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1.
Sci Adv ; 6(26): eaba4353, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32637608

RESUMO

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.

2.
Cell Metab ; 32(2): 243-258.e6, 2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32516575

RESUMO

Adipose tissue invariant natural killer T (iNKT) cells are phenotypically different from other iNKT cells because they produce IL-10 and control metabolic homeostasis. Why that is the case is unclear. Here, using single-cell RNA sequencing, we found several adipose iNKT clusters, which we grouped into two functional populations based on NK1.1 expression. NK1.1NEG cells almost exclusively produced IL-10 and other regulatory cytokines, while NK1.1POS iNKT cells predominantly produced IFNγ. Mechanistically, biochemical fractionation revealed that free fatty acids drive IL-10 production primarily in NK1.1NEG iNKT cells via the IRE1α-XBP1s arm of the unfolded protein response. Correspondingly, adoptive transfer of adipose tissue NK1.1NEG iNKT cells selectively restored metabolic function in obese mice. Further, we found an unexpected role for NK1.1POS iNKT cells in lean adipose tissue, as IFNγ licenses natural killer cell-mediated macrophage killing to limit pathological macrophage expansion. Together, these two iNKT cell populations utilize non-redundant pathways to preserve metabolic integrity.

3.
Nature ; 582(7811): 259-264, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499639

RESUMO

The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.


Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Receptor Notch3/metabolismo , Transdução de Sinais , Membrana Sinovial/patologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Células Endoteliais/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Receptor Notch3/antagonistas & inibidores , Receptor Notch3/deficiência , Receptor Notch3/genética , Antígenos Thy-1/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(16): 8900-8911, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32253314

RESUMO

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Artrite Reumatoide/tratamento farmacológico , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/cirurgia , Linhagem Celular , Fibroblastos , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Pulmão/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Piperidinas/uso terapêutico , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Quinazolinonas/uso terapêutico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Transdução de Sinais/imunologia , Membrana Sinovial/citologia , Membrana Sinovial/patologia , Sinoviócitos , Transativadores/genética , Transativadores/metabolismo
5.
Cell Rep ; 31(1): 107466, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32268091

RESUMO

Interleukin-1ß (IL-1ß) is a key orchestrator of anti-microbial immunity whose secretion is typically dependent on activation of inflammasomes. However, many pathogens have evolved strategies to evade inflammasome activation. Here we describe an alternative, two-cell model for IL-1ß release where invariant natural killer T (iNKT) cells use the death receptor pathway to instruct antigen-presenting cells to secrete IL-1ß. Following cognate interactions with TLR-primed bone marrow-derived dendritic cells (BMDCs), iNKT cells rapidly translocate intracellular Fas ligand to the surface to engage Fas on BMDCs. Fas ligation activates a caspase-8-dependent signaling cascade in BMDCs that drives IL-1ß release largely independent of inflammasomes. The apoptotic program initiated by Fas ligation rapidly transitions into a pyroptosis-like form of cell death mediated by gasdermin D. Together, our findings support a two-cell model for IL-1ß secretion that may supersede inflammasome activation when cytosolic triggers fail.

6.
J Clin Invest ; 130(6): 3238-3252, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32154791

RESUMO

As treatment of the early, inflammatory phase of sepsis improves, post-sepsis immunosuppression and secondary infection have increased in importance. How early inflammation drives immunosuppression remains unclear. Although IFN-γ typically helps microbial clearance, we found that increased plasma IFN-γ in early clinical sepsis was associated with the later development of secondary Candida infection. Consistent with this observation, we found that exogenous IFN-γ suppressed macrophage phagocytosis of zymosan in vivo, and antibody blockade of IFN-γ after endotoxemia improved survival of secondary candidemia. Transcriptomic analysis of innate lymphocytes during endotoxemia suggested that NKT cells drove IFN-γ production by NK cells via mTORC1. Activation of invariant NKT (iNKT) cells with glycolipid antigen drove immunosuppression. Deletion of iNKT cells in Cd1d-/- mice or inhibition of mTOR by rapