Inhibiting CSF1R Signaling Accelerates Scar Tissue Remodeling By Modulating Macrophage Polarization

Background:Hypertrophic scar is characterized by excessive deposition of extracellular matrix,and there is still a lack of effective treatment at present.The essence of hypertrophic scar is the imbalance between collagen formation and catabolism.As the pioneer of tissue repair,macrophage-mediated changes in the immune microenvironment have always been the focus of research in tissue healing and regeneration.A large number of studies have confirmed that long-term abnormal polarization of macrophages is the essential factor leading to hypertrophic scar.In this state,macrophages secrete a large number of fibrosis factors,triggering a cascade that eventually leads to tissue fibrosis.Colony stimulation factor-1 receptor(CSFIR)is a receptor tyrosine kinases(RTKs)expressed on the surface of macrophages.Signal changes caused by the combination of colony stimulation factor-1(CSF1)and CSF1R have been proved to be an crucial pathway to regulate macrophage growth,proliferation and differentiation.The role of CSF1/CSF1R signaling pathway in hypertrophic scar formation has not been reported.Considering that this signaling pathway has shown effective anti-inflammatory function in regulating the phenotypic transformation of macrophages,and the abnormal activation of M2 macrophages has long been observed in the formation of hypertrophic scar,therefore,we plan to take CSF1R as the focus,to regulate the polarization of macrophages through specific inhibition of CSF1R,and to accelerate the remodeling process of scar tissue,so as to achieve the ideal anti-scarring effects.Objective:(1)To determine whether CSF1R is a key factor in the formation of hypertrophic scar.(2)To clarify how the CSF1R inhibitor regulates macrophages and thus plays an anti-scarring role.Methods:1.Expression characteristics of CSF1R in hypertrophic scar tissue(1)The expression of CSF1R in hypertrophic scar and normal skin was detected by qPCR and ELISA;(2)Immunostaining was used to detect the difference in the distribution of M2 macrophages between hypertrophic scar and normal skin;(3)The animal model of wound repair was constructed,and the samples were collected at multiple time points after the injury,and the dynamic expression of Csflr was detected by qPCR.2.Effects of inhibiting CSF1R signal in vivo on the formation of hypertrophic scar(1)The models of hypertrophic scar in mouse tails and rabbit ears were constructed and grouped;(2)After wound epithelialization,the experimental group(Group GW2580)was treated with CSF1R inhibitors;(3)One month after surgery,the scar was assessed:Vancouver scar scale(VSS)was used to assess the features of scar,HE and Masson’s trichrome stain were used to observe the histological morphology of the scar,immunofluorescence and immunohistochemistry techniques were used to detect the expression of CSF1R and fibrosis markers,qPCR was used to detect the expression of fibrosin-related genes;3.Effects of inhibiting CSF1R signal in vitro on the bioactivity of hypertrophic scar fibroblasts(1)Effects of the polarization of Raw 264.7 cells treated with different stimuli were determined:Raw 264.7 cells were stimulated with CSF1,CSF1+GW2580 and CSF1+GDC0068 respectively.Genes related to macrophages polarization were detected by qPCR,and markers of M1/M2 macrophages,CD86/CD206,were detected by flow cytometry;(2)Effects of macrophages in different polarized states on the proliferation and migration of hypertrophic scar fibroblasts were determined:through cell co-culture techniques,macrophages subjected to different stimuli were co-cultured with hypertrophic scar fibroblasts,CCK-8 was used to detect the proliferation of scar fibroblasts,migration of scar fibroblasts was observed by scraping line method.Results:1.Expression characteristics of CSF1R in hypertrophic scar tissue(1)The expression of CSF1R in adult hypertrophic scar tissue was significantly higher than that in adult normal skin.CSF1R protein in adult hypertrophic scar tissue was significantly higher than that in adult normal skin.(2)Immunofluorescence staining showed that the density of CD206,a specific marker of M2 macrophages,was higher in adult hypertrophic scar than in normal adult skin.(3)During wound healing in mice,the expression of Csflr gradually increased with time,reached the peak 16 days after surgery.Up to 30 days after surgery,the expression of Csflr was still at a high level.2.Effects of inhibiting CSF1R signal in vivo on hypertrophic scar formation(1)One month after surgery,the appearance of scar in the Control group of mice was more obvious:the scar was red with hyperemia,higher than the surrounding skin,and it feels hard,which was significantly different from the surrounding skin.However,the appearance of scar in Group GW2580 was similar to that of the surrounding skin,not higher than the surface of the surroundings,and it feels soft.After inhibiting CSF1R,the VSS of the hypertrophic scar in mouse tails decreased significantly.In rabbit ear models:after inhibiting CSF1R signal,the VSS of hypertrophic scar in rabbit ears decreased significantly.