before use as substrate for efferocytosis. Aged neutrophils (5 × 10⁵ cells per well) were labelled with DAPI (10 mg ml⁻¹) and incubated with macrophages for 60 or 90 min. Where indicated, CCN1 was preincubated with neutrophils for 1 h before incubation with macrophages. After incubation, cells were washed with ice-cold PBS four times fixed with 4% paraformaldehyde for 5 min. Efferocytosis was measured using the fluorescence microscopy by counting macrophages containing >2 neutrophils in 12 randomly selected high-powered fields and efferocytosis index was expressed as a percentage positive macrophages over total counted (∼300 cells). For inhibition of efferocytosis, PS-containing liposomes were prepared as described²² and preloaded on to CCN1 for 30 min. For fibroblasts efferocytosis, human normal skin fibroblasts (1077SK; ATCC CRL-2094) were cultured, labelled with CellTracker Blue 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (5 μM, Invitrogen) and induced to undergo apoptosis by exposure to staurosporine (1 μM; Invitrogen) for 16 h. Efferocytosis assay was performed as described above.

Depletion of neutrophils in mice

Neutrophils depletion was achieved as described³⁸. In brief, anti-Ly6G mAb (hybridoma clone# RB6-8C5; originally developed by Dr Robert I. Coffman, DNAX Research Institute, Palo Alto, CA and obtained from Dr David Sibley, Washington University, St Louis, MO) was produced under serum-free conditions using a CELLine bioreactor (CL350; Argos technologies, IL). Anti-Ly6G mAb (0.5 mg per mouse) was delivered in a single i.p. injection 1 day before wounding. Neutrophils depletion was confirmed by whole blood count on day 2 post wounding.

Immunofluorescence

Frozen wound tissues were cold acetone-fixed, washed in PBS-Tween-20 (PBST) and incubated in normal goat serum blocking solution containing 2% goat serum, 1% bovine serum albumin, 0.1% cold fish skin gelatin, 0.1% Triton-X-100, 0.05% Tween-20, 0.05% sodium azide and 0.01M PBS (pH 7.2) for 1 h. For labelling, the single primary antibody or antibody mixes were applied for 16 h at 4 °C. After washing (5 × ) in PBST, slides were incubated with the secondary antibodies (Texas Red-conjugated anti-rabbit IgG and Alexafluor488-conjugated anti-rat IgG; Invitrogen, 1:500 dilution each) for 30 min, followed by washing (5 × ) in PBST. DAPI was used as counterstain (1 mg ml⁻¹). Fluorescence images were acquired using a Leica DM4000B microscope and processed with Photoshop CS2 (Adobe). Primary antibodies used for immunofluorescence staining are followed: anti-Ly6G rat mAb (1A8; BD Pharmingen, 1:100 dilution), anti-CD68 rat mAb (FA-11; Abcam, 1:100 dilution), anti-CD68 rabbit polyclonal antibody (Abcam, 1:200 dilution), anti-CD31 rat mAb (MEC 7.46; Abcam, 1:200 dilution), anti-NG2 rabbit polyclonal antibody (AB5320; Millipore, 1:100 dilution), anti-desmin rabbit polyclonal antibody (Abcam, 1:200 dilution), anti-CK14 mouse mAb (LL002; Abcam, 1:200 dilution), anti-vimentin rabbit polyclonal antibody (Abcam, 1:200 dilution) and anti-CCN1 rabbit polyclonal antibody (from the house, 1:200 dilution).

Cell migration assay

Human immortalized HaCaT keratinocytes were plated on to non-TC-treated dishes that were precoated with rat tail collagen I (4.3 mg ml⁻¹; BD Bioscience) and serum starved for 48 h. Cells were pretreated with mitomycin C (10 μg ml⁻¹; Sigma) for 30 min, followed by treatment of human epidermal growth factor (10 ng ml⁻¹) or recombinant CCN1 protein (either WT or D125A mutants; 2 μg ml⁻¹ each). Cell migration was assessed after 48 h and quantified using Image J software.

