While hypoxic signaling has been shown to play a role in many cellular processes,its role in metabolism-linked extracellular matrix(ECM)organization and downstream processes of cell fate after musculoskeletal injury r...While hypoxic signaling has been shown to play a role in many cellular processes,its role in metabolism-linked extracellular matrix(ECM)organization and downstream processes of cell fate after musculoskeletal injury remains to be determined.Heterotopicossification(HO)is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues.Hypoxia andhypoxia-inducible factor 1α(HIF-1α)activation have been shown to promote HO.However,the underlying molecular mechanisms bywhich the HIF-1αpathway in mesenchymal progenitor cells(MPCs)contributes to pathologic bone formation remain to beelucidated.Here,we used a proven mouse injury-induced HO model to investigate the role of HIF-1αon aberrant cell fate.Usingsingle-cell RNA sequencing(scRNA-seq)and spatial transcriptomics analyses of the HO site,we found that collagen ECM organizationis the most highly up-regulated biological process in MPCs.Zeugopod mesenchymal cell-specific deletion of Hif1α(Hoxa11-CreER^(T2);Hif1a^(fl/fl))significantly mitigated HO in vivo.ScRNA-seq analysis of these Hoxa11-CreER^(T2);Hif1a^(fl/fl)mice identified the PLOD2/LOXpathway for collagen cross-linking as downstream of the HIF-1αregulation of HO.Importantly,our scRNA-seq data and mechanisticstudies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1αdeletion.From a translational aspect,a pan-LOX inhibitor significantly decreased HO.A newly screened compound revealed that the inhibition of PLOD2 activity in MPCssignificantly decreased osteogenic differentiation and glycolytic metabolism.This suggests that the HIF-1α/PLOD2/LOX axis linked tometabolism regulates HO-forming MPC fate.These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promisingstrategy to mitigate HO formation.展开更多
Heterotopic ossification(HO)is a pathological process that commonly arises following severe polytrauma,characterized by the anomalous differentiation of mesenchymal progenitor cells and resulting in the formation of e...Heterotopic ossification(HO)is a pathological process that commonly arises following severe polytrauma,characterized by the anomalous differentiation of mesenchymal progenitor cells and resulting in the formation of ectopic bone in non-skeletal tissues.This abnormal bone growth contributes to pain and reduced mobility,especially when adjacent to a joint.Our prior observations suggested an essential role of NGF(Nerve Growth Factor)-responsive TrkA(Tropomyosin Receptor Kinase A)-expressing peripheral nerves in regulating abnormal osteochondral differentiation following tendon injury.Here,we utilized a recently developed mouse model of hip arthroplasty-induced HO to further validate the role of peripheral nerve regulation of traumatic HO.Nerve ingrowth was either modulated using a knockin transgenic animals with point mutation in TrkA,or local treatment with an FDA-approved formulation of long acting Bupivacaine which prevents peripheral nerve growth.Results demonstrate exuberant sensory and sympathetic nerve growth within the peri-articular HO site,and that both methods to reduce local innervation significantly reduced heterotopic bone formation.TrkA inhibition led to a 34%reduction in bone volume,while bupivacaine treatment resulted in a 50%decrease.Mechanistically,alterations in TGFβand FGF signaling activation accompanied both methods of local denervation,and a shift in macrophages from M1 to M2 phenotypes was observed.In sum,these studies reinforce the observations that peripheral nerves play a role in the etiopathogenesis of HO,and that targeting local nerves represents a potential therapeutic approach for disease prevention.展开更多
Osteoblasts,which are the bone-forming cells,operate in a hypoxic environment.The transcription factors hypoxia-inducible factor-1α(HIF1)and HIF2 are key mediators of the cellular response to hypoxia.Both are express...Osteoblasts,which are the bone-forming cells,operate in a hypoxic environment.The transcription factors hypoxia-inducible factor-1α(HIF1)and HIF2 are key mediators of the cellular response to hypoxia.Both are expressed in osteoblasts.HIF1 is known to be a positive regulator of bone formation.Conversely,the role of HIF2 in the control osteoblast biology is still poorly understood.In this study,we used mouse genetics to demonstrate that HIF2 is an inhibitor of osteoblastogenesis and bone mass accrual.Moreover,we provided evidence that HIF2 impairs osteoblast differentiation at least in part,by upregulating the transcription factor Sox9.Our findings constitute a paradigm shift,as activation of the hypoxia-signaling pathway has traditionally been associated with increased bone formation through HIF1.Inhibiting HIF2 could thus represent a therapeutic approach for the treatment of the low bone mass observed in chronic diseases,osteoporosis,or aging.展开更多