(2)Scars in mouse tail:HE staining showed that the collagen fibers inside the scar in the Control group were thick,overlapping and disordered.A large number of fibroblasts proliferated and infiltrated among collagen fibers.Collagen fibers of Group GW2580 were relatively thin and neatly arranged,with a small number of fibroblast infiltration.The scar elevation index(SEI)of Group GW2580 was lower than that of Control group.Masson’s trichrome stain showed that a large number of collagen fibers were dyed blue inside the scar in the Control group,and the structure were thick and disordered.However,the collagen fibers in the scars of Group GW2580 were fine and neatly arranged,and the collagen content of Group GW2580 was significantly lower than that of the Control group.Scars in rabbit ears:HE staining showed that the collagen fibers inside the scar in the Control group were thick and disordered.The collagen fibers in the scar of Group GW2580 were relatively thin and neatly arranged.Masson trichrome staining showed that the collagen fibers were dyed blue in the scars of the Control group,with a thick and disordered structure,while the collagen fibers in the scars of Group GW2580 were slender and neatly arranged,and the collagen content was significantly lower than that of the Control group.(3)In mouse tail scars,the expression of fibrosis-related genes such as Collal,Ctgf,Tgfβl and Acta2 in Group GW2580 were significantly lower than those in the Control group.In rabbit ear scars,the expression of fibrosis-related genes such as Collal,Fnl and Tnc in Group GW2580 were significantly lower than those in the Control group.(4)Immunofluorescence results showed that the expression of TGF-β,α-SMA and CD206 in the Control group of mice were higher than those in Group GW2580.In rabbit ear scars,the expression of Collagen 1 and α-SMA in the Control group were higher than those of Group GW2580.(5)The results of immunohistochemistry showed that the expression of CSF1R and Smad2 in the Control group of mice were higher than those in Group GW2580.3.Effects of inhibiting CSF1R signal in vitro on the bioactivity of hypertrophic scar fibroblasts(1)Raw 264.7 cells were stimulated by CSF1,the expression of Cd206(marker of M2)was significantly up-regulated,and Cd86(marker of M1)was decreased.In Group CSF1+GW2580,no significant change was observed in Cd206 and Cd86.In Group CSF1+GDC00 68,there was no significant change in Cd206 and Cd86.(2)The positive rate of CD86 in Raw 264.7 cells stimulated by CSF1 was 88.9%,and that of CD206 was 99.9%.In Group CSF1+GW2580,the positive rate of CD86 was 100%,and that of CD206 was 88.2%.In Group CSF1+GDC0068,the positive rate of CD86 was 99.5%,and that of CD206 was 89.0%.(3)Co-culture of macrophages and hypertrophic scar fibroblasts:from 12 h,the proliferation rate of fibroblasts in Group CSF1 began to increase,and reached the peak at 48 h,and then gradually slowed down.After 24 h,the speed of Group CSF1 was significantly faster than that of Group CSF1+GW2580,Group CSF1+GDC0068 and the Control Group(hypertrophic scar fibroblasts cultured in normal medium).The growth curves of Group CSF1+GW2580 and Group CSF1+GDC0068 were similar to those of the Control group.The proliferative activity of all cells decreased after 60 h.At 12 h,the migration rate of hypertrophic scar fibroblasts in the Group CSF1 was significantly faster than that in the other three groups,and at 24 h,the migration was completed in Group CSF1.Compared with the Control group,Group CSF1+GW2580 and Group CSF1+GDC0068 showed no significant difference in the migration of fibroblasts.Conclusions:1.Compared with normal skin,the expression of CSF1R and CD206 were increased in hypertrophic scar,suggesting that CSF1R may be a factor involved in the formation of hypertrophic scar.During the process of wound healing,the expression of CSF1R gradually increases with time and remains at a relatively high level in the later stage,suggesting that CSF1R is involved in regulating the process of wound healing,especially in remodeling stage.2.Inhibition of CSF1R signal in the hypertrophic scar in mouse tails and rabbit ears can significantly reduce the level of M2 macrophages and the expression of fibrosis related genes,accelerate the remodeling of scar tissue,and thereby reduce the formation of hypertrophic scar;3.The M2-type phenotypic polarization of macrophages is regulated by CSF1/CSF1R signaling.By inhibiting CSF1R signal,the ability of M2 macrophages to promote the proliferation and migration of hypertrophic scar fibroblasts will be inhibited at the same time,which may be related to the blocking of the Akt pathway.