Transcription factor c‐Maf drives macrophages to promote hypertrophic scar formation

Hypertrophic scar (HS) is caused by the abnormal proliferation of fibroblasts and excessive deposition of extracellular matrix (ECM). Emerging evidence demonstrates that c‐Maf positive M2 macrophages were mainly located in the hypertrophic scar tissues of proliferative phase. But whether c‐Maf positive M2 macrophages can promote hypertrophic scar formation through modulating hypertrophic scar fibroblasts remains elusive.


Aims:
The aim of this study is to investigate the effects of c-Maf positive M2 macrophages on the biological behaviors and functions of hypertrophic scar fibroblasts and the potential mechanism.
Methods: HE and Masson trichrome staining were used to examine the histological features of human hypertrophic scar.Immunofluorescence staining was employed to label and quantify the c-Maf + /CD68 + M2 macrophages.CCK8, wound healing, and transwell assays were utilized to test the effects of c-Maf overexpressed M2 macrophages or the cell culture supernatants on the proliferation and migration of hypertrophic scar derived fibroblasts (HFBs) and normal skin derived fibroblasts (NFBs).
Western blot and qPCR were harnessed to test the expressions of COL1, COL3, and α-SMA in the co-cultivated fibroblasts and TGF-β1 in the c-Maf overexpressed M2 macrophages.
Results: Increased number of c-Maf + /CD68 + M2 macrophages were found in HS in contrast to the normal skin (NS).Elevated proliferation and migration were observed in the HFBs or NFBs co-cultured with c-Maf overexpressed macrophages or the cell culture supernatants.A higher mRNA and protein expressions of COL1, COL3, and α-SMA were recorded in the HFBs co-cultured with c-Maf overexpressed macrophages or treated with its culture supernatants.In addition, augmented mRNA and protein expressions of TGF-β1 were also investigated in the c-Maf overexpressed macrophages.

| INTRODUC TI ON
Hypertrophic scar is a kind of fibrotic diseases secondary to skin injury.The main cause of hypertrophic scar is the excessive wound healing, which results from the abnormal proliferation of fibroblasts and excessive deposit of extracellular matrix (ECM). 1 It is reported that the United States even spends about $12 billion on the treatment of skin scars annually. 2Patients fearing of hypertrophic scar often originate from a cosmetic perspective, but they rarely recognize that scars can also lead to the psychological and functional problems such as pruritus and pain. 3Therefore, based on the consideration of the economic factors and people's urgent needs, it is necessary to explore the factors that contribute to hypertrophic scar pathogenesis.
During hypertrophic scar formation, alternatively activated macrophages (M2 macrophages) play an important role in regulating the biological behaviors and ECM production of fibroblasts. 4It is reported that M2 macrophages were mainly located in the developing hypertrophic scar tissues at 3-4 weeks post wounding, systemic depletion of macrophages in the subacute phase of wound healing will attenuate hypertrophic scar formation. 5In addition, M2 macrophages polarized from THP-1 cells can increase the fibrogenic activities of co-cultured human dermal fibroblasts by promoting cell proliferation, elevating the collagen content and number of alphasmooth muscle actin (α-SMA) expressed cells, and augmenting the expression of the pro-fibrotic genes. 6However, the mechanisms of how M2 macrophages promote hypertrophic scar formation remain unclear.
M2 macrophages are the main immunomodulatory cells characterized by the expression of MAF BZIP transcription factor (c-Maf), Arg1, CD163, or CD209. 7c-Maf is a member of transcription factor proteins that belong to the activated protein-1 super family.
Existed evidence showed that c-Maf could regulate the expression of connective tissue growth factor (CTGF), 8 interleukin-4 (IL-4), and interleukin-13 (IL-13) in macrophages, thereby inducing the differentiation of fibroblast into myofibroblast. 9In addition, bone marrowderived macrophages from c-Maf knockout chimeric mice showed a decreased expression of transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF). 10 More importantly, we previously found that M2 macrophages marked by c-Maf and CD68 were mainly distributed in the hypertrophic scar tissues of proliferative phase and dispersed in the dermis. 11These suggest that M2 macrophages may have promoted hypertrophic scar formation through modulating c-Maf.In this study, we explored the effects of c-Maf overexpressed macrophages on hypertrophic scar derived fibroblasts (HFBs) and its mechanism, which may provide a novel intervention target for hypertrophic scar treatment.

| Patient sample collection
The human hypertrophic scar samples and normal skin tissues were collected from patients receiving the operation of scar revision at the department of plastic surgery, Xijing Hospital.The patients were diagnosed of hypertrophic scar clinically and histologically.All patients offered their written consents and agreed to provide their surgically removed tissues for study.This study has been approved by the Ethics Committee of Xijing hospital (KY20233251-1) and was carried out with strict adherence to the Declaration of Helsinki Principles.

| Histological analysis
The hypertrophic scar and normal skin tissues were fixed with paraformaldehyde and embedded in paraffin.Sections of hypertrophic scar and normal skin were subjected to hematoxylin and eosin (HE)   or Masson trichrome staining and examined under the light microscope (Olympus, FSX100).

| Primary cell isolation and cell culture
Immediately after the tissues were transferred aseptically to the laboratory, the epidermis and fatty tissues were removed, the remaining dermal tissues were cut into small pieces.Then, the pieces of tissues were incubated in medium including 89% DMEM (Gibco, Invitrogen), 10% fetal bovine serum (FBS, Yeasen), and 1% penicillin/streptomycin (Gibco, 15 140 122).All cultures were placed at a 5% CO

| Plasmid transfection
The plasmid of pcDNA3.1-EGFP-c-Mafwas obtained from Hanheng biology.When Raw264.7 grew to 60%-80% cell confluence, the plasmid of pcDNA3.1-EGFP-c-Maf(8 μg/μL) was transfected into it with advanced DNA transfection reagent (ZETA LIFE INC, AD600025.).In addition, the negative control plasmid was transfected too.Twelve hours later, the culture medium was removed and changed with new medium.After another culture of 24 h, the cell culture supernatant was harvested and kept for further study.
Quantitative real-time PCR (qPCR) and western blot were applied to assess the mRNA and protein expressions of c-Maf.

| Cell proliferation
The HFBs and NFBs were seeded in 96-well plates with a density of 3 × 10 3 cells/well and cultured for 24 h.Then, the medium was removed, and new medium containing 0%, 10%, 50%, or 100% c-Maf overexpressed macrophages Raw 264.7 culture supernatants was added to treat the fibroblast cells.Cell proliferation was assessed by CCK8 assay (MCE, HY-K0301) 24 h and 48 h later.The absorbance of OD 450 nm was measured using microplate reader (TECAN GENIOS, INFINITE M200 PRO, Tecan Group, Ltd).

| Wound healing assay
The HFBs and NFBs were cultured in six-well plates.When the cell density reached 100% confluency, HFBs and NFBs were starved for 12 h.Then, the cell monolayer was scratched using a 200 μL sterile tip and washed with phosphate buffered saline (PBS).Medium containing 0%, 10%, 50%, or 100% c-Maf overexpressed macrophages Raw 264.7 culture supernatants was added to treat cells.The areas of scratch were measured by ImageJ software (1.6.0 20) 12 h and 18 h later.

| Transwell assay
The transwell assay was conducted in the 24-well plates.The upper chamber harbors a membrane with a pore size of 8 μm (Corning, NY), which was applied for the culture of NFBs or HFBs with a density of 5 × 10 4 /well in 500 μL cell suspension.The negative control cells (Raw264.7 cells transfected with pcDNA3.1)and the c-Maf overexpressed Raw264.7 cells (Raw264.7 cells transfected with pcDNA3.1-EGFP-c-MAF)were cultured in the lower chamber.Following a co-culture for 8 h, the NFBs or HFBs were fixed with methanol for 25 min, washed with PBS for three times, and stained with 0.5% crystal violet solution for 30 min.Then, the cells on the upper side of membrane were removed, cells on the lower side of membrane were examined and photographed using a microscope (Olympus, FSX100).

| Total RNA extraction and qPCR
The extraction of total RNA was the same with our previous descriptions of Lin Chen et al. 11 Superscript II® (yeasen, Superscript®Reverse Transcriptase) was used for the production of cDNA.The Real-time PCR System (BIO-RAD, CFX Connect, US) was employed to perform quantitative polymerase chain reaction (qPCR) using Platinum SYBR Green (Yeasen, 11184ES08).The mRNA expressions of c-Maf, Collagen 1 (COL1), Collagen 3 (COL3), Transforming growth factor beta 1 (TGF-β1), and α-Smooth muscle actin (α-SMA) were calculated using the 2 −ΔΔCt (threshold cycle) method.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.The primes used for qPCR were showed in Table 1.

| Statistical analysis
All data were displayed as means ± standard deviation (SD).The data were analyzed with Graphpad Prism 6.01 software.Statistical differences were assessed using two-way ANOVA for multiple group comparisons.Student's t-test or Mann-Whitney U-test were used to compare the difference between two groups.p < 0.05 was considered as statistically significant.

| The number of c-Maf + /CD68 + cells was increased in the hypertrophic scar tissues
To reveal the functions of c-Maf + /CD68 + M2 macrophages in hypertrophic scar formation, human hypertrophic scar and normal skin tissues were collected.Histologically, lots of dense and irregular collagen fibers and absence of hair follicles were examined in the hypertrophic scar tissues, and more extracellular matrix deposition was observed in the hypertrophic scar tissues (Figure 1A, p < 0.001).
Compared with the normal skin tissues, a significantly larger number of c-Maf + /CD68 + M2 macrophages (Figure 1B, p < 0.001) were observed in the hypertrophic scar tissues.
This indicated that the transfection of pcDNA3.1-EGFP-c-Mafcan transcriptionally regulate the expressions of downstream genes.

NFBs and HFBs
CCK8 assay was employed to test the effects of c-Maf overexpressed macrophages supernatants on the proliferation of NFBs and HFBs; the result showed that 100% c-Maf overexpressed macrophages supernatants significantly promoted the proliferation of NFBs at 48 h (Figure 3A).With regard to HFBs, in contrast to medium containing 0% c-Maf overexpressed macrophages supernatants, medium containing 50% c-Maf overexpressed macrophages supernatants can significantly facilitate the proliferation of cells at 24 h, while medium containing 10%, 50%, or 100% c-Maf overexpressed macrophages supernatants can significantly promote the proliferation of HFBs at 48 h (Figure 3B).

| c-Maf overexpressed macrophages facilitated the migration of NFBs and HFBs
Further, wound healing and transwell assay were employed to investigate the effects of c-Maf overexpressed macrophages on fibroblasts migration.In contrast to the control group (0%), medium containing 10%, 50%, or 100% c-Maf overexpressed macrophages culture supernatants can significantly promote the horizontal migration of NFBs at 12 h and 18 h after scratch (Figure 4A).Regarding the HFBs, medium containing 50% and 100% c-Maf overexpressed macrophages culture supernatants can significantly promote the horizontal migration relative to the control cells at 12 h and 18 h, and the pro-migration effect was also found at 18 h when containing 10% c-Maf overexpressed macrophages culture supernatants (Figure 4B).Compared with the negative control group (NC), co-culture with c-Maf overexpressed macrophages can facilitate the vertical migration of both NFBs (Figure 4C) and HFBs (Figure 4D).

| c-Maf overexpressed macrophages upregulated the expressions of genes related to extracellular matrix synthesis in HFBs
To further study, the effects of c-Maf overexpressed macrophages on the expressions of genes related to extracellular matrix synthesis, qPCR, and western blot were performed.The results of qPCR showed that c-Maf overexpressed macrophages significantly increased the mRNA levels of COL1, COL3, and α-SMA in HFBs co-culture with c-Maf overexpressed macrophages (Figure 5A).
Correspondingly, the culture supernatants of c-Maf overexpressed macrophages can also augmented the protein expressions of COL1, COL3, and α-SMA in HFBs (Figure 5B).In addition, the results of qPCR and western blot demonstrated that c-Maf overexpression can elevate the mRNA and protein expressions of TGF-β1 in macrophages (Figure 6A,B).

