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1.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34479991

RESUMO

COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNß directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNß reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNß/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.


Assuntos
Coronavirus/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/virologia , Macrófagos/metabolismo , Animais , COVID-19/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Feminino , Histona-Lisina N-Metiltransferase/genética , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais
2.
J Exp Med ; 218(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33779682

RESUMO

Abdominal aortic aneurysms (AAAs) are a life-threatening disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by macrophage infiltration, and the mechanisms regulating macrophage-mediated inflammation remain undefined. Recent evidence suggests that an epigenetic enzyme, JMJD3, plays a critical role in establishing macrophage phenotype. Using single-cell RNA sequencing of human AAA tissues, we identified increased JMJD3 in aortic monocyte/macrophages resulting in up-regulation of an inflammatory immune response. Mechanistically, we report that interferon-ß regulates Jmjd3 expression via JAK/STAT and that JMJD3 induces NF-κB-mediated inflammatory gene transcription in infiltrating aortic macrophages. In vivo targeted inhibition of JMJD3 with myeloid-specific genetic depletion (JMJD3f/fLyz2Cre+) or pharmacological inhibition in the elastase or angiotensin II-induced AAA model preserved the repressive H3K27me3 on inflammatory gene promoters and markedly reduced AAA expansion and attenuated macrophage-mediated inflammation. Together, our findings suggest that cell-specific pharmacologic therapy targeting JMJD3 may be an effective intervention for AAA expansion.


Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Macrófagos/metabolismo , Angiotensina II/farmacologia , Animais , Modelos Animais de Doenças , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia
3.
JCI Insight ; 5(17)2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32879137

RESUMO

Macrophages are a primary immune cell involved in inflammation, and their cell plasticity allows for transition from an inflammatory to a reparative phenotype and is critical for normal tissue repair following injury. Evidence suggests that epigenetic alterations play a critical role in establishing macrophage phenotype and function during normal and pathologic wound repair. Here, we find in human and murine wound macrophages that cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) is elevated in diabetes and regulates downstream macrophage-mediated inflammation and host defense. Using single-cell RNA sequencing of human wound tissue, we identify increased NF-κB-mediated inflammation in diabetic wounds and show increased COX-2/PGE2 in diabetic macrophages. Further, we identify that COX-2/PGE2 production in wound macrophages requires epigenetic regulation of 2 key enzymes in the cytosolic phospholipase A2/COX-2/PGE2 (cPLA2/COX-2/PGE2) pathway. We demonstrate that TGF-ß-induced miRNA29b increases COX-2/PGE2 production via inhibition of DNA methyltransferase 3b-mediated hypermethylation of the Cox-2 promoter. Further, we find mixed-lineage leukemia 1 (MLL1) upregulates cPLA2 expression and drives COX-2/PGE2. Inhibition of the COX-2/PGE2 pathway genetically (Cox2fl/fl Lyz2Cre+) or with a macrophage-specific nanotherapy targeting COX-2 in tissue macrophages reverses the inflammatory macrophage phenotype and improves diabetic tissue repair. Our results indicate the epigenetically regulated PGE2 pathway controls wound macrophage function, and cell-targeted manipulation of this pathway is feasible to improve diabetic wound repair.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus/fisiopatologia , Dinoprostona/farmacologia , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Cicatrização , Idoso , Animais , Ciclo-Oxigenase 2/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Ocitócicos/farmacologia , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Transdução de Sinais
4.
Eur J Immunol ; 50(12): 1929-1940, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32662520

