Efficacy And Mechanism Of Macrophages-TREM2 In Regulating Diabetic Wound Healing

BackgroundDiabetes mellitus is a prevalent and serious chronic metabolic disorder showing a rapidly increasing incidence in recent years due to changing LIFestyle habits.Diabetic cutaneous ulcers are one of the most severe complications of diabetes,often exhibiting slow or challenging healing processes.They are characterized by high rates of recurrence and amputation,placing a significant burden on patients and their families.Despite the diverse clinical treatment modalities available for nonhealing diabetic ulcers,the overall clinical efficacy remains suboptimal,leading to a poor prognosis.Cutaneous wound healing is a complex physiological process involving stages of inflammatory response,tissue pro LIFeration,and tissue remodeling.Each stage relies on the coordinated interaction of various cellular components and factors within the human body.However,the hyperglycemic microenvironment that exists in patients with diabetes often causes abnormal local inflammation and oxidative stress reactions,hindering the synthesis of new granulation tissue and epithelial cells,as well as angiogenesis.Thus,the slow healing of diabetic wounds results from multifactorial interactions,necessitating in-depth investigation of the formation and repair mechanisms of diabetic cutaneous wounds.The identification of novel disease markers and therapeutic approaches capable of intervening in the repair processes of diabetic cutaneous wounds holds significant promise from various perspectives,ultimately contributing to improved clinical diagnosis,treatment,and patient outcomes.Triggering receptor expressed on myeloid cells 2(TREM2),initially discovered in2001,is a member of the Trem family.TREM2 is crucial not only for the regulation of inflammatory diseases of the nervous system but also for macrophage-related immune responses,organ fibrosis,epithelial cell repair,lipid metabolism,hair growth,and tumor progression.The skin wound healing process primarily involves stages of inflammatory response,tissue pro LIFeration,and tissue remodeling.It also encompasses responses,such as angiogenesis,extracellular matrix synthesis,wound contraction,and re-epithelialization.Therefore,investigating the potential involvement of the molecular mechanisms of TREM2 in the healing process of diabetic cutaneous wounds is extremely feasible and innovative.TREM2 may emerge as a potential novel molecular target in the clinical diagnosis and treatment of diabetic cutaneous wound healing in the future,providing valuable insights into the clinical management and improvement of outcomes in diabetic cutaneous wounds and other nonhealing wounds.ObjectivesPart One:Explore the differential expression of TREM2 in normal and diabetic skin wound tissues;determine the trend of TREM2 expression in the diabetic skin wound repair process;and validate and assess the role of TREM2 in myeloid cells.Part Two:Investigate the impact of macrophage TREM2 deletion on diabetic skin wound healing;compare the effects of macrophage TREM2 deletion on inflammatory and repair-related indicators in tissues and cells;and explore the direct functional impact of macrophage TREM2 deletion on dermal fibroblasts.Part Three:Elucidate the upstream and downstream mechanisms of TREM2involved in promoting diabetic skin wound repair and validate them using relevant experiments.Part Four:Explore the possibility of using hydrogel-encapsulated TREM2-related proteins to clinically treat diabetic skin wounds.MethodsPart One:(1)Use the Gene Expression Omnibus(GEO)datasets to identify the differential expression of TREM2 in human skin tissues by analyzing sequencing data from human diabetic skin ulcers and normal skin.(2)Utilize C57BL/6 mouse models of diabetic skin wounds,collect samples at different time points for quantitative reverse transcription-polymerase chain reaction(q RT-PCR)assay,western blot(WB),immunohistochemistry analysis,and transcriptome sequencing to investigate the temporal expression trends of TREM2 during wound healing.