PS binding

Standard binding assays usually coat plastic wells with phospholipids, followed by the addition of binding protein and immunodetection of the binding protein. However, CCN1 is a cell adhesion molecule and characteristically coats plastic surfaces efficiently even in a short incubation period, making it impossible to detect specific binding using this protocol. Thus, we precoated 96-well plates with serial dilution of CCN1 proteins (WT, DM and D125A) and laminin at 4 °C overnight. After washing with PBST (2 × ), uncoated area was blocked with bovine serum albumin (10 mg ml⁻¹) for 1 h. A total of 300 ng of PS was then added to each well for 1 h, followed by washing with PBST (3 × ). Anti-PS mAb (Abcam; 1 μg per well) was added, and standard enzyme-linked immunosorbent assay protocol was employed using 3,3′,5,5′-Tetramethylbenzine as a substrate and measured at OD 450 nm (ref. 20). Alternatively, lipid membranes (Echelon biosciences) were incubated overnight with CCN1 proteins (WT, CCN1-N, CCN1-ΔCT and D125A-ΔCT; 10 μg each), and anti-CCN1 vWC antibodies were used for detection.

Co-immunoprecipitation

Murine macrophages (Raw264.7) were treated with recombinant WT CCN1 protein or CCN1-D125A (5 μg each) for 30 or 60 min. Cells were lysed with RIPA buffer at 4 °C for 4 h with gentle agitation, followed by centrifugation at 13,000g for 30 min. Supernatants were carefully removed and incubated with 1 μg of either anti-CrkII (C-18; Santa Cruz), anti-p130^Cas (C-20; Santa Cruz), or anti-FAK (C-20; Santa Cruz) at 4 °C for overnight. Protein G agarose beads (5 μl; Amersham) were added for 1 h, then followed by centrifugation at 10,000g for 1 min. Pellets were washed four times with RIPA buffer and lysates were prepared with Laemmli sample buffer (3 × ). Western blot analyses of immune complex were performed for anti-CrkII (C-18; 1:1,000 dilution), anti-p130^Cas (C-20; 1:1,000 dilution), anti-FAK (C-20; 1:500 dilution) and anti-phophoFAK (Tyr397; #3283, Cell Signaling; 1:250 dilution). Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. S11.

GTP-Rac1 pull-down assay

Activated, GTP-bound Rac1 was detected using Rac1 activation assay kit (Cell Biolabs Inc.) according to the manufacturer’s instruction. In brief, bone marrow-derived macrophages were treated with CCN1-WT or CCN1-D125A (4 μg each) for 60 min. Cells were then washed with cold PBS twice and lysed. The cell lysates were incubated with 20 μg of PAK1-PBD (p21-binding domain of p21-activated protein kinase) agarose for 1 h at 4 °C. The precipitation of GTP-Rac1 was detected by immunoblotting.

Statistics

Results are expressed as the mean±s.d., and statistical analysis was performed by one-sided two-sample t-tests and one-way analysis of variance (for multiple group comparison) with Bonferroni post hoc tests. A P<0.05 was considered significant. All experiments were performed in triplicate, unless otherwise indicated.

Additional information

How to cite this article: Jun, J.-I. et al. The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing. Nat. Commun. 6:7386 doi: 10.1038/ncomms8386 (2015).

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Acknowledgements

We thank Dr David Sibley for the generous gift of hybridoma clone RB6-8C5, Seung Won Shin and Guoqiang Yan for excellent assistance and members of the laboratory for helpful discussion. This work was supported by grants from the National Institutes of Health (R01AR061791 and R01GM78492) to L.F.L.

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Authors and Affiliations

Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, 60607, Illinois, USA
Joon-Il Jun, Ki-Hyun Kim & Lester F. Lau

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Joon-Il Jun
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Ki-Hyun Kim
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Lester F. Lau
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Contributions

J.-I.J. conducted the experiments, K.-H.K. contributed knockin mice construction; J.-I.J. and L.F.L. designed the experimental plan, analysed the data and wrote the paper.

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Correspondence to Lester F. Lau.

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The authors declare no competing financial interests.

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Supplementary Figures 1-11 and Supplementary Table 1 (PDF 5306 kb)

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Jun, JI., Kim, KH. & Lau, L. The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing. Nat Commun 6, 7386 (2015). https://doi.org/10.1038/ncomms8386

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Received: 30 September 2014
Accepted: 04 May 2015
Published: 16 June 2015
DOI: https://doi.org/10.1038/ncomms8386

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