Heterotopic ossification(HO)is a debilitating condition characterized by the pathologic formation of ectopic bone.HO occurs commonly following orthopedic surgeries,burns,and neurologic injuries.While surgical excision...Heterotopic ossification(HO)is a debilitating condition characterized by the pathologic formation of ectopic bone.HO occurs commonly following orthopedic surgeries,burns,and neurologic injuries.While surgical excision may provide palliation,the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone.Based on these clinical observations,we set out to examine the role of vascular signaling in HO.Vascular endothelial growth factor A(VEGFA)has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis.Our findings,using a validated mouse model of HO,demonstrate that HO lesions are highly vascular,and that VEGFA is critical to ectopic bone formation,despite lacking a contribution of endothelial cells within the developing anlagen.展开更多
Heterotopic ossification(HO)is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues.During this process,mesenchymal progenitor cells undergo endochondral ossification.N...Heterotopic ossification(HO)is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues.During this process,mesenchymal progenitor cells undergo endochondral ossification.Nonetheless,the specific cell phenotypes and mechanisms driving this process are not well understood,in part due to the high degree of heterogeneity of the progenitor cells involved.Here,using a combination of lineage tracing and single-cell RNA sequencing(sc RNA-seq),we investigated the extent to which synovial/tendon sheath progenitor cells contribute to heterotopic bone formation.For this purpose,Tppp3(tubulin polymerization-promoting protein family member 3)-inducible reporter mice were used in combination with either Scx(Scleraxis)or Pdgfra(platelet derived growth factor receptor alpha)reporter mice.Both tendon injury-and arthroplasty-induced mouse experimental HO models were utilized.Sc RNA-seq of tendon-associated traumatic HO suggested that Tppp3 is an early progenitor cell marker for either tendon or osteochondral cells.Upon HO induction,Tppp3 reporter^(+)cells expanded in number and partially contributed to cartilage and bone formation in either tendon-or joint-associated HO.In double reporter animals,both Pdgfra^(+)Tppp3^(+)and Pdgfra^(+)Tppp3^(-) progenitor cells gave rise to HO-associated cartilage.Finally,analysis of human samples showed a substantial population of TPPP3^(-) expressing cells overlapping with osteogenic markers in areas of heterotopic bone.Overall,these data demonstrate that synovial/tendon sheath progenitor cells undergo aberrant osteochondral differentiation and contribute to HO after trauma.展开更多
The functional interdependence of nerves and blood vessels is a well-established concept during tissue morphogenesis, yet the role of neurovascular coupling in proper and aberrant tissue repair is an emerging field of...The functional interdependence of nerves and blood vessels is a well-established concept during tissue morphogenesis, yet the role of neurovascular coupling in proper and aberrant tissue repair is an emerging field of interest. Here, we sought to define the regulatory relationship of peripheral nerves on vasculature in a severe extremity trauma model in mice, which results in aberrant cell fate and heterotopic ossification(HO). First, a high spatial degree of neurovascular congruency was observed to exist within extremity injury associated heterotopic ossification. Vascular and perivascular cells demonstrate characteristic responses to injury,as assessed by single cell RNA sequencing. This vascular response to injury was blunted in neurectomized mice, including a decrease in endothelial proliferation and type H vessel formation, and a downregulation of key transcriptional networks associated with angiogenesis. Independent mechanisms to chemically or genetically inhibit axonal ingrowth led to similar deficits in HO site angiogenesis, a reduction in type H vessels, and heterotopic bone formation. Finally, a combination of single cell transcriptomic approaches within the dorsal root ganglia identified key neural-derived angiogenic paracrine factors that may mediate neuron-to-vascular signaling in HO. These data provide further understanding of nerve-to-vessel crosstalk in traumatized soft tissues, which may reflect a key determinant of mesenchymal progenitor cell fate after injury.展开更多
Self-renewal and differentiation of skeletal stem and progenitor cells(SSPCs)are tightly regulated processes,with SSPC dysregulation leading to progressive bone disease.While the application of single-cell RNA sequenc...Self-renewal and differentiation of skeletal stem and progenitor cells(SSPCs)are tightly regulated processes,with SSPC dysregulation leading to progressive bone disease.While the application of single-cell RNA sequencing(scRNAseq)to the bone field has led to major advancements in our understanding of SSPC heterogeneity,stem cells are tightly regulated by their neighboring cells which comprise the bone marrow niche.However,unbiased interrogation of these cells at the transcriptional level within their native niche environment has been challenging.Here,we combined spatial transcriptomics and scRNAseq using a predictive modeling pipeline derived from multiple deconvolution packages in adult mouse femurs to provide an endogenous,in vivo context of SSPCs within the niche.This combined approach localized SSPC subtypes to specific regions of the bone and identified cellular components and signaling networks utilized within the niche.Furthermore,the use of spatial transcriptomics allowed us to identify spatially restricted activation of metabolic and major morphogenetic signaling gradients derived from the vasculature and bone surfaces that establish microdomains within the marrow cavity.Overall,we demonstrate,for the first time,the feasibility of applying spatial transcriptomics to fully mineralized tissue and present a combined spatial and single-cell transcriptomic approach to define the cellular components of the stem cell niche,identify cell-cell communication,and ultimately gain a comprehensive understanding of local and global SSPC regulatory networks within calcified tissue.展开更多
Macrophage plasticity is critical for normal tissue repair following injury.In pathologic states such as diabetes,macrophage plasticity is impaired,and macrophages remain in a persistent proinflammatory state;however,...Macrophage plasticity is critical for normal tissue repair following injury.In pathologic states such as diabetes,macrophage plasticity is impaired,and macrophages remain in a persistent proinflammatory state;however,the reasons for this are unknown.Here,using single-cell RNA sequencing of human diabetic wounds,we identified increased JMJD3 in diabetic wound macrophages,resulting in increased inflammatory gene expression.Mechanistically,we report that in wound healing,JMJD3 directs early macrophage-mediated inflammation via JAK1,3/STAT3 signaling.However,in the diabetic state,we found that IL-6,a cytokine increased in diabetic wound tissue at later time points post-injury,regulates JMJD3 expression in diabetic wound macrophages via the JAK1,3/STAT3 pathway and that this late increase in JMJD3 induces NFκB-mediated inflammatory gene transcription in wound macrophages via an H3K27me3 mechanism.Interestingly,RNA sequencing of wound macrophages isolated from mice with JMJD3-deficient myeloid cells(Jmjd3f/fLyz2Cre+)identified that the STING gene(Tmem173)is regulated by JMJD3 in wound macrophages.STING limits inflammatory cytokine production by wound macrophages during healing.However,in diabetic mice,its role changes to limit wound repair and enhance inflammation.This finding is important since STING is associated with chronic inflammation,and we found STING to be elevated in human and murine diabetic wound macrophages at late time points.Finally,we demonstrate that macrophage-specific,nanoparticle inhibition of JMJD3 in diabetic wounds significantly improves diabetic wound repair by decreasing inflammatory cytokines and STING.Taken together,this work highlights the central role of JMJD3 in tissue repair and identifies cell-specific targeting as a viable therapeutic strategy for nonhealing diabetic wounds.展开更多
文摘While hypoxic signaling has been shown to play a role in many cellular processes,its role in metabolism-linked extracellular matrix(ECM)organization and downstream processes of cell fate after musculoskeletal injury remains to be determined.Heterotopicossification(HO)is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues.Hypoxia andhypoxia-inducible factor 1α(HIF-1α)activation have been shown to promote HO.However,the underlying molecular mechanisms bywhich the HIF-1αpathway in mesenchymal progenitor cells(MPCs)contributes to pathologic bone formation remain to beelucidated.Here,we used a proven mouse injury-induced HO model to investigate the role of HIF-1αon aberrant cell fate.Usingsingle-cell RNA sequencing(scRNA-seq)and spatial transcriptomics analyses of the HO site,we found that collagen ECM organizationis the most highly up-regulated biological process in MPCs.Zeugopod mesenchymal cell-specific deletion of Hif1α(Hoxa11-CreER^(T2);Hif1a^(fl/fl))significantly mitigated HO in vivo.ScRNA-seq analysis of these Hoxa11-CreER^(T2);Hif1a^(fl/fl)mice identified the PLOD2/LOXpathway for collagen cross-linking as downstream of the HIF-1αregulation of HO.Importantly,our scRNA-seq data and mechanisticstudies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1αdeletion.From a translational aspect,a pan-LOX inhibitor significantly decreased HO.A newly screened compound revealed that the inhibition of PLOD2 activity in MPCssignificantly decreased osteogenic differentiation and glycolytic metabolism.This suggests that the HIF-1α/PLOD2/LOX axis linked tometabolism regulates HO-forming MPC fate.These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promisingstrategy to mitigate HO formation.
基金supported by NIH/NIAMS (P01 AG066603,R01 AR079171,R01 AR079171-07S1,R21AR078919)NIH/NIDCR (R01 DE031488,R01 DE031028)+4 种基金Alex’s Lemonade Stand Foundation (22-26743)American Cancer Society (DBG-23-1155131-01-IBCD)the Maryland Stem Cell Research Foundation (2021-MSCRFD-5641)Department of Defense (USAMRAA HT9425-24-1-0051)supported by NIH/NIAMS (R01 AR079171-07,R01 AR079171-07S1)。
文摘Heterotopic ossification(HO)is a pathological process that commonly arises following severe polytrauma,characterized by the anomalous differentiation of mesenchymal progenitor cells and resulting in the formation of ectopic bone in non-skeletal tissues.This abnormal bone growth contributes to pain and reduced mobility,especially when adjacent to a joint.Our prior observations suggested an essential role of NGF(Nerve Growth Factor)-responsive TrkA(Tropomyosin Receptor Kinase A)-expressing peripheral nerves in regulating abnormal osteochondral differentiation following tendon injury.Here,we utilized a recently developed mouse model of hip arthroplasty-induced HO to further validate the role of peripheral nerve regulation of traumatic HO.Nerve ingrowth was either modulated using a knockin transgenic animals with point mutation in TrkA,or local treatment with an FDA-approved formulation of long acting Bupivacaine which prevents peripheral nerve growth.Results demonstrate exuberant sensory and sympathetic nerve growth within the peri-articular HO site,and that both methods to reduce local innervation significantly reduced heterotopic bone formation.TrkA inhibition led to a 34%reduction in bone volume,while bupivacaine treatment resulted in a 50%decrease.Mechanistically,alterations in TGFβand FGF signaling activation accompanied both methods of local denervation,and a shift in macrophages from M1 to M2 phenotypes was observed.In sum,these studies reinforce the observations that peripheral nerves play a role in the etiopathogenesis of HO,and that targeting local nerves represents a potential therapeutic approach for disease prevention.
基金supported by NIH grants AR067330 (to E.S.) and NIH1R01AR071379 (to B.L.).C.M.attributed a grant by the University of Michigan Orthopaedic Research Advisory Committee (RAC G020329)
文摘Osteoblasts,which are the bone-forming cells,operate in a hypoxic environment.The transcription factors hypoxia-inducible factor-1α(HIF1)and HIF2 are key mediators of the cellular response to hypoxia.Both are expressed in osteoblasts.HIF1 is known to be a positive regulator of bone formation.Conversely,the role of HIF2 in the control osteoblast biology is still poorly understood.In this study,we used mouse genetics to demonstrate that HIF2 is an inhibitor of osteoblastogenesis and bone mass accrual.Moreover,we provided evidence that HIF2 impairs osteoblast differentiation at least in part,by upregulating the transcription factor Sox9.Our findings constitute a paradigm shift,as activation of the hypoxia-signaling pathway has traditionally been associated with increased bone formation through HIF1.Inhibiting HIF2 could thus represent a therapeutic approach for the treatment of the low bone mass observed in chronic diseases,osteoporosis,or aging.