RESUMO

Chronic macrophage inflammation is a hallmark of type 2 diabetes (T2D) and linked to the development of secondary diabetic complications. T2D is characterized by excess concentrations of saturated fatty acids (SFA) that activate innate immune inflammatory responses, however, mechanism(s) by which SFAs control inflammation is unknown. Using monocyte-macrophages isolated from human blood and murine models, we demonstrate that palmitate (C16:0), the most abundant circulating SFA in T2D, increases expression of the histone demethylase, Jmjd3. Upregulation of Jmjd3 results in removal of the repressive histone methylation (H3K27me3) mark on NFκB-mediated inflammatory gene promoters driving macrophage-mediated inflammation. We identify that the effects of palmitate are fatty acid specific, as laurate (C12:0) does not regulate Jmjd3 and the associated inflammatory profile. Further, palmitate-induced Jmjd3 expression is controlled via TLR4/MyD88-dependent signaling mechanism, where genetic depletion of TLR4 (Tlr4-/- ) or MyD88 (MyD88-/- ) negated the palmitate-induced changes in Jmjd3 and downstream NFκB-induced inflammation. Pharmacological inhibition of Jmjd3 using a small molecule inhibitor (GSK-J4) reduced macrophage inflammation and improved diabetic wound healing. Together, we conclude that palmitate contributes to the chronic Jmjd3-mediated activation of macrophages in diabetic peripheral tissue and a histone demethylase inhibitor-based therapy may represent a novel treatment for nonhealing diabetic wounds.


Assuntos
Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Macrófagos/metabolismo , Palmitatos/metabolismo , Receptor 4 Toll-Like/metabolismo , Cicatrização/fisiologia , Animais , Diabetes Mellitus Tipo 2 , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
5.
J Immunol ; 204(9): 2503-2513, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32205424

RESUMO

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound healing. In diabetes, macrophages display a prolonged inflammatory phenotype preventing tissue repair. TLRs, particularly TLR4, have been shown to regulate myeloid-mediated inflammation in wounds. We examined macrophages isolated from wounds of patients afflicted with diabetes and healthy controls as well as a murine diabetic model demonstrating dynamic expression of TLR4 results in altered metabolic pathways in diabetic macrophages. Further, using a myeloid-specific mixed-lineage leukemia 1 (MLL1) knockout (Mll1f/fLyz2Cre+ ), we determined that MLL1 drives Tlr4 expression in diabetic macrophages by regulating levels of histone H3 lysine 4 trimethylation on the Tlr4 promoter. Mechanistically, MLL1-mediated epigenetic alterations influence diabetic macrophage responsiveness to TLR4 stimulation and inhibit tissue repair. Pharmacological inhibition of the TLR4 pathway using a small molecule inhibitor (TAK-242) as well as genetic depletion of either Tlr4 (Tlr4-/- ) or myeloid-specific Tlr4 (Tlr4f/fLyz2Cre+) resulted in improved diabetic wound healing. These results define an important role for MLL1-mediated epigenetic regulation of TLR4 in pathologic diabetic wound repair and suggest a target for therapeutic manipulation.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , Epigênese Genética/genética , Macrófagos/fisiologia , Receptor 4 Toll-Like/genética , Cicatrização/genética , Idoso , Animais , Epigênese Genética/imunologia , Feminino , Histonas/genética , Histonas/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Mediadores da Inflamação/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/imunologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Receptor 4 Toll-Like/imunologia , Cicatrização/imunologia
6.
JCI Insight ; 5(5)2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32069267

RESUMO

A critical component of wound healing is the transition from the inflammatory phase to the proliferation phase to initiate healing and remodeling of the wound. Macrophages are critical for the initiation and resolution of the inflammatory phase during wound repair. In diabetes, macrophages display a sustained inflammatory phenotype in late wound healing characterized by elevated production of inflammatory cytokines, such as TNF-α. Previous studies have shown that an altered epigenetic program directs diabetic macrophages toward a proinflammatory phenotype, contributing to a sustained inflammatory phase. Males absent on the first (MOF) is a histone acetyltransferase (HAT) that has been shown be a coactivator of TNF-α signaling and promote NF-κB-mediated gene transcription in prostate cancer cell lines. Based on MOF's role in TNF-α/NF-κB-mediated gene expression, we hypothesized that MOF influences macrophage-mediated inflammation during wound repair. We used myeloid-specific Mof-knockout (Lyz2Cre Moffl/fl) and diet-induced obese (DIO) mice to determine the function of MOF in diabetic wound healing. MOF-deficient mice exhibited reduced inflammatory cytokine gene expression. Furthermore, we found that wound macrophages from DIO mice had elevated MOF levels and higher levels of acetylated histone H4K16, MOF's primary substrate of HAT activity, on the promoters of inflammatory genes. We further identified that MOF expression could be stimulated by TNF-α and that treatment with etanercept, an FDA-approved TNF-α inhibitor, reduced MOF levels and improved wound healing in DIO mice. This report is the first to our knowledge to define an important role for MOF in regulating macrophage-mediated inflammation in wound repair and identifies TNF-α inhibition as a potential therapy for the treatment of chronic inflammation in diabetic wounds.