(3)Validate the functional role of TREM2 in myeloid cells using flow cytometry and immunofluorescence colocalization.Part Two:(1)Establish a full-thickness skin wound model in conditional TREM2 knockout(Trem2-c KO)and wild-type mice(Trem2-flox)to evaluate the impact of macrophage TREM2 deletion on diabetic skin wound healing using various techniques,including hematoxylin and eosin(HE)staining,Masson trichrome staining,WB(collagenⅠ,collagenⅢ,α-smooth muscle actin[α-SMA],interleukin(IL)-6,IL-1β,NADPH oxidase2[NOX2],and thioredoxin binding protein),immunofluorescent staining(CD31,α-SMA,F4/80,CD206,IL-1β,tumor necrosis factorα[TNF-α],dihydroethidium[DHE]Kit,and glutathione peroxidase 4[GPX4]),and flow cytometry(CD11b,F4/80,CD86,and CD206).(2)Assess the influence of TREM2 deletion on macrophage polarization and the secretion of repair-related cytokines by extracting bone marrow-derived macrophages(BMDMs)from Trem2-c KO and Trem2-flox mice following IL-4 stimulation.(3)Investigate the impact of macrophage TREM2 deletion on skin fibroblast migration,pro LIFeration,and oxidative stress by coculturing primary skin fibroblasts with BMDMs from Trem2-c KO and Trem2-flox mice and assessing differences between them using scratch assays,Transwell assays,5-ethynyl 2?-deoxyuridine(Ed U)pro LIFeration assays,WB(α-SMA,transforming growth factor?1[TGF-β1],vimentin,and NOX2),and immunofluorescence staining(DHE kit andα-SMA).Part Three:(1)Identify and validate upstream ligands directly binding to TREM2 using protein mass spectrometry,molecular docking,coimmunoprecipitation(Co-IP),GST-pulldown,and immunofluorescence colocalization experiments.(2)Perform RNA-seq on skin wound tissues from Trem2-c KO and Trem2-flox mice,analyze differential gene expression,and perform functional enrichment analysis to explore potential mechanisms through which TREM2 exerts its effects in diabetic skin wound healing.(3)Perform RNA-seq on BMDMs derived from Trem2-c KO and Trem2-flox mice after IL-4 stimulation.Analyze differential gene expression and perform functional enrichment analysis to identify potential mechanisms through which TREM2 influences macrophage polarization and cytokine secretion.(4)Validate key factors and potential functions of candidate signaling pathways using q RT-PCR,WB,and immunofluorescence experiments.(5)Employ lentiviruses,pathway inhibitors,and functional antibodies to selectively antagonize potential signaling pathways.Confirm the downstream mechanisms of TREM2involved in promoting diabetic skin wound healing through reverse confirmation at animal and cellular levels.Part Four:(1)Synthesize gelatin methacryloyl(Gel MA)hydrogels with and without soluble TREM2(s TREM2)encapsulation for use in subsequent experiments.(2)Perform skin sensitivity tests on C57BL/6 mice to evaluate the sensitivity to blank Gel MA and Gel MA-s TREM2.(3)Model skin wounds in C57BL/6 mice,dividing them into three wound treatment groups(phosphate-buffered saline[PBS],blank Gel MA hydrogel,and Gel MA-s TREM2)and periodically observe and collect samples after light curing.Assess wound healing rates,re-epithelialization,collagen deposition,the inflammatory microenvironment,neovascularization,andα-SMA synthesis using HE staining,Masson trichome staining,immunohistochemistry(IL-6 and IL-1β),and immunofluorescence(F4/80,CD86,α-SMA,and CD31).ResultsPart One:(1)Analysis of sequencing and WB results from the GEO database and C57BL/6mouse skin wound experiments,respectively,revealed that,compared with normal human and mouse skin,diabetic skin wounds showed a significant upregulation of TREM2expression.(2)q RT-PCR and immunohistochemistry results from diabetic mouse skin wounds showed a temporal increase in TREM2 expression,with the most significant increase observed on the third day.(3)Flow cytometry analysis and immunofluorescence colocalization staining demonstrated that TREM2 primarily functioned in macrophages.Part Two:(1)Compared with Trem-flox mice,Trem-c KO diabetic mice showed significantly slower skin wound healing.Histopathological analysis revealed slower re-epithelialization and granulation tissue synthesis rates,with suboptimal collagen deposition.