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1.
Clin J Am Soc Nephrol ; 16(2): 213-224, 2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33462083

RESUMO

BACKGROUND AND OBJECTIVES: IgA nephropathy is the most common form of primary GN worldwide. The evidence of geographic and ethnic differences, as well as familial aggregation of the disease, supports a strong genetic contribution to IgA nephropathy. Evidence for genetic factors in IgA nephropathy comes also from genome-wide association patient-control studies. However, few studies have systematically evaluated the contribution of coding variation in IgA nephropathy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a two-stage exome chip-based association study in 13,242 samples, including 3363 patients with IgA nephropathy and 9879 healthy controls of Han Chinese ancestry. Common variant functional annotation, gene-based low-frequency variants analysis, differential mRNA expression, and gene network integration were also explored. RESULTS: We identified three non-HLA gene regions (FBXL21, CCR6, and STAT3) and one HLA gene region (GABBR1) with suggestive significance (P meta <5×10-5) in single-variant associations. These novel non-HLA variants were annotated as expression-associated single-nucleotide polymorphisms and were located in enhancer regions enriched in histone marks H3K4me1 in primary B cells. Gene-based low-frequency variants analysis suggests CFB as another potential susceptibility gene. Further combined expression and network integration suggested that the five novel susceptibility genes, TGFBI, CCR6, STAT3, GABBR1, and CFB, were involved in IgA nephropathy. CONCLUSIONS: Five novel gene regions with suggestive significance for IgA nephropathy were identified and shed new light for further mechanism investigation.

2.
Arthritis Rheumatol ; 2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33305541

RESUMO

OBJECTIVE: Skin inflammation heralds systemic disease in juvenile myositis (JM), yet we lack an understanding of pathogenic mechanisms driving skin inflammation in JM. The goal of this study is to define cutaneous gene expression signatures in JM and identify key genes and pathways that differentiate skin disease in JM from childhood-onset systemic lupus erythematosus (cSLE). METHODS: We utilized formalin-fixed paraffin-embedded (FFPE) skin biopsy samples from 15 JM (9 lesional, 6 non-lesional), 5 cSLE, and 8 control patients to perform transcriptomic analysis and identify significantly differentially expressed genes (DEGs; q-value ≤ 5%) between patient groups. We utilized Ingenuity Pathway Analysis (IPA) to highlight enriched biological pathways and validated DEGs using immunohistochemistry and quantitative real-time PCR. RESULTS: Comparison of lesional JM to control revealed 221 DEGs, with the majority of upregulated genes representing interferon-stimulated genes. CXCL10, CXCL9 and IFI44L represented the top three DEGs (fold-change respectively = 23.2, 13.3, 13.0, q-value < 0.0001). IPA revealed interferon (IFN) signaling as the top canonical pathway. When compared to cSLE, JM lesional skin shared a similar gene expression pattern, with only 28 unique DEGs, including FBLN2, CHKA and SLURP1. Notably, JM patients with NXP2 autoantibodies exhibited the strongest IFN signature and also demonstrated the most extensive MX1 immunostaining, both in keratinocytes and perivascular regions. CONCLUSION: JM lesional skin demonstrates a striking IFN signature similar to that previously reported in muscle and peripheral blood. Further investigation into the association of a higher IFN score with NXP2 autoantibodies may lend insight into disease endotypes and pathogenesis.

3.
Kidney Int ; 98(6): 1502-1518, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33038424

RESUMO

COVID-19 morbidity and mortality are increased via unknown mechanisms in patients with diabetes and kidney disease. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Because ACE2 is a susceptibility factor for infection, we investigated how diabetic kidney disease and medications alter ACE2 receptor expression in kidneys. Single cell RNA profiling of kidney biopsies from healthy living donors and patients with diabetic kidney disease revealed ACE2 expression primarily in proximal tubular epithelial cells. This cell-specific localization was confirmed by in situ hybridization. ACE2 expression levels were unaltered by exposures to renin-angiotensin-aldosterone system inhibitors in diabetic kidney disease. Bayesian integrative analysis of a large compendium of public -omics datasets identified molecular network modules induced in ACE2-expressing proximal tubular epithelial cells in diabetic kidney disease (searchable at hb.flatironinstitute.org/covid-kidney) that were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing. The diabetic kidney disease ACE2-positive proximal tubular epithelial cell module overlapped with expression patterns seen in SARS-CoV-2-infected cells. Similar cellular programs were seen in ACE2-positive proximal tubular epithelial cells obtained from urine samples of 13 hospitalized patients with COVID-19, suggesting a consistent ACE2-coregulated proximal tubular epithelial cell expression program that may interact with the SARS-CoV-2 infection processes. Thus SARS-CoV-2 receptor networks can seed further research into risk stratification and therapeutic strategies for COVID-19-related kidney damage.

4.
JCI Insight ; 5(16)2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32644977

RESUMO

Skin lesions in dermatomyositis (DM) are common, are frequently refractory, and have prognostic significance. Histologically, DM lesions appear similar to cutaneous lupus erythematosus (CLE) lesions and frequently cannot be differentiated. We thus compared the transcriptional profile of DM biopsies with CLE lesions to identify unique features. Type I IFN signaling, including IFN-κ upregulation, was a common pathway in both DM and CLE; however, CLE also exhibited other inflammatory pathways. Notably, DM lesions could be distinguished from CLE by a 5-gene biomarker panel that included IL18 upregulation. Using single-cell RNA-sequencing, we further identified keratinocytes as the main source of increased IL-18 in DM skin. This study identifies a potentially novel molecular signature, with significant clinical implications for differentiating DM from CLE lesions, and highlights the potential role for IL-18 in the pathophysiology of DM skin disease.

5.
medRxiv ; 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32511461

RESUMO

COVID-19 morbidity and mortality is significantly increased in patients with diabetes and kidney disease via unknown mechanisms. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into human host cells, and ACE2 levels in target cells may influence SARS-CoV-2 susceptibility. We investigated how pre-existing conditions and drug treatments alter receptor expression in kidney tissue. Using single cell RNA profiling (scRNAseq) to assess ACE2 and associated SARS-CoV-2 proteases in healthy living donors (LD) kidneys, diabetic kidney disease (DKD), and in kidney injury during viral infection, ACE2 expression was primarily associated with proximal tubular epithelial cells (PTEC). ACE2 mRNA expression levels were significantly upregulated in DKD versus LD, however, ACE2 levels were not altered by exposures to renin angiotensin aldosterone system (RAAS) inhibitors. ACE2+ expression signatures were defined by differential expression analysis and characterized by Bayesian integrative analysis of a large compendium of public -omics datasets, resulting in the identification of network modules induced in ACE2 positive PTEC in DKD and BK virus nephropathy. These ACE2 upregulated cell programs were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing and overlapped significantly with the cellular responses induced by SARS-CoV-2 infection. Similar cellular programs were activated in ACE2-positive PTEC isolated in a urine sample from a COVID19 patient with acute kidney injury, suggesting a consistent ACE2-coregulated expression program that may interact with SARS-Cov-2 infection processes. The SARS-CoV-2 receptor associated gene signatures could seed further research into therapeutic strategies for COVID-19. Functional networks of gene expression signatures are available for further exploration to researchers at HumanBase (hb.flatironinstitute.org/covid-kidney).

6.
JCI Insight ; 5(12)2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32396533

RESUMO

Lupus nephritis, one of the most serious manifestations of systemic lupus erythematosus (SLE), has a heterogeneous clinical and pathological presentation. For example, proliferative nephritis identifies a more aggressive disease class that requires immunosuppression. However, the current classification system relies on the static appearance of histopathological morphology, which does not capture differences in the inflammatory response. Therefore, a biomarker grounded in the disease biology is needed in order to understand the molecular heterogeneity of lupus nephritis and identify immunologic mechanism and pathways. Here, we analyzed the patterns of 1000 urine protein biomarkers in 30 patients with active lupus nephritis. We found that patients stratify over a chemokine gradient inducible by IFN-γ. Higher values identified patients with proliferative lupus nephritis. After integrating the urine proteomics with the single-cell transcriptomics of kidney biopsies, we observed that the urinary chemokines defining the gradient were predominantly produced by infiltrating CD8+ T cells, along with natural killer and myeloid cells. The urine chemokine gradient significantly correlated with the number of kidney-infiltrating CD8+ cells. These findings suggest that urine proteomics can capture the complex biology of the kidney in lupus nephritis. Patient-specific pathways could be noninvasively tracked in the urine in real time, enabling diagnosis and personalized treatment.

7.
JCI Insight ; 5(6)2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32107344

RESUMO

To define cellular mechanisms underlying kidney function and failure, the KPMP analyzes biopsy tissue in a multicenter research network to build cell-level process maps of the kidney. This study aimed to establish a single cell RNA sequencing strategy to use cell-level transcriptional profiles from kidney biopsies in KPMP to define molecular subtypes in glomerular diseases. Using multiple sources of adult human kidney reference tissue samples, 22,268 single cell profiles passed KPMP quality control parameters. Unbiased clustering resulted in 31 distinct cell clusters that were linked to kidney and immune cell types using specific cell markers. Focusing on endothelial cell phenotypes, in silico and in situ hybridization methods assigned 3 discrete endothelial cell clusters to distinct renal vascular beds. Transcripts defining glomerular endothelial cells (GEC) were evaluated in biopsies from patients with 10 different glomerular diseases in the NEPTUNE and European Renal cDNA Bank (ERCB) cohort studies. Highest GEC scores were observed in patients with focal segmental glomerulosclerosis (FSGS). Molecular endothelial signatures suggested 2 distinct FSGS patient subgroups with α-2 macroglobulin (A2M) as a key downstream mediator of the endothelial cell phenotype. Finally, glomerular A2M transcript levels associated with lower proteinuria remission rates, linking endothelial function with long-term outcome in FSGS.

8.
Arthritis Care Res (Hoboken) ; 72(2): 233-242, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31502417

RESUMO

The Accelerating Medicines Partnership (AMP) Lupus Network was established as a partnership between the National Institutes of Health, pharmaceutical companies, nonprofit stakeholders, and lupus investigators across multiple academic centers to apply high-throughput technologies to the analysis of renal tissue, urine, and blood from patients with lupus nephritis (LN). The AMP network provides publicly accessible data to the community with the goal of generating new scientific hypotheses and improving diagnostic and therapeutic tools so as to improve disease outcomes. We present here a description of the structure of the AMP Lupus Network and a summary of the preliminary results from the phase 1 studies. The successful completion of phase 1 sets the stage for analysis of a large cohort of LN samples in phase 2 and provides a model for establishing similar discovery cohorts.


Assuntos
Centros Médicos Acadêmicos/organização & administração , Ensaios Clínicos Fase I como Assunto/métodos , Indústria Farmacêutica/organização & administração , Nefrite Lúpica/metabolismo , National Institutes of Health (U.S.)/organização & administração , Dados Preliminares , Parcerias Público-Privadas/organização & administração , Biomarcadores/metabolismo , Humanos , Nefrite Lúpica/epidemiologia , Nefrite Lúpica/genética , Análise de Sequência de RNA/métodos , Estados Unidos/epidemiologia
9.
J Invest Dermatol ; 140(5): 1066-1074.e4, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31877319

RESUMO

Cutaneous inflammation is recurrent in systemic lupus erythematosus (SLE), yet mechanisms that drive cutaneous inflammation in SLE are not well defined. Type I IFNs are elevated in nonlesional SLE skin and promote inflammatory responses. Staphylococcus aureus, known to induce IFN production, could play a role in cutaneous inflammation in SLE. We show here that active cutaneous lupus erythematosus lesions are highly colonized (∼50%) by S. aureus. To define the impact of IFNs on S. aureus colonization, we examined the effects of type I and type II IFNs on S. aureus adherence and invasion. An increase in adherent S. aureus was observed after exposure to both IFN-α and -γ, whereas IFN-γ appeared to inhibit invasion of S. aureus. Cutaneous lupus erythematosus lesional skin microarray data and RNA sequencing data from SLE keratinocytes identified repression of barrier gene expression, such as filaggrin and loricrin, and SLE keratinocytes exhibited increased S. aureus-binding integrins. These SLE-associated changes could be replicated by IFN treatment of keratinocytes. Further, SLE keratinocytes exhibited increased binding to S. aureus. Together, these data suggest that chronic exposure to IFNs induces barrier disruption that allows for higher S. aureus colonization in SLE skin.