Assuntos
Diabetes Mellitus Experimental/imunologia , Histona Acetiltransferases/metabolismo , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Diabetes Mellitus Experimental/fisiopatologia , Etanercepte/farmacologia , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Cicatrização/fisiologia
7.
Arterioscler Thromb Vasc Biol ; 39(11): 2353-2366, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31644352

RESUMO

OBJECTIVE: Sepsis represents an acute life-threatening disorder resulting from a dysregulated host response. For patients who survive sepsis, there remains long-term consequences, including impaired inflammation, as a result of profound immunosuppression. The mechanisms involved in this long-lasting deficient immune response are poorly defined. Approach and Results: Sepsis was induced using the murine model of cecal ligation and puncture. Following a full recovery period from sepsis physiology, mice were subjected to our wound healing model and wound macrophages (CD11b+, CD3-, CD19-, Ly6G-) were sorted. Post-sepsis mice demonstrated impaired wound healing and decreased reepithelization in comparison to controls. Further, post-sepsis bone marrow-derived macrophages and wound macrophages exhibited decreased expression of inflammatory cytokines vital for wound repair (IL [interleukin]-1ß, IL-12, and IL-23). To evaluate if decreased inflammatory gene expression was secondary to epigenetic modification, we conducted chromatin immunoprecipitation on post-sepsis bone marrow-derived macrophages and wound macrophages. This demonstrated decreased expression of Mll1, an epigenetic enzyme, and impaired histone 3 lysine 4 trimethylation (activation mark) at NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells)-binding sites on inflammatory gene promoters in bone marrow-derived macrophages and wound macrophages from postcecal ligation and puncture mice. Bone marrow transplantation studies demonstrated epigenetic modifications initiate in bone marrow progenitor/stem cells following sepsis resulting in lasting impairment in peripheral macrophage function. Importantly, human peripheral blood leukocytes from post-septic patients demonstrate a significant reduction in MLL1 compared with nonseptic controls. CONCLUSIONS: These data demonstrate that severe sepsis induces stable mixed-lineage leukemia 1-mediated epigenetic modifications in the bone marrow, which are passed to peripheral macrophages resulting in impaired macrophage function and deficient wound healing persisting long after sepsis recovery.


Assuntos
Epigênese Genética , Inflamação/fisiopatologia , Macrófagos/fisiologia , Sepse/genética , Sepse/fisiopatologia , Cicatrização/fisiologia , Animais , Células da Medula Óssea/fisiologia , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Tolerância Imunológica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Proteína de Leucina Linfoide-Mieloide/genética , NF-kappa B/genética , Regiões Promotoras Genéticas , Sepse/metabolismo
8.
Immunity ; 51(2): 258-271.e5, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31350176

RESUMO

Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to the proliferative phase of healing. We examined macrophages isolated from wounds of patients afflicted with diabetes and of healthy controls and found differential expression of the methyltransferase Setdb2. Myeloid-specific deletion of Setdb2 impaired the transition of macrophages from an inflammatory phenotype to a reparative one in normal wound healing. Mechanistically, Setdb2 trimethylated histone 3 at NF-κB binding sites on inflammatory cytokine gene promoters to suppress transcription. Setdb2 expression in wound macrophages was regulated by interferon (IFN) ß, and under diabetic conditions, this IFNß-Setdb2 axis was impaired, leading to a persistent inflammatory macrophage phenotype in diabetic wounds. Setdb2 regulated the expression of xanthine oxidase and thereby the uric acid (UA) pathway of purine catabolism in macrophages, and pharmacologic targeting of Setdb2 or the UA pathway improved healing. Thus, Setdb2 regulates macrophage plasticity during normal and pathologic wound repair and is a target for therapeutic manipulation.