(2)Tissue immunofluorescence revealed that compared with Trem2-flox mice,Trem2-c KO mouse skin wounds showed a significant decrease in repair-related indicators(F4/80~+CD206~+,α-SMA,CD31,GPX4)and an increase in proinflammatory factors(IL-1β,TNF-α).Flow cytometry and WB results showed decreased M2 macrophages in Trem2-c KO skin wounds and reduced collagen I,collagen II,andα-SMA synthesis,respectively.(3)Immunofluorescence staining showed weakened M2 polarization ability in BMDMs derived from Trem2-c KO mice;enzyme-linked immunosorbent assay(ELISA)results showed decreased expression of anti-inflammatory factor IL-10 and growth factors(TGF-β1,platelet-derived growth factor,and vascular endothelial growth factor)in BMDMs from Trem2-c KO mice.(4)Immunofluorescence results revealed reduced levels ofα-SMA expression and enhanced levels of reactive oxygen species(ROS)in skin fibroblasts cocultured with BMDMs from Trem2-c KO mice.Furthermore,scratch,Transwell,and Ed U assays demonstrated reduced migration and pro LIFeration capabilities,and WB showed decreased expression levels ofα-SMA,TGF-β1,and vimentin and increased NOX2 activity.Part Three:(1)Protein mass spectrometry showed IL-4 as a potential ligand that directly binds to TREM2.Additionally.molecular docking experiments showed a strong possibility of direct binding between IL-4 and TREM2,predominantly at extracellular sites.Co-IP and GST-pulldown results showed direct binding of IL-4 to endogenous and exogenous TREM2,primarily in the 1–132-aa segment.Co-IP results in IL-4 receptorα-knockout RAW264.7 cells indicated the direct binding of IL-4 to TREM2 independent of its classical receptor.Immunofluorescence results confirmed the direct binding of IL-4 to TREM2,predominantly on macrophage membranes.(2)RNA-seq results revealed 1283 and 1156 differentially expressed genes in skin wound tissues and BMDMs,respectively,from Trem2-c KO and Trem2-flox mice.Enrichment analysis identified potential pathways,and upon combining the sequencing results with our background research,we propose that TREM2 regulates the mitogen-activated protein kinase/activator protein 1/leukemia inhibitory factor(MAPK/AP-1/LIF)pathway during diabetic skin wound healing.q RT-PCR and WB results confirmed the key factors in this pathway.(3)Macrophage TREM2 deletion inhibited M2 polarization,which was reversed by inhibition of the MAPK signaling pathway and suppression of AP-1 transcription factor expression.(4)Lentiviral inhibition of AP-1 family transcription factors improved the wound healing rate in Trem2-c KO mice,promoted macrophage M2 polarization,accelerated neovascularization,α-SMA synthesis,re-epithelialization,and collagen deposition,and suppressed excessive ROS production.(5)Specific inhibitors of the MAPK signaling pathway and lentiviral inhibition of AP-1 expression reversed the effects of macrophage TREM2 deletion on skin fibroblast function.Part Four:(1)Blank Gel MA and Gel MA-s TREM2 were nonallergenic in mouse skin sensitivity tests.(2)Gel MA-s TREM2 increased the rate of mouse skin wound repair.(3)Histopathological examination revealed that,compared with blank Gel MA and PBS,Gel MA-s TREM2 promoted re-epithelialization,increased granulation tissue synthesis,and resulted in a tighter arrangement of collagen.Immunohistochemistry results showed improved suppression of local inflammatory factors with Gel MA-s TREM2.Immunofluorescence results revealed that,compared with Gel MA and PBS,Gel MA-s TREM2 improved the inhibition of local M1 polarization,significantly enhanced neovascularization,and increasedα-SMA synthesis.ConclusionsThis study confirms that TREM2,serving as a nonspecific receptor for IL-4,facilitates the healing of diabetic skin wounds.The underlying mechanism involves the downstream signaling of MAPK/AP-1/LIF,regulating the phenotypic transformation of macrophages.This modulation suppresses local inflammation at the wound site and promotes neovascularization,α-SMA synthesis,collagen deposition,and relevant activities in skin fibroblasts.Future interventions related to TREM2 could serve as potential novel clinical treatments for diabetic skin wounds.TREM2 is expected to be a new target for clinical treatment of diabetic skin wounds in the future.