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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.
Semin Cell Dev Biol ; 119: 111-118, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34183242

RESUMO

Non-healing wounds in Type 2 Diabetes (T2D) patients represent the most common cause of amputation in the US, with an associated 5-year mortality of nearly 50%. Our lab has examined tissue from both T2D murine models and human wounds in order to explore mechanisms contributing to impaired wound healing. Current published data in the field point to macrophage function serving a pivotal role in orchestrating appropriate wound healing. Wound macrophages in mice and patients with T2D are characterized by a persistent inflammatory state; however, the mechanisms that control this persistent inflammatory state are unknown. Current literature demonstrates that gene regulation through histone modifications, DNA modifications, and microRNA can influence macrophage plasticity during wound healing. Further, accumulating studies reveal the importance of cells such as adipocytes, infiltrating immune cells (PMNs and T cells), and keratinocytes secrete factors that may help drive macrophage polarization. This review will examine the role of macrophages in the wound healing process, along with their function and interactions with other cells, and how it is perturbed in T2D. We also explore epigenetic factors that regulate macrophage polarization in wounds, while highlighting the emerging role of other cell types that may influence macrophage phenotype following tissue injury.

3.
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
4.
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
5.
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
6.
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
7.
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
8.
J Autoimmun ; 103: 102291, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31248690

RESUMO

Ultraviolet (UV) light is a known trigger of skin and possibly systemic inflammation in systemic lupus erythematosus (SLE) patients. Although type I interferons (IFN) are upregulated in SLE skin after UV exposure, the mechanisms to explain increased UVB-induced inflammation remain unclear. This paper compares the role of type I IFNs in regulating immune cell activation between wild-type and lupus-prone mice following UVB exposure. 10-week old female lupus-prone (NZM2328), wild-type (BALB/c) and iNZM mice (lack a functional type I IFN receptor on NZM2328 background) were treated on their dorsal skin with 100 mJ/cm2 of UVB for 5 consecutive days. Following UVB treatment, draining lymph node cell populations were characterized via flow cytometry and suppression assays; treated skin was examined for changes in expression of type I IFN genes. Only NZM2328 mice showed an increase in T cell numbers and activation 2 weeks post UVB exposure. This was preceded by a significant increase in UVB-induced type I IFN expression in NZM2328 mice compared to BALB/c mice. Following UVB exposure, both BALB/c and iNZM mice demonstrated an increase in functional T regulatory (TReg) cells; however, this was not seen in NZM2328 mice. These data suggest a skewed UVB-mediated T cell response in lupus-prone mice where activation of T cells is enhanced secondary to a type I IFN-dependent suppression of TReg cells. Thus, we propose type I IFNs are important for UVB-induced inflammation in lupus-prone mice and may be an effective target for prevention of UVB-mediated flares.


Assuntos
Inflamação/imunologia , Interferon Tipo I/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Pele/patologia , Linfócitos T Reguladores/imunologia , Raios Ultravioleta/efeitos adversos , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Exposição à Radiação , Pele/efeitos da radiação
9.
JCI Insight ; 4(8)2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30996136

RESUMO

Autoimmune disease is 4 times more common in women than men. This bias is largely unexplained. Female skin is "autoimmunity prone," showing upregulation of many proinflammatory genes, even in healthy women. We previously identified VGLL3 as a putative transcription cofactor enriched in female skin. Here, we demonstrate that skin-directed overexpression of murine VGLL3 causes a severe lupus-like rash and systemic autoimmune disease that involves B cell expansion, autoantibody production, immune complex deposition, and end-organ damage. Excess epidermal VGLL3 drives a proinflammatory gene expression program that overlaps with both female skin and cutaneous lupus. This includes increased B cell-activating factor (BAFF), the only current biologic target in systemic lupus erythematosus (SLE); IFN-κ, a key inflammatory mediator in cutaneous lupus; and CXCL13, a biomarker of early-onset SLE and renal involvement. Our results demonstrate that skin-targeted overexpression of the female-biased factor VGLL3 is sufficient to drive cutaneous and systemic autoimmune disease that is strikingly similar to SLE. This work strongly implicates VGLL3 as a pivotal orchestrator of sex-biased autoimmunity.


Assuntos
Autoimunidade/genética , Regulação da Expressão Gênica/imunologia , Lúpus Eritematoso Cutâneo/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fatores de Transcrição/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Lúpus Eritematoso Cutâneo/genética , Lúpus Eritematoso Cutâneo/patologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fatores Sexuais , Pele/imunologia , Pele/patologia , Fatores de Transcrição/genética
10.
Front Immunol ; 9: 2430, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405625

RESUMO

Ultraviolet (UV) light is an important environmental trigger for systemic lupus erythematosus (SLE) patients, yet the mechanisms by which UV light impacts disease are not fully known. This review covers evidence in both human and murine systems for the impacts of UV light on DNA damage, apoptosis, autoantigen exposure, cytokine production, inflammatory cell recruitment, and systemic flare induction. In addition, the role of the circadian clock is discussed. Evidence is compared in healthy individuals and SLE patients as well as in wild-type and lupus-prone mice. Further research is needed into the effects of UV light on cutaneous and systemic immune responses to understand how to prevent UV-light mediated lupus flares.


Assuntos
Lúpus Eritematoso Sistêmico/imunologia , Transtornos de Fotossensibilidade/imunologia , Raios Ultravioleta/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Autoimunidade/efeitos dos fármacos , Relógios Circadianos , Citocinas/metabolismo , Dano ao DNA/efeitos da radiação , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Camundongos
11.
J Immunol ; 201(2): 393-405, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29884703

RESUMO

Systemic lupus erythematosus is an autoimmune disease characterized by increased type I IFNs, autoantibodies, and inflammatory-mediated multiorgan damage. TLR7 activation is an important contributor to systemic lupus erythematosus pathogenesis, but the mechanisms by which type I IFNs participate in TLR7-driven pathologic conditions remain uncertain. In this study, we examined the requirement for type I IFNs in TLR7-stimulated lupus nephritis. Lupus-prone NZM2328, INZM (which lack a functional type I IFN receptor), and NZM2328 IL-1ß-/- mice were treated at 10 wk of age on the right ear with R848 (TLR7 agonist) or control (DMSO). Autoantibody production and proteinuria were assessed throughout treatment. Multiorgan inflammation was assessed at the time of decline in health. Renal infiltrates and mRNA expression were also examined after 14 d of treatment. Both NZM2328 and INZM mice exhibited a decline in survival after 3-4 wk of R848 but not vehicle treatment. Development of splenomegaly and liver inflammation were dependent on type I IFN. Interestingly, autoantibody production, early renal infiltration of dendritic cells, upregulation of IL-1ß, and lupus nephritis occurred independent of type I IFN signaling. Development of TLR7-driven lupus nephritis was not abolished by the deletion of IL-1ß. Thus, although IFN-α is sufficient to induce nephritis acceleration, our data emphasize a critical role for IFN-independent signaling in TLR7-mediated lupus nephritis. Further, despite upregulation of IL-1ß after TLR7 stimulation, deletion of IL-1ß is not sufficient to reduce lupus nephritis development in this model.


Assuntos
Interferon Tipo I/imunologia , Nefrite Lúpica/imunologia , Glicoproteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Autoanticorpos/imunologia , Células Dendríticas/imunologia , Feminino , Inflamação/metabolismo , Interleucina-1beta/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , RNA Mensageiro/imunologia
12.
Methods Mol Biol ; 1382: 21-39, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26611576

RESUMO

Gene delivery using recombinant adeno-associated virus (rAAV) has emerged to the forefront demonstrating safe and effective phenotypic correction of diverse diseases including hemophilia B and Leber's congenital amaurosis. In addition to rAAV's high efficiency of transduction and the capacity for long-term transgene expression, the safety profile of rAAV remains unsoiled in humans with no deleterious vector-related consequences observed thus far. Despite these favorable attributes, rAAV vectors have a major disadvantage preventing widespread therapeutic applications; as the AAV capsid is the smallest described to date, it cannot package "large" genomes. Currently, the packaging capacity of rAAV has yet to be definitively defined but is approximately 5 kb, which has served as a limitation for large gene transfer. There are two main approaches that have been developed to overcome this limitation, split AAV vectors, and fragment AAV (fAAV) genome reassembly (Hirsch et al., Mol Ther 18(1):6-8, 2010). Split rAAV vector applications were developed based upon the finding that rAAV genomes naturally concatemerize in the cell post-transduction and are substrates for enhanced homologous recombination (HR) (Hirsch et al., Mol Ther 18(1):6-8, 2010; Duan et al., J Virol 73(1):161-169, 1999; Duan et al., J Virol 72(11):8568-8577, 1998; Duan et al., Mol Ther 4(4):383-391, 2001; Halbert et al., Nat Biotechnol 20(7):697-701, 2002). This method involves "splitting" the large transgene into two separate vectors and upon co-transduction, intracellular large gene reconstruction via vector genome concatemerization occurs via HR or nonhomologous end joining (NHEJ). Within the split rAAV approaches there currently exist three strategies: overlapping, trans-splicing, and hybrid trans-splicing (Duan et al., Mol Ther 4(4):383-391, 2001; Halbert et al., Nat Biotechnol 20(7):697-701, 2002; Ghosh et al., Mol Ther 16(1):124-130, 2008; Ghosh et al., Mol Ther 15(4):750-755, 2007). The other major strategy for AAV-mediated large gene delivery is the use of fragment AAV (fAAV) (Dong et al., Mol Ther 18(1):87-92, 2010; Hirsch et al., Mol Ther 21(12):2205-2216, 2013; Lai et al., Mol Ther 18(1):75-79, 2010; Wu et al., Mol Ther 18(1):80-86, 2010). This strategy developed following the observation that the attempted encapsidation of transgenic cassettes exceeding the packaging capacity of the AAV capsid results in the packaging of heterogeneous single-strand genome fragments (<5 kb) of both polarities (Dong et al., Mol Ther 18(1):87-92, 2010; Hirsch et al., Mol Ther 21(12):2205-2216, 2013; Lai et al., Mol Ther 18(1):75-79, 2010; Wu et al., Mol Ther 18(1):80-86, 2010). After transduction by multiple fAAV particles, the genome fragments can undergo opposite strand annealing, followed by host-mediated DNA synthesis to reconstruct the intended oversized genome within the cell. Although, there appears to be growing debate as to the most efficient method of rAAV-mediated large gene delivery, it remains possible that additional factors including the target tissue and the transgenomic sequence factor into the selection of a particular approach for a specific application (Duan et al., Mol Ther 4(4):383-391, 2001; Ghosh et al., Mol Ther 16(1):124-130, 2008; Hirsch et al., Mol Ther 21(12):2205-2216, 2013; Trapani et al., EMBO Mol Med 6(2):194-211, 2014; Ghosh et al., Hum Gene Ther 22(1):77-83, 2011). Herein we discuss the design, production, and verification of the leading rAAV large gene delivery strategies.


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Transgenes , Empacotamento do DNA , Vetores Genéticos/administração & dosagem , Células HEK293 , Humanos , Transdução Genética
13.
J Autoimmun ; 65: 38-48, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26305061

RESUMO

Systemic lupus erythematosus is clinically characterized by episodes of flare and remission. In patients, cutaneous exposure to ultraviolet light has been proposed as a flare trigger. However, induction of flare secondary to cutaneous exposure has been difficult to emulate in many murine lupus models. Here, we describe a system in which epidermal injury is able to trigger the development of a lupus nephritis flare in New Zealand Mixed (NZM) 2328 mice. 20-week old NZM2328 female mice underwent removal of the stratum corneum via duct tape, which resulted in rapid onset of proteinuria and death when compared to sham-stripped littermate control NZM2328 mice. This was coupled with a drop in serum C3 concentrations and dsDNA antibody levels and enhanced immune complex deposition in the glomeruli. Recruitment of CD11b(+)CD11c(+)F4/80(high) macrophages and CD11b(+)CD11c(+)F4/80(low) dendritic cells was noted prior to the onset of proteinuria in injured mice. Transcriptional changes within the kidney suggest a burst of type I IFN-mediated and inflammatory signaling which is followed by upregulation of CXCL13 following epidermal injury. Thus, we propose that tape stripping of lupus-prone NZM2328 mice is a novel model of lupus flare induction that will allow for the study of the role of cutaneous inflammation in lupus development and how crosstalk between dermal and systemic immune systems can lead to lupus flare.


Assuntos
Epiderme/lesões , Glomérulos Renais/imunologia , Nefrite Lúpica/imunologia , Animais , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/metabolismo , Quimiocina CXCL13/metabolismo , Complemento C3/análise , Complemento C3/metabolismo , DNA/imunologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Epiderme/imunologia , Feminino , Glomérulos Renais/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteinúria/etiologia , Proteinúria/imunologia , Exacerbação dos Sintomas
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