Assuntos
Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Macrófagos/fisiologia , Proteínas Nucleares/metabolismo , Idoso , Animais , Proteínas de Transporte/genética , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Fenótipo , Ácido Úrico/metabolismo , Cicatrização
9.
J Invest Dermatol ; 139(12): 2528-2537.e2, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31207226

RESUMO

Control of inflammation is critical for the treatment of nonhealing wounds, but a delicate balance exists between early inflammation that is essential for normal tissue repair and the pathologic inflammation that can occur later in the repair process. This necessitates the development of novel therapies that can target inflammation at the appropriate time during repair. Here, we found that SIRT3 is essential for normal healing and regulates inflammation in wound macrophages after injury. Under prediabetic conditions, SIRT3 was decreased in wound macrophages and resulted in dysregulated inflammation. In addition, we found that FABP4 regulates SIRT3 in human blood monocytes, and inhibition of FABP4 in wound macrophages decreases inflammatory cytokine expression, making FABP4 a viable target for the regulation of excess inflammation and wound repair in diabetes. Using a series of ex vivo and in vivo studies with genetically engineered mouse models and diabetic human monocytes, we showed that FABP4 expression is epigenetically upregulated in diabetic wound macrophages and, in turn, diminishes SIRT3 expression, thereby promoting inflammation. These findings have significant implications for controlling inflammation and promoting tissue repair in diabetic wounds.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Sirtuína 3/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL
10.
J Immunol ; 202(6): 1777-1785, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30710046

RESUMO

Myeloid cells are critical for orchestrating regulated inflammation during wound healing. TLRs, particularly TLR4, and its downstream-signaling MyD88 pathway play an important role in regulating myeloid-mediated inflammation. Because an initial inflammatory phase is vital for tissue repair, we investigated the role of TLR4-regulated, myeloid-mediated inflammation in wound healing. In a cutaneous tissue injury murine model, we found that TLR4 expression is dynamic in wound myeloid cells during the course of normal wound healing. We identified that changes in myeloid TLR4 during tissue repair correlated with increased expression of the histone methyltransferase, mixed-lineage leukemia 1 (MLL1), which specifically trimethylates the histone 3 lysine 4 (H3K4me3) position of the TLR4 promoter. Furthermore, we used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+ ) to determine MLL1 drives Tlr4 expression during wound healing. To understand the critical role of myeloid-specific TLR4 signaling, we used mice deficient in Tlr4 (Tlr4-/- ), Myd88 (Myd88 -/-), and myeloid-specific Tlr4 (Tlr4f/fLyz2Cre+) to demonstrate delayed wound healing at early time points postinjury. Furthermore, in vivo wound myeloid cells isolated from Tlr4-/- and Myd88 -/- wounds demonstrated decreased inflammatory cytokine production. Importantly, adoptive transfer of monocyte/macrophages from wild-type mice trafficked to wounds with restoration of normal healing and myeloid cell function in Tlr4-deficient mice. These results define a role for myeloid-specific, MyD88-dependent TLR4 signaling in the inflammatory response following cutaneous tissue injury and suggest that MLL1 regulates TLR4 expression in wound myeloid cells.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Pele/metabolismo , Receptor 4 Toll-Like/biossíntese , Cicatrização/fisiologia , Animais , Metilação de DNA/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/fisiologia , Pele/lesões
11.
Eur J Immunol ; 48(9): 1445-1455, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29879295

