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1.
Artigo em Inglês | MEDLINE | ID: mdl-32035984

RESUMO

BACKGROUND: Atopic dermatitis (AD) is a prevalent inflammatory skin disease with a complex pathogenesis involving immune cell and epidermal abnormalities. Despite whole tissue biopsy studies that have advanced the mechanistic understanding of AD, single cell-based molecular alterations are largely unknown. OBJECTIVE: Our aims were to construct a detailed, high-resolution atlas of cell populations and assess variability in cell composition and cell-specific gene expression in the skin of patients with AD versus in controls. METHODS: We performed single-cell RNA sequencing on skin biopsy specimens from 5 patients with AD (4 lesional samples and 5 nonlesional samples) and 7 healthy control subjects, using 10× Genomics. RESULTS: We created transcriptomic profiles for 39,042 AD (lesional and nonlesional) and healthy skin cells. Fibroblasts demonstrated a novel COL6A5+COL18A1+ subpopulation that was unique to lesional AD and expressed CCL2 and CCL19 cytokines. A corresponding LAMP3+ dendritic cell (DC) population that expressed the CCL19 receptor CCR7 was also unique to AD lesions, illustrating a potential role for fibroblast signaling to immune cells. The lesional AD samples were characterized by expansion of inflammatory DCs (CD1A+FCER1A+) and tissue-resident memory T cells (CD69+CD103+). The frequencies of type 2 (IL13+)/type 22 (IL22+) T cells were higher than those of type 1 (IFNG+) in lesional AD, whereas this ratio was slightly diminished in nonlesional AD and further diminished in controls. CONCLUSION: AD lesions were characterized by expanded type 2/type 22 T cells and inflammatory DCs, and by a unique inflammatory fibroblast that may interact with immune cells to regulate lymphoid cell organization and type 2 inflammation.

2.
Arthritis Care Res (Hoboken) ; 72(2): 233-242, 2020 Feb.
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.

4.
Lupus Sci Med ; 6(1): e000329, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31245017

RESUMO

Single-cell RNA sequencing (scRNA-seq) has recently undergone rapid advances in the development of this technology, leading to high throughput and accelerating discovery in many biological systems and diseases. The single-cell resolution of the technique allows for the investigation of heterogeneity in cell populations, and the pinpointing of pathological populations contributing to disease. Here we review the development of scRNA-seq technology and the analysis that has evolved with the ever-increasing throughput. Finally, we highlight recent applications of scRNA-seq to understand the molecular pathogenesis of lupus and lupus nephritis.

5.
JCI Insight ; 4(11)2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31167973

RESUMO

The central nervous system manifestations of systemic lupus erythematosus (SLE) remain poorly understood. Given the well-defined role of autoantibodies in other lupus manifestations, extensive work has gone into the identification of neuropathic autoantibodies. However, attempts to translate these findings to patients with SLE have yielded mixed results. We used the MRL/MpJ-Faslpr/lpr mouse, a well-established, spontaneous model of SLE, to establish the immune effectors responsible for brain disease. Transcriptomic analysis of the MRL/MpJ-Faslpr/lpr choroid plexus revealed an expression signature driving tertiary lymphoid structure formation, including chemokines related to stromal reorganization and lymphocyte compartmentalization. Additionally, transcriptional profiles indicated various stages of lymphocyte activation and germinal center formation. The extensive choroid plexus infiltrate present in MRL/MpJ-Faslpr/lpr mice with overt neurobehavioral deficits included locally proliferating B and T cells, intercellular interactions between lymphocytes and antigen-presenting cells, as well as evidence for in situ somatic hypermutation and class switch recombination. Furthermore, the choroid plexus was a site for trafficking lymphocytes into the brain. Finally, histological evaluation in human lupus patients with neuropsychiatric manifestations revealed increased leukocyte migration through the choroid plexus. These studies identify a potential new pathway underlying neuropsychiatric lupus and support tertiary lymphoid structure formation in the choroid plexus as a novel mechanism of brain-immune interfacing.

6.
Nat Immunol ; 20(7): 915-927, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31110316

RESUMO

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.


Assuntos
Perfilação da Expressão Gênica , Interferon Tipo I/metabolismo , Queratinócitos/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Transcriptoma , Biópsia , Linhagem da Célula/genética , Biologia Computacional/métodos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Perfilação da Expressão Gênica/métodos , Humanos , Nefrite Lúpica/patologia , Ligação Proteica , Transdução de Sinais , Análise de Célula Única , Pele/imunologia , Pele/metabolismo , Pele/patologia
7.
Front Immunol ; 9: 2189, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319641

RESUMO

About 40% of patients with systemic lupus erythematosus experience diffuse neuropsychiatric manifestations, including impaired cognition and depression. Although the pathogenesis of diffuse neuropsychiatric SLE (NPSLE) is not fully understood, loss of brain barrier integrity, autoreactive antibodies, and pro-inflammatory cytokines are major contributors to disease development. Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator, prevents lymphocyte egress from lymphoid organs through functional antagonism of S1P receptors. In addition to reducing the circulation of autoreactive lymphocytes, fingolimod has direct neuroprotective effects such as preserving brain barrier integrity and decreasing pro-inflammatory cytokine secretion by astrocytes and microglia. Given these effects, we hypothesized that fingolimod would attenuate neurobehavioral deficits in MRL-lpr/lpr (MRL/lpr) mice, a validated neuropsychiatric lupus model. Fingolimod treatment was initiated after the onset of disease, and mice were assessed for alterations in cognitive function and emotionality. We found that fingolimod significantly attenuated spatial memory deficits and depression-like behavior in MRL/lpr mice. Immunofluorescent staining demonstrated a dramatic lessening of brain T cell and macrophage infiltration, and a significant reduction in cortical leakage of serum albumin, in fingolimod treated mice. Astrocytes and endothelial cells from treated mice exhibited reduced expression of inflammatory genes, while microglia showed differential regulation of key immune pathways. Notably, cytokine levels within the cortex and hippocampus were not appreciably decreased with fingolimod despite the improved neurobehavioral profile. Furthermore, despite a reduction in splenomegaly, lymphadenopathy, and circulating autoantibody titers, IgG deposition within the brain was unaffected by treatment. These findings suggest that fingolimod mediates attenuation of NPSLE through a mechanism that is not dependent on reduction of autoantibodies or cytokines, and highlight modulation of the S1P signaling pathway as a novel therapeutic target in lupus involving the central nervous system.


Assuntos
Depressão/imunologia , Cloridrato de Fingolimode/farmacologia , Vasculite Associada ao Lúpus do Sistema Nervoso Central/psicologia , Lisofosfolipídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Autoanticorpos/imunologia , Técnicas de Observação do Comportamento , Comportamento Animal/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/imunologia , Encéfalo/fisiologia , Cognição/efeitos dos fármacos , Cognição/fisiologia , Citocinas/imunologia , Depressão/tratamento farmacológico , Depressão/psicologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Feminino , Cloridrato de Fingolimode/uso terapêutico , Humanos , Vasculite Associada ao Lúpus do Sistema Nervoso Central/tratamento farmacológico , Vasculite Associada ao Lúpus do Sistema Nervoso Central/genética , Vasculite Associada ao Lúpus do Sistema Nervoso Central/imunologia , Lisofosfolipídeos/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Microglia/efeitos dos fármacos , Microglia/imunologia , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/imunologia , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais/imunologia , Esfingosina/imunologia , Esfingosina/metabolismo , Resultado do Tratamento
8.
JCI Insight ; 2(9)2017 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28469080

RESUMO

Lupus nephritis is a leading cause of mortality among systemic lupus erythematosus (SLE) patients, and its heterogeneous nature poses a significant challenge to the development of effective diagnostics and treatments. Single cell RNA sequencing (scRNA-seq) offers a potential solution to dissect the heterogeneity of the disease and enables the study of similar cell types distant from the site of renal injury to identify novel biomarkers. We applied scRNA-seq to human renal and skin biopsy tissues and demonstrated that scRNA-seq can be performed on samples obtained during routine care. Chronicity index, IgG deposition, and quantity of proteinuria correlated with a transcriptomic-based score composed of IFN-inducible genes in renal tubular cells. Furthermore, analysis of cumulative expression profiles of single cell keratinocytes dissociated from nonlesional, non-sun-exposed skin of patients with lupus nephritis also revealed upregulation of IFN-inducible genes compared with keratinocytes isolated from healthy controls. This indicates the possible use of scRNA-seq analysis of skin biopsies as a biomarker of renal disease. These data support the potential utility of scRNA-seq to provide new insights into the pathogenesis of lupus nephritis and pave the way for exploiting a readily accessible tissue to reflect injury in the kidney.

9.
Nature ; 543(7646): 568-572, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28297718

RESUMO

The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice. Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFß superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.


Assuntos
Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Espermatogônias/citologia , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Apoptose/genética , Sequência de Bases , Sítios de Ligação , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Inflamação/genética , Masculino , Camundongos , Complexos Multiproteicos/química , Proteínas de Neoplasias/deficiência , Motivos de Nucleotídeos , Células-Tronco Pluripotentes/citologia , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ribonucleases/química , Transdução de Sinais/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Transcrição Genética/genética , Fator de Crescimento Transformador beta/genética
10.
Sci Rep ; 6: 26164, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27192942

RESUMO

Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton's tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1, either prophylactically or therapeutically. When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease, which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells, as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Inibidores Enzimáticos/administração & dosagem , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/prevenção & controle , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia , Animais , Análise Química do Sangue , Complemento C3/análise , Citocinas/análise , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Rim/patologia , Nefrite Lúpica/induzido quimicamente , Nefrite Lúpica/patologia , Camundongos , Análise de Sequência de RNA , Resultado do Tratamento
11.
J Immunol ; 192(4): 1570-6, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24442428

RESUMO

Systemic lupus erythematosus is an autoimmune disease characterized by elevated production of autoreactive Abs. The disease has a much higher prevalence in women than in men. Although testosterone has been shown to be protective in the disease, and estrogens exacerbating, the discrepancy in prevalence between men and women is still not well understood and the mechanism behind it is unknown. We have recently described that male (New Zealand black [NZB] × New Zealand white [NZW])F1 mice have higher levels of Gr1(+)CD11b(+) cells, and that these cells suppress autoantibody production in vivo. In this article, we extend our findings to show that similarly to humans, female lupus-prone (NZB × NZW)F1 mice also respond with stronger Ab responses to thymus-dependent Ag immunization than male littermates. Furthermore, the presence or absence of Gr1-expressing cells not only control Ag-specific Ab responses in male, but not female, (NZB × NZW)F1 mice, but also significantly alter the activation and differentiation of CD4(+) T cells in vitro and in vivo. In particular, we found that Gr1(+) cells from male (NZB × NZW)F1 mice suppress the differentiation and effector function of CXCR5(+)PD-1(+) T follicular helper cells, thereby controlling germinal center formation and plasma cell differentiation. This new finding strongly supports efforts to develop new drugs that target myeloid cell subsets in a number of T and B cell-mediated diseases with a female predominance.


Assuntos
Formação de Anticorpos/imunologia , Centro Germinativo/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Superfície Celular/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Antígeno CD11b/metabolismo , Diferenciação Celular/imunologia , Proliferação de Células , Feminino , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NZB , Plasmócitos/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR5/metabolismo , Receptores de Superfície Celular/imunologia , Fatores Sexuais
12.
Arthritis Rheum ; 65(9): 2392-402, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23754362

RESUMO

OBJECTIVE: Systemic lupus erythematosus (SLE) develops much more readily in females than in males. Previous research has focused primarily on identifying mechanisms pertinent to the pathology in females. The aim of the current study was to delineate active protective mechanisms in males. We present evidence of a new male-associated mechanism of protection against the development of lupus-like disease in lupus-prone (NZB × NZW)F1 mice. METHODS: We identified previously uncharacterized cellular and functional differences in myeloid cells between male and female (NZB × NZW)F1 mice, with the use of flow cytometry, confocal imaging, in vivo antibody-mediated depletion, and in vitro cell coculture assays. RESULTS: A population of Gr-1(high) Ly-6G+CD11b+ myeloid cells was found to be constitutively increased in male (NZB × NZW)F1 mice as compared with female mice and was regulated by testosterone. The cells were located adjacent to spleen B cell follicles in vivo and were found to directly inhibit cytokine-induced differentiation of naive B cells into antibody-secreting cells in vitro. Most notably, treatment with anti-Gr-1-depleting antibodies increased the spontaneous production of antinuclear autoantibodies in male (NZB × NZW)F1 mice, while a similar approach in female mice had no effect on disease development. CONCLUSION: Male lupus-prone (NZB × NZW)F1 mice harbor elevated levels of a population of myeloid cells with pronounced immunosuppressive capacities that specifically target B cells and the production of antibodies in vivo. We suggest that these cells represent a male-driven inhibitory mechanism involved in the control of B cell pathogenesis, delaying (or preventing) lupus-like disease development in otherwise genetically predisposed male (NZB × NZW)F1 mice.


Assuntos
Linfócitos B/imunologia , Antígeno CD11b/metabolismo , Diferenciação Celular/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Células Mieloides/imunologia , Receptores de Quimiocinas/imunologia , Animais , Autoanticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Feminino , Rim/efeitos dos fármacos , Rim/imunologia , Rim/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NZB , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo
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