基金B.L.:Supported by funding from NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases NIH1R01AR071379American College of Surgeons Clowes Award.D.M.S.:Supported by Plastic Surgery Foundation Resident Research Award+6 种基金M.S.:Supported by Plastic Surgery Foundation National Endowment AwardC.H.:Supported by Howard Hughes Medical Institute Medical Research FellowshipJ.L.:Supported by Vascular Surgery T32 5-T32-HL-076123–14A.W.J.:Supported by the NIH/NIAMS(R01 AR070773,K08 AR068316,S10OD016374)the Orthopedic Research and Education Foundation with funding provided by the Maryland Stem Cell Research Foundation,and the Musculoskeletal Transplant FoundationP.B.Y.:Supported by funding from NIH/NIAMS R01 AR057374 and NHLBI R01 HL131910Y.M.:Supported by funding from NIH/NIDCR R01 DE020843 and DE027662
文摘Heterotopic ossification(HO)is a debilitating condition characterized by the pathologic formation of ectopic bone.HO occurs commonly following orthopedic surgeries,burns,and neurologic injuries.While surgical excision may provide palliation,the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone.Based on these clinical observations,we set out to examine the role of vascular signaling in HO.Vascular endothelial growth factor A(VEGFA)has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis.Our findings,using a validated mouse model of HO,demonstrate that HO lesions are highly vascular,and that VEGFA is critical to ectopic bone formation,despite lacking a contribution of endothelial cells within the developing anlagen.
基金funded by the NIH/NIAMS (R01 AR070773,R01 AR068316,R01 DE031028,R21 AR078919)USAMRAA through the Peer Reviewed Medical Research Program (W81XWH-18-1-0121,W81XWH-18-1-0336)+4 种基金the Peer Reviewed Orthopaedic Research Program (W81XWH-20-10795)Broad Agency Announcement (W81XWH-1810613)the American Cancer Society (Research Scholar Grant,RSG-18-027-01-CSM)the Maryland Stem Cell Research Foundationfunded by the NIH (R01 AR079171,R01 AR078324,and R01 AR071379)。
文摘Heterotopic ossification(HO)is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues.During this process,mesenchymal progenitor cells undergo endochondral ossification.Nonetheless,the specific cell phenotypes and mechanisms driving this process are not well understood,in part due to the high degree of heterogeneity of the progenitor cells involved.Here,using a combination of lineage tracing and single-cell RNA sequencing(sc RNA-seq),we investigated the extent to which synovial/tendon sheath progenitor cells contribute to heterotopic bone formation.For this purpose,Tppp3(tubulin polymerization-promoting protein family member 3)-inducible reporter mice were used in combination with either Scx(Scleraxis)or Pdgfra(platelet derived growth factor receptor alpha)reporter mice.Both tendon injury-and arthroplasty-induced mouse experimental HO models were utilized.Sc RNA-seq of tendon-associated traumatic HO suggested that Tppp3 is an early progenitor cell marker for either tendon or osteochondral cells.Upon HO induction,Tppp3 reporter^(+)cells expanded in number and partially contributed to cartilage and bone formation in either tendon-or joint-associated HO.In double reporter animals,both Pdgfra^(+)Tppp3^(+)and Pdgfra^(+)Tppp3^(-) progenitor cells gave rise to HO-associated cartilage.Finally,analysis of human samples showed a substantial population of TPPP3^(-) expressing cells overlapping with osteogenic markers in areas of heterotopic bone.Overall,these data demonstrate that synovial/tendon sheath progenitor cells undergo aberrant osteochondral differentiation and contribute to HO after trauma.
基金JHU microscopy facility.A.W.J.was funded by NIH/NIAMS(R01 AR070773),NIH/NIDCR(R21 DE027922)USAMRAA through the Peer-Reviewed Medical Research Program(W81XWH-18–1–0121,W81XWH-18–1–0336)+7 种基金Broad Agency Announcement(W81XWH-18–10613)American Cancer Society(Research Scholar Grant,RSG-18–027–01-CSM)the Maryland Stem Cell Research Foundation.B.L.funded by the NIH(1R01 AR071379)funded by NIH(R01 AR079171)Do D(W81XWH-20–1–0795)supported by the NIH/NIAMS(R01 AR068934)NIH/NIDCR(R21 DE027922)the VA(Merit Award and Senior Research Career Scientist Award)。
文摘The functional interdependence of nerves and blood vessels is a well-established concept during tissue morphogenesis, yet the role of neurovascular coupling in proper and aberrant tissue repair is an emerging field of interest. Here, we sought to define the regulatory relationship of peripheral nerves on vasculature in a severe extremity trauma model in mice, which results in aberrant cell fate and heterotopic ossification(HO). First, a high spatial degree of neurovascular congruency was observed to exist within extremity injury associated heterotopic ossification. Vascular and perivascular cells demonstrate characteristic responses to injury,as assessed by single cell RNA sequencing. This vascular response to injury was blunted in neurectomized mice, including a decrease in endothelial proliferation and type H vessel formation, and a downregulation of key transcriptional networks associated with angiogenesis. Independent mechanisms to chemically or genetically inhibit axonal ingrowth led to similar deficits in HO site angiogenesis, a reduction in type H vessels, and heterotopic bone formation. Finally, a combination of single cell transcriptomic approaches within the dorsal root ganglia identified key neural-derived angiogenic paracrine factors that may mediate neuron-to-vascular signaling in HO. These data provide further understanding of nerve-to-vessel crosstalk in traumatized soft tissues, which may reflect a key determinant of mesenchymal progenitor cell fate after injury.
基金funded by R01HD107034 and R21HD106162 by the NIH/NICHD(MCS)the Faculty of Surgery Pilot Research Award and grant HT94252310327 from the DoD(R.J.T.)。
文摘Self-renewal and differentiation of skeletal stem and progenitor cells(SSPCs)are tightly regulated processes,with SSPC dysregulation leading to progressive bone disease.While the application of single-cell RNA sequencing(scRNAseq)to the bone field has led to major advancements in our understanding of SSPC heterogeneity,stem cells are tightly regulated by their neighboring cells which comprise the bone marrow niche.However,unbiased interrogation of these cells at the transcriptional level within their native niche environment has been challenging.Here,we combined spatial transcriptomics and scRNAseq using a predictive modeling pipeline derived from multiple deconvolution packages in adult mouse femurs to provide an endogenous,in vivo context of SSPCs within the niche.This combined approach localized SSPC subtypes to specific regions of the bone and identified cellular components and signaling networks utilized within the niche.Furthermore,the use of spatial transcriptomics allowed us to identify spatially restricted activation of metabolic and major morphogenetic signaling gradients derived from the vasculature and bone surfaces that establish microdomains within the marrow cavity.Overall,we demonstrate,for the first time,the feasibility of applying spatial transcriptomics to fully mineralized tissue and present a combined spatial and single-cell transcriptomic approach to define the cellular components of the stem cell niche,identify cell-cell communication,and ultimately gain a comprehensive understanding of local and global SSPC regulatory networks within calcified tissue.
基金This work was supported in part by National Institute of Health grants R01-HL137919(KAG),R01-DK124290-01(KAG,BBM),R01-AR 07986301(KAG),R01-HL156274-01A1(KAG),R01-DK 12753101 A1(KAG),Doris Duke Foundation CSDA 2017079(KAG),NIH F32-DK126471(COA)the Vascular and Endovascular Surgical Society Resident Research Award(COA),the Society for Vascular Surgery Resident Research Award(COA)and the Coller Surgical Society Resident Research Award(COA)。
文摘Macrophage plasticity is critical for normal tissue repair following injury.In pathologic states such as diabetes,macrophage plasticity is impaired,and macrophages remain in a persistent proinflammatory state;however,the reasons for this are unknown.Here,using single-cell RNA sequencing of human diabetic wounds,we identified increased JMJD3 in diabetic wound macrophages,resulting in increased inflammatory gene expression.Mechanistically,we report that in wound healing,JMJD3 directs early macrophage-mediated inflammation via JAK1,3/STAT3 signaling.However,in the diabetic state,we found that IL-6,a cytokine increased in diabetic wound tissue at later time points post-injury,regulates JMJD3 expression in diabetic wound macrophages via the JAK1,3/STAT3 pathway and that this late increase in JMJD3 induces NFκB-mediated inflammatory gene transcription in wound macrophages via an H3K27me3 mechanism.Interestingly,RNA sequencing of wound macrophages isolated from mice with JMJD3-deficient myeloid cells(Jmjd3f/fLyz2Cre+)identified that the STING gene(Tmem173)is regulated by JMJD3 in wound macrophages.STING limits inflammatory cytokine production by wound macrophages during healing.However,in diabetic mice,its role changes to limit wound repair and enhance inflammation.This finding is important since STING is associated with chronic inflammation,and we found STING to be elevated in human and murine diabetic wound macrophages at late time points.Finally,we demonstrate that macrophage-specific,nanoparticle inhibition of JMJD3 in diabetic wounds significantly improves diabetic wound repair by decreasing inflammatory cytokines and STING.Taken together,this work highlights the central role of JMJD3 in tissue repair and identifies cell-specific targeting as a viable therapeutic strategy for nonhealing diabetic wounds.