RESUMO

Macrophages play a critical role in the establishment of a regulated inflammatory response following tissue injury. Following injury, CCR2+ monocytes are recruited from peripheral blood to wound tissue, and direct the initiation and resolution of inflammation that is essential for tissue repair. In pathologic states where chronic inflammation prevents healing, macrophages fail to transition to a reparative phenotype. Using a murine model of cutaneous wound healing, we found that CCR2-deficient mice (CCR2-/- ) demonstrate significantly impaired wound healing at all time points postinjury. Flow cytometry analysis of wounds from CCR2-/- and WT mice revealed a significant decrease in inflammatory, Ly6CHi recruited monocyte/macrophages in CCR2-/- wounds. We further show that wound macrophage inflammatory cytokine production is decreased in CCR2-/- wounds. Adoptive transfer of mT/mG monocyte/macrophages into CCR2+/+ and CCR2-/- mice demonstrated that labeled cells on days 2 and 4 traveled to wounds in both CCR2+/+ and CCR2-/- mice. Further, adoptive transfer of monocyte/macrophages from WT mice restored normal healing, likely through a restored inflammatory response in the CCR2-deficient mice. Taken together, these data suggest that CCR2 plays a critical role in the recruitment and inflammatory response following injury, and that wound repair may be therapeutically manipulated through modulation of CCR2.


Assuntos
Macrófagos/transplante , Receptores CCR2/genética , Cicatrização/genética , Cicatrização/fisiologia , Animais , Inflamação/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR2/metabolismo
12.
Arterioscler Thromb Vasc Biol ; 38(5): 1102-1114, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29496661

RESUMO

OBJECTIVE: Wound monocyte-derived macrophage plasticity controls the initiation and resolution of inflammation that is critical for proper healing, however, in diabetes mellitus, the resolution of inflammation fails to occur. In diabetic wounds, the kinetics of blood monocyte recruitment and the mechanisms that control in vivo monocyte/macrophage differentiation remain unknown. APPROACH AND RESULTS: Here, we characterized the kinetics and function of Ly6CHi [Lin- (CD3-CD19-NK1.1-Ter-119-) Ly6G-CD11b+] and Ly6CLo [Lin- (CD3-CD19-NK1.1-Ter-119-) Ly6G-CD11b+] monocyte/macrophage subsets in normal and diabetic wounds. Using flow-sorted tdTomato-labeled Ly6CHi monocyte/macrophages, we show Ly6CHi cells transition to a Ly6CLo phenotype in normal wounds, whereas in diabetic wounds, there is a late, second influx of Ly6CHi cells that fail transition to Ly6CLo. The second wave of Ly6CHi cells in diabetic wounds corresponded to a spike in MCP-1 (monocyte chemoattractant protein-1) and selective administration of anti-MCP-1 reversed the second Ly6CHi influx and improved wound healing. To examine the in vivo phenotype of wound monocyte/macrophages, RNA-seq-based transcriptome profiling was performed on flow-sorted Ly6CHi [Lin-Ly6G-CD11b+] and Ly6CLo [Lin-Ly6G-CD11b+] cells from normal and diabetic wounds. Gene transcriptome profiling of diabetic wound Ly6CHi cells demonstrated differences in proinflammatory and profibrotic genes compared with controls. CONCLUSIONS: Collectively, these data identify kinetic and functional differences in diabetic wound monocyte/macrophages and demonstrate that selective targeting of CD11b+Ly6CHi monocyte/macrophages is a viable therapeutic strategy for inflammation in diabetic wounds.