| DISCUSS ION
During the process of hypertrophic scar formation, macrophages can work in distinct ways: Directly, macrophages function through its manipulation on ECM composition by regulating matrix metalloproteinases (MMPs); indirectly, macrophages work through modulating the activation and stimulation of myofibroblast and collagen production. 12,22In this study, we found increased number of M2 macrophages marked by c-Maf and CD68 in hypertrophic scar tissues.This is consistent with our previous During the process of wound healing, fibroblasts nearby the wound site are activated by macrophages and transform into myofibroblasts, which then migrate into the wound beds and produce lots of ECM to promote wound repair. 15However, abnormal proliferation, migration, and excessive production of ECM in fibroblasts can lead to the formation of hypertrophic scars. 16In this process, 20 222 530) was used to stain the nuclear.Images were acquired using Nikon C2 Confocal microscope (Nikon).
Conclusion: c-Maf positive macrophages promote hypertrophic scar formation through regulating HFBs proliferation, migration, and ECM deposition via the secreted TGF-β1.K E Y W O R D S c-Maf, fibroblast, hypertrophic scar, macrophage | 641 YANG et al.
2 incubator at 37°C.All the procedures were conducted in an aseptic environment.Followed by a culture of 12-15 days, hypertrophic scar derived fibroblasts or normal skin derived fibroblasts (NFBs) grew out and were passaged routinely.The HFBs and NFBs of passages 5-8 were used for this study.Mouse macrophage Raw264.7 cells were purchased from the National Collection of Authenticated Cell Cultures of China.The culture methods for Raw264.7 were the same as HFBs and NFBs.

1
The histological features of hypertrophic scar and quantification of c-Maf + /CD68 + M2 macrophages.(A) The HE staining images (upper) and Masson trichrome staining images (lower) of hypertrophic scar and normal tissues and quantification of extracellular matrix using Masson trichrome staining images.The black arrows indicated the hair follicles.(B) Representative images of c-Maf + /CD68 + M2 macrophages and quantitative analysis in hypertrophic scar and normal tissues.***p < 0.001.NS, normal skin; HS, hypertrophic scar.F I G U R E 2 Transfection of pcDNA3.1-EGFP-c-Mafplasmid into Raw264.7 macrophages.(A) Representative images of Raw264.7 transfected with pcDNA3.1-EGFP-c-Maf(c-Maf) and control plasmids (NC).Green fluorescence can be seen in the c-Maf overexpressed macrophages, whereas no fluorescence was examined in the negative control macrophages.(B) The mRNA expression of c-Maf in macrophages detected by qPCR and quantified using Mann-Whitney U-test.More c-Maf mRNA expression was found in the c-Maf overexpressed macrophages.(C) More c-Maf protein expression was observed in the c-Maf overexpressed macrophages relative to the control macrophages.*p < 0.05.F I G U R E 3 The effects of c-Maf overexpressed macrophages supernatants on the proliferation of NFBs (A) and HFBs (B) at individual time points.*p < 0.05, **p < 0.01, ***p < 0.001.research which showed that M2 macrophages play a vital role in anti-inflammatory process and predominate in the proliferation stage of hypertrophic scar. 11While c-Maf is a critical transcription factor facilitating the expressions of M2 macrophage-associated genes, overexpression of c-Maf in macrophages is conducive to macrophage polarization toward M2 type.13The prolonged infiltration of M2 macrophages in hypertrophic scar coincides with an extended increased expression of Col3A1.14These implied that c-Maf positive M2 macrophages have contributed to the formation of hypertrophic scar.

F I G U R E 4
c-Maf overexpressed macrophages promoted the migration of NFBs and HFBs.Wound healing assay revealed that the c-Maf overexpressed macrophages culture supernatants enhanced the horizontal migration of NFBs (A) and HFBs (B).Transwell assay showed the c-Maf overexpressed macrophages promote the vertical migration of NFBs (C) and HFBs (D).*p < 0.05, **p < 0.01, ***p < 0.001.M2 macrophages serve an important role in facilitating hypertrophic scar formation.Presently, we found that transfection of c-Maf into macrophages led to the elevated expression of TGF-β1 in the c-Maf overexpressed M2 macrophages.Macrophages with overexpressed c-Maf promoted the proliferation and migration of co-cultured HFBs.More importantly, HFBs co-cultured with c-Maf overexpressed macrophages and HFBs cultured in c-Maf overexpressed macrophages culture supernatants expressed more COL1, COL3, and α-SMA.While activation of fibroblasts and accumulation of collagens are the typical marks of ongoing tissue fibrogenesis, alternative activated macrophages (M2) produce elevated mRNA and protein of growth factors including TGF-β1 and PDGF to promote the activation of fibroblasts and excessive production of collagens.17In addition, c-Maf is one of the markers of M2 macrophages.These suggest that transfection of c-Maf to Raw264.7 macrophages leads to the formation of M2 macrophages and contributes to hypertrophic scar pathogenesis through regulating fibroblasts by paracrine.Mahdavian Delavary B exhibited that in the proliferation phase of abnormal wound healing, M2 macrophages synthesize a large amount of TGF-β1 to facilitate myofibroblasts formation from fibroblasts, which then result in the production of collagens and contribute to the excessive deposition of ECM.18 Sheng J et al. showed that the fibroblasts to myofibroblast transition can be induced in fibroblasts from the early-progressed benign prostatic hyperplasia samples using the M2 macrophage-fibroblast co-culture system.19Diana Ta Ploeger et al. revealed that fibroblasts stimulated with the conditioned medium of M2 macrophages displayed increased levels of α-SMA relative to the fibroblasts stimulated with conditioned medium of M1 macrophages. 20More importantly, Nakamura R et al. demonstrated that THP-1 monocyte-derived macrophages treated with TGF-β1 stimulated the production of connective tissue growth factor (CTGF), α-SMA, and fibronectin 1 (FN1) in the co-cultivated fibroblasts. 21These implied that the TGF-β1 secreted by c-Maf overexpressed M2 macrophages not only stimulate the fibroblasts to myofibroblasts transformation and the production of collagens, but also advance the co-cultured fibroblasts to produce more fibrogenic factors.In addition, it also demonstrated the precise function of c-Maf positive M2 macrophages in facilitating hypertrophic scarring and fibrosis.In clinical practice, hypertrophic scar seriously affects the patients.Searching for a target to control hypertrophic scarring is of great significance.M2 macrophages work as one the types of macrophages, their fibrogenic function in hypertrophic scar has been extensively studied. 12In this study, we found increased number of c-Maf positive M2 macrophages in hypertrophic scar tissues, and macrophages with overexpressed c-Maf can produce lots of TGF-β1 to stimulate the fibroblast to myofibroblast transition, while myofibroblasts are the major effector cells for hypertrophic scarring.These suggest that targeting c-Maf in macrophages using small molecule inhibitors, gene interference, or virus of different kinds may serve as potential candidate methods to control hypertrophic scarring through dampening fibroblast activation and myofibroblast differentiation.In addition, targeted intervention of c-Maf positive macrophages in hypertrophic scar may also inhibit hypertrophic scar formation.However, there exist no available F I G U R E 5 c-Maf overexpressed macrophages and its culture supernatant upregulated the expressions of genes associated with extracellular matrix synthesis in HFBs.(A) c-Maf overexpressed macrophages increased the mRNA expression of COL1, COL3, and α-SMA in HFBs co-cultured with c-Maf overexpressed macrophages.(B) c-Maf overexpressed macrophages culture supernatants augmented the protein expression of COL1, COL3, and α-SMA in HFBs.*p < 0.05, **p < 0.01, ***p < 0.001.F I G U R E 6 c-Maf overexpression upregulated the expression of TGF-β1 in macrophages.The results of qPCR and western blot showed that c-Maf overexpression augmented the mRNA (A) and protein (B) expressions of TGF-β1 in macrophages.*p < 0.05, **p < 0.01.drugs or methods to achieve such goal; further researches are still needed.In summary, we for the first time demonstrate that c-Maf positive macrophages promote hypertrophic scar fibroblasts proliferation, migration, and production of ECM through upregulating TGF-β1, thus contributing to hypertrophic scar formation.This may provide a candidate intervention target and theoretical reference for the treatment of hypertrophic scar.