10.
Sci Transl Med ; 11(511)2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554739

RESUMO

Lichen planus (LP) is a chronic debilitating inflammatory disease of unknown etiology affecting the skin, nails, and mucosa with no current FDA-approved treatments. It is histologically characterized by dense infiltration of T cells and epidermal keratinocyte apoptosis. Using global transcriptomic profiling of patient skin samples, we demonstrate that LP is characterized by a type II interferon (IFN) inflammatory response. The type II IFN, IFN-γ, is demonstrated to prime keratinocytes and increase their susceptibility to CD8+ T cell-mediated cytotoxic responses through MHC class I induction in a coculture model. We show that this process is dependent on Janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1), but not JAK1 or STAT2 signaling. Last, using drug prediction algorithms, we identify JAK inhibitors as promising therapeutic agents in LP and demonstrate that the JAK1/2 inhibitor baricitinib fully protects keratinocytes against cell-mediated cytotoxic responses in vitro. In summary, this work elucidates the role and mechanisms of IFN-γ in LP pathogenesis and provides evidence for the therapeutic use of JAK inhibitors to limit cell-mediated cytotoxicity in patients with LP.


Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Interferon gama/farmacologia , Janus Quinase 2/metabolismo , Queratinócitos/imunologia , Líquen Plano/imunologia , Fator de Transcrição STAT1/metabolismo , Apoptose/efeitos dos fármacos , Epiderme/patologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Inflamação/patologia , Queratinócitos/efeitos dos fármacos , Líquen Plano/genética , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genética
12.
J Clin Med ; 8(8)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426521

RESUMO

Cutaneous lupus erythematosus (CLE) is a common manifestation of systemic lupus erythematosus (SLE), and CLE can also develop without systemic involvement. CLE can be difficult to treat and negatively contributes to quality of life. Despite the importance of CLE, our knowledge of what differentiates cutaneous lupus subtypes is limited. Here, we utilized a large cohort of 90 CLE lesional biopsies to compare discoid lupus erythematosus (DLE) and subacute cutaneous lupus (SCLE) in patients with and without associated SLE in order to discern the drivers of disease activity and possibly uncover better treatment targets. Overall, we found that DLE and SCLE share many differentially expressed genes (DEG) reflecting type I interferon (IFN) signaling and repression of EGFR pathways. No differences between CLE only and SLE-associated CLE lesions were found. Of note, DLE uniquely expresses an IFN-γ node. Unbiased cluster analysis of the DEGs identified two groups separated by neutrophilic vs. monocytic signatures that did not sort the patients based on clinical phenotype or disease activity. This suggests that unbiased analysis of the pathobiology of CLE lesions may be important for personalized medicine and targeted therapeutic decision making.

13.
Nat Immunol ; 20(7): 902-914, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209404

RESUMO

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.


Assuntos
Rim/imunologia , Nefrite Lúpica/imunologia , Biomarcadores , Biópsia , Análise por Conglomerados , Biologia Computacional/métodos , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Interferons/metabolismo , Rim/metabolismo , Rim/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Anotação de Sequência Molecular , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Célula Única , Transcriptoma
14.
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
15.
J Immunol ; 202(7): 2121-2130, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30745462

RESUMO

Systemic lupus erythematosus (SLE) is a complex autoimmune disease in which 70% of patients experience disfiguring skin inflammation (grouped under the rubric of cutaneous lupus erythematosus [CLE]). There are limited treatment options for SLE and no Food and Drug Administration-approved therapies for CLE. Studies have revealed that IFNs are important mediators for SLE and CLE, but the mechanisms by which IFNs lead to disease are still poorly understood. We aimed to investigate how IFN responses in SLE keratinocytes contribute to development of CLE. A cohort of 72 RNA sequencing samples from 14 individuals (seven SLE and seven healthy controls) were analyzed to study the transcriptomic effects of type I and type II IFNs on SLE versus control keratinocytes. In-depth analysis of the IFN responses was conducted. Bioinformatics and functional assays were conducted to provide implications for the change of IFN response. A significant hypersensitive response to IFNs was identified in lupus keratinocytes, including genes (IFIH1, STAT1, and IRF7) encompassed in SLE susceptibility loci. Binding sites for the transcription factor PITX1 were enriched in genes that exhibit IFN-sensitive responses. PITX1 expression was increased in CLE lesions based on immunohistochemistry, and by using small interfering RNA knockdown, we illustrated that PITX1 was required for upregulation of IFN-regulated genes in vitro. SLE patients exhibit increased IFN signatures in their skin secondary to increased production and a robust, skewed IFN response that is regulated by PITX1. Targeting these exaggerated pathways may prove to be beneficial to prevent and treat hyperinflammatory responses in SLE skin.


Assuntos
Regulação da Expressão Gênica/imunologia , Interferons/imunologia , Queratinócitos/imunologia , Lúpus Eritematoso Cutâneo/imunologia , Fatores de Transcrição Box Pareados/imunologia , Adulto , Feminino , Humanos , Masculino
16.
Ann Rheum Dis ; 77(11): 1653-1664, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30021804

RESUMO

OBJECTIVE: Skin inflammation and photosensitivity are common in patients with cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE), yet little is known about the mechanisms that regulate these traits. Here we investigate the role of interferon kappa (IFN-κ) in regulation of type I interferon (IFN) and photosensitive responses and examine its dysregulation in lupus skin. METHODS: mRNA expression of type I IFN genes was analysed from microarray data of CLE lesions and healthy control skin. Similar expression in cultured primary keratinocytes, fibroblasts and endothelial cells was analysed via RNA-seq. IFNK knock-out (KO) keratinocytes were generated using CRISPR/Cas9. Keratinocytes stably overexpressing IFN-κ were created via G418 selection of transfected cells. IFN responses were assessed via phosphorylation of STAT1 and STAT2 and qRT-PCR for IFN-regulated genes. Ultraviolet B-mediated apoptosis was analysed via TUNEL staining. In vivo protein expression was assessed via immunofluorescent staining of normal and CLE lesional skin. RESULTS: IFNK is one of two type I IFNs significantly increased (1.5-fold change, false discovery rate (FDR) q<0.001) in lesional CLE skin. Gene ontology (GO) analysis showed that type I IFN responses were enriched (FDR=6.8×10-04) in keratinocytes not in fibroblast and endothelial cells, and this epithelial-derived IFN-κ is responsible for maintaining baseline type I IFN responses in healthy skin. Increased levels of IFN-κ, such as seen in SLE, amplify and accelerate responsiveness of epithelia to IFN-α and increase keratinocyte sensitivity to UV irradiation. Notably, KO of IFN-κ or inhibition of IFN signalling with baricitinib abrogates UVB-induced apoptosis. CONCLUSION: Collectively, our data identify IFN-κ as a critical IFN in CLE pathology via promotion of enhanced IFN responses and photosensitivity. IFN-κ is a potential novel target for UVB prophylaxis and CLE-directed therapy.


Assuntos
Epiderme/imunologia , Interferon Tipo I/biossíntese , Lúpus Eritematoso Cutâneo/complicações , Transtornos de Fotossensibilidade/etiologia , Adulto , Células Cultivadas , Células Dendríticas/imunologia , Feminino , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Queratinócitos/imunologia , Lúpus Eritematoso Cutâneo/imunologia , Masculino , Pessoa de Meia-Idade , Transtornos de Fotossensibilidade/imunologia , RNA Mensageiro/genética , Pele/imunologia , TYK2 Quinase/imunologia , Regulação para Cima/imunologia
17.
Rheumatology (Oxford) ; 56(11): 1970-1981, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28968684

RESUMO

Objectives: SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Methods: Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-ß. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. Results: SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-ß. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Conclusion: Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-ß-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy.


Assuntos
Diferenciação Celular , Fibroblastos/citologia , Queratinócitos/citologia , Escleroderma Sistêmico/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Adulto , Idoso , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Meios de Cultivo Condicionados , Regulação para Baixo , Feminino , Fibroblastos/metabolismo , Fibrose , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , PPAR gama/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Esclerodermia Difusa , Esclerodermia Localizada , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Pele/patologia , Regulação para Cima
18.
Am J Pathol ; 187(12): 2799-2810, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28935578

RESUMO

Transcription factor NF-κB regulates expression of numerous genes that control inflammation and is activated in glomerular cells in glomerulonephritis (GN). We previously identified genetic variants for a NF-κB regulatory, ubiquitin-binding protein ABIN1 as risk factors for GN in systemic autoimmunity. The goal was to define glomerular inflammatory events controlled by ABIN1 function in GN. Nephrotoxic serum nephritis was induced in wild-type (WT) and ubiquitin-binding deficient ABIN1[D485N] mice, and renal pathophysiology and glomerular inflammatory phenotypes were assessed. Proteinuria was also measured in ABIN1[D485N] mice transplanted with WT mouse bone marrow. Inflammatory activation of ABIN1[D472N] (D485N homolog) cultured human-derived podocytes, and interaction with primary human neutrophils were also assessed. Disruption of ABIN1 function exacerbated proteinuria, podocyte injury, glomerular NF-κB activity, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in antibody-mediated nephritis. Transplantation of WT bone marrow did not prevent the increased proteinuria in ABIN1[D845N] mice. Tumor necrosis factor-stimulated enhanced expression and secretion of NF-κB-targeted proinflammatory mediators in ABIN1[D472N] cultured podocytes compared with WT cells. Supernatants from ABIN1[D472N] podocytes accelerated chemotaxis of human neutrophils, and ABIN1[D472N] podocytes displayed a greater susceptibility to injurious morphologic findings induced by neutrophil granule contents. These studies define a novel role for ABIN1 dysfunction and NF-κB in mediating GN through proinflammatory activation of podocytes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Glomerulonefrite/patologia , NF-kappa B/metabolismo , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Glomerulonefrite/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Mutantes
19.
Arthritis Rheumatol ; 69(9): 1840-1849, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28564495

RESUMO

OBJECTIVE: The inflammasome complex is a driver of organ damage in patients with systemic lupus erythematosus (SLE). Although type I interferons (IFNs) are well established as mediators of SLE pathogenesis, their role in inflammasome activation in SLE has not been assessed. The aim of this study was to examine type I IFNs as regulators of the inflammasome. METHODS: SLE patients fulfilled ≥4 American College of Rheumatology criteria and were recruited from the University of Michigan Lupus Cohort. Primary monocytes were isolated from SLE patients or healthy controls by negative selection, treated with inflammasome activators in the presence or absence of IFNα, and IL-1ß secretion was measured by enzyme-linked immunosorbent assay. Expression levels of IFN and inflammasome-related molecules were assessed by real-time polymerase chain reaction and Western blotting. IFN regulatory factor 1 (IRF-1) expression was specifically down-regulated by small interfering RNA (siRNA) transfection and a chemical inhibitor. RESULTS: Monocytes from patients with SLE exhibited increased expression and enhanced activation of the inflammasome by ATP when compared with control monocytes. Expression of inflammasome and IFN-regulated genes was significantly correlated in monocytes from SLE patients but not in control monocytes. Inflammasome activity was increased after prolonged exposure to IFNα. Reduction of IRF-1 expression via siRNA blocked caspase 1 up-regulation after treatment with IFNα. Importantly, hyperactivity of the inflammasome in the monocytes of SLE patients was significantly reduced after knockdown or inhibition of IRF-1. CONCLUSION: Prolonged type I IFN exposure, as seen in SLE patients, primes monocytes for robust inflammasome activation in an IRF-1-dependent manner. IRF-1 inhibition may serve as a novel target for treatment of SLE-associated inflammation and organ damage.


Assuntos
Inflamassomos/fisiologia , Fator Regulador 1 de Interferon/fisiologia , Interferon Tipo I/fisiologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Regulação para Cima , Adulto , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real
20.
J Am Soc Nephrol ; 28(10): 2961-2972, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28646076

RESUMO

IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type-specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell-positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell-positive standard genes. Additionally, patient clinical parameters (serum creatinine values and eGFRs) significantly correlated with Z scores derived from the expression profile of mesangial cell-positive standard genes. Among patients grouped according to Oxford MEST score, patients with segmental glomerulosclerosis had a significantly higher mesangial cell-positive standard gene Z score than patients without segmental glomerulosclerosis. By investigating mesangial cell proteomics and glomerular transcriptomics, we identified 22 common pathways induced in mesangial cells by gd-IgA, most of which mediate inflammation. The genes, proteins, and corresponding pathways identified provide novel insights into the pathophysiologic mechanisms leading to IgAN.


Assuntos
Glomerulonefrite por IGA/metabolismo , Células Mesangiais/metabolismo , Adulto , Estudos de Casos e Controles , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Glomerulonefrite por IGA/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma , Transcriptoma
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