Assuntos
Antígenos Ly/metabolismo , Diabetes Mellitus Tipo 2/sangue , Angiopatias Diabéticas/sangue , Inflamação/sangue , Macrófagos/metabolismo , Monócitos/metabolismo , Úlcera Cutânea/sangue , Cicatrização , Animais , Plasticidade Celular , Quimiocina CCL2/metabolismo , Doença Crônica , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/patologia , Angiopatias Diabéticas/fisiopatologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Fibrose , Inflamação/genética , Inflamação/patologia , Inflamação/fisiopatologia , Mediadores da Inflamação/metabolismo , Cinética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Transdução de Sinais , Úlcera Cutânea/genética , Úlcera Cutânea/patologia
13.
Diabetes ; 66(9): 2459-2471, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28663191

RESUMO

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Mixed-lineage leukemia-1 (MLL1) has been shown to direct gene expression by regulating nuclear factor-κB (NF-κB)-mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+ ) to determine the function of MLL1 in wound healing. Mll1f/fLyz2Cre+ mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared with control animals. Furthermore, wound macrophages from Mll1f/fLyz2Cre+ mice demonstrated decreased histone H3 lysine 4 trimethylation (H3K4me3) (activation mark) at NF-κB binding sites on inflammatory gene promoters. Of note, early wound macrophages from prediabetic mice displayed similarly decreased MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines compared with controls. Late wound macrophages from prediabetic mice demonstrated an increase in MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines. Prediabetic macrophages treated with an MLL1 inhibitor demonstrated reduced inflammation. Finally, monocytes from patients with type 2 diabetes had increased Mll1 compared with control subjects without diabetes. These results define an important role for MLL1 in regulating macrophage-mediated inflammation in wound repair and identify a potential target for the treatment of chronic inflammation in diabetic wounds.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Macrófagos/fisiologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Obesidade/metabolismo , Cicatrização/fisiologia , Animais , Células Cultivadas , Dieta Hiperlipídica , Histona-Lisina N-Metiltransferase/genética , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Estado Pré-Diabético/metabolismo
14.
J Immunol ; 199(5): 1865-1874, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28733487

RESUMO

Macrophages are critical immune cells for the clearance of microbial pathogens and cellular debris from peripheral tissues. Macrophage inflammatory responses are governed by gene expression patterns, and these patterns are often subject to epigenetic control. Chromatin modifications, such as histone methylation, regulate gene accessibility in macrophages, and macrophage polarization is governed in part by the expression and function of chromatin-modifying enzymes. The histone methyltransferase mixed-lineage leukemia 1 (MLL1) preferentially modifies lysine residue 4 on the unstructured protein tail of histone H3. MLL1 expression and function have been shown to be governed by signal transduction pathways that are activated by inflammatory stimuli, such as NF-κB. Therefore, we sought to investigate the role of MLL1 in mediating macrophage inflammatory responses. Bone marrow-derived macrophages from mice with a targeted MLL1 gene knockout (Lys2-Cre+/- MLL1fx/fx) exhibited decreased proinflammatory gene expression with concurrent decreases in activating histone methylation. However, MLL1-deficient macrophages also exhibited increased phagocytic and bacterial killing activity in vitro. RNA profiling of MLL1-knockout macrophages identified numerous genes involved with inflammatory responses whose expression was altered in response to TLR ligands or proinflammatory cytokines, including STAT4. STAT4-dependent cytokines, such as type I IFNs were able to drive MLL1 expression in macrophages, and MLL1-knockout macrophages exhibited decreased activating histone methylation in the STAT4 promoter. These results implicate an important role for MLL1-dependent epigenetic regulation of macrophage antimicrobial functions.


Assuntos
Epigênese Genética/imunologia , Histona-Lisina N-Metiltransferase/metabolismo , Infecções/imunologia , Macrófagos/imunologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Fator de Transcrição STAT4/metabolismo , Animais , Bacteriólise , Células Cultivadas , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , NF-kappa B/metabolismo , Fator de Transcrição STAT4/genética , Transdução de Sinais , Transcriptoma
15.
Front Immunol ; 8: 635, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28620387

RESUMO

Macrophages are essential immune cells necessary for regulated inflammation during wound healing. Recent studies have identified that Notch plays a role in macrophage-mediated inflammation. Thus, we investigated the role of Notch signaling on wound macrophage phenotype and function during normal and diabetic wound healing. We found that Notch receptor and ligand expression are dynamic in wound macrophages during normal healing. Mice with a myeloid-specific Notch signaling defect (DNMAMLfloxedLyz2Cre+ ) demonstrated delayed early healing (days 1-3) and wound macrophages had decreased inflammatory gene expression. In our physiologic murine model of type 2 diabetes (T2D), Notch receptor expression was significantly increased in wound macrophages on day 6, following the initial inflammatory phase of wound healing, corresponding to increased inflammatory cytokine expression. This increase in Notch1 and Notch2 was also observed in human monocytes from patients with T2D. Further, in prediabetic mice with a genetic Notch signaling defect (DNMAMLfloxedLyz2Cre+ on a high-fat diet), improved wound healing was seen at late time points (days 6-7). These findings suggest that Notch is critical for the early inflammatory phase of wound healing and directs production of macrophage-dependent inflammatory mediators. These results identify that canonical Notch signaling is important in directing macrophage function in wound repair and define a translational target for the treatment of non-healing diabetic wounds.

16.
Cell Immunol ; 314: 63-72, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28242024

RESUMO

Macrophage polarization plays a central role in both protective immunity and immunopathology. While the role of cytokines in driving macrophage polarization is well characterized, less is understood about the role of chemokines. The purpose of this study was to determine if CC chemokine 2 (CCL2/MCP1) could influence macrophage polarization in response to subsequent activation with cytokines and microbial products. Treatment of bone marrow-derived macrophages with CCL2 alone did not result in increased expression of either classical or alternatively-activated macrophage genes as compared to standard skewing cytokines or Toll-like receptor agonists. However, subsequent stimulation of CCL2 pre-treated macrophages with classical activation stimuli resulted in enhanced expression of genes associated with classical activation. This enhancement correlated with increased phosphorylation of ERK1/2 kinases, a decrease in expression of the ERK phosphatase Dusp6 and enhanced expression of miR-9. These results indicate that CCL2 supports the classical activation of macrophages, with miR-9 mediated down-regulation of Dusp6 and enhanced ERK-mediated signal transduction possibly mediating this enhanced pro-inflammatory gene expression.


Assuntos
Fosfatase 6 de Especificidade Dupla/metabolismo , Inflamação/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , MicroRNAs/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Fosfatase 6 de Especificidade Dupla/genética , Feminino , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Células Th1/imunologia , Células Th2/imunologia , Receptores Toll-Like/metabolismo
17.
Diabetes ; 64(4): 1420-30, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25368099

RESUMO

Classically activated (M1) macrophages are known to play a role in the development of chronic inflammation associated with impaired wound healing in type 2 diabetes (T2D); however, the mechanism responsible for the dominant proinflammatory (M1) macrophage phenotype in T2D wounds is unknown. Since epigenetic enzymes can direct macrophage phenotypes, we assessed the role of histone methylation in bone marrow (BM) stem/progenitor cells in the programming of macrophages toward a proinflammatory phenotype. We have found that a repressive histone methylation mark, H3K27me3, is decreased at the promoter of the IL-12 gene in BM progenitors and this epigenetic signature is passed down to wound macrophages in a murine model of glucose intolerance (diet-induced obese). These epigenetically "preprogrammed" macrophages result in poised macrophages in peripheral tissue and negatively impact wound repair. We found that in diabetic conditions the H3K27 demethylase Jmjd3 drives IL-12 production in macrophages and that IL-12 production can be modulated by inhibiting Jmjd3. Using human T2D tissue and murine models, we have identified a previously unrecognized mechanism by which macrophages are programmed toward a proinflammatory phenotype, establishing a pattern of unrestrained inflammation associated with nonhealing wounds. Hence, histone demethylase inhibitor-based therapy may represent a novel treatment option for diabetic wounds.


Assuntos
Células da Medula Óssea/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Epigênese Genética , Células-Tronco/metabolismo , Cicatrização/fisiologia , Animais , Humanos , Inflamação , Interleucina-12/metabolismo , Macrófagos/metabolismo , Camundongos , Fenótipo , Regiões Promotoras Genéticas
18.
mBio ; 4(1): e00620-12, 2013 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-23404401

RESUMO

UNLABELLED: When microbes contaminate the macrophage cytoplasm, leukocytes undergo a proinflammatory death that is initiated by nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) that bind and activate caspase-1. We report that these inflammasome components also regulate autophagy, a vesicular pathway to eliminate cytosolic debris. In response to infection with flagellate Legionella pneumophila, C57BL/6J mouse macrophages equipped with caspase-1 and the NLR proteins NAIP5 and NLRC4 stimulated autophagosome turnover. A second trigger of inflammasome assembly, K(+) efflux, also rapidly activated autophagy in macrophages that produced caspase-1. Autophagy protects infected macrophages from pyroptosis, since caspase-1-dependent cell death occurred more frequently when autophagy was dampened pharmacologically by either 3-methyladenine or an inhibitor of the Atg4 protease. Accordingly, in addition to coordinating pyroptosis, both (pro-) caspase-1 protein and NLR components of inflammasomes equip macrophages to recruit autophagy, a disposal pathway that raises the threshold of contaminants necessary to trigger proinflammatory leukocyte death. IMPORTANCE: An exciting development in the innate-immunity field is the recognition that macrophages enlist autophagy to protect their cytoplasm from infection. Nutrient deprivation has long been known to induce autophagy; how infection triggers this disposal pathway is an active area of research. Autophagy is encountered by many of the intracellular pathogens that are known to trigger pyroptosis, an inflammatory cell death initiated when nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) activate caspase-1 within inflammasome complexes. Therefore, we tested the hypothesis that NLR proteins and caspase-1 also coordinate autophagy as a barrier to cytosolic infection. By exploiting classical bacterial and mouse genetics and kinetic assays of autophagy, we demonstrate for the first time that, when confronted with cytosolic contamination, primary mouse macrophages rely not only on the NLR proteins NAIP5 and NLRC4 but also on (pro-)caspase-1 protein to mount a rapid autophagic response that wards off proinflammatory cell death.


Assuntos
Autofagia , Inflamassomos/metabolismo , Legionella pneumophila/imunologia , Macrófagos/imunologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 1/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Inibidora de Apoptose Neuronal/metabolismo
19.
Front Microbiol ; 2: 138, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21743811

RESUMO

To proliferate within phagocytes, Legionella pneumophila relies on Type IV secretion to modulate host cellular pathways. Autophagy is an evolutionarily conserved degradative pathway that captures and transfers a variety of microbes to lysosomes. Biogenesis of L. pneumophila-containing vacuoles and autophagosomes share several features, including endoplasmic reticulum (ER)-derived membranes, contributions by the host GTPases Rab1, Arf1 and Sar1, and a final destiny in lysosomes. We discuss morphological, molecular genetic, and immunological data that support the model that, although A/J mouse macrophages efficiently engulf L. pneumophila within autophagosomal membranes, the Type IV effector proteins DrrA/SidM, LidA, and RalF prolong association with the ER. By inhibiting immediately delivery to lysosomes, the bacteria persist in immature autophagosomal vacuoles for a period sufficient to differentiate into an acid-resistant, replicative form. Subsequent secretion of the Type IV effector LepB releases the block to autophagosome maturation, and the adapted progeny continue to replicate within autophagolysosomes. Accordingly, L. pneumophila can be exploited as a genetic tool to analyze the recruitment and function of the macrophage autophagy pathway.

20.
PLoS One ; 6(6): e21253, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21712985

RESUMO

BACKGROUND: Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF. METHODOLOGY AND PRINCIPAL FINDINGS: miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts. CONCLUSION: These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing.


Assuntos
Progressão da Doença , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/fisiopatologia , MicroRNAs/metabolismo , Idoso , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/citologia , Pulmão/patologia , Pulmão/fisiologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Ribonuclease III/genética , Ribonuclease III/metabolismo , Adulto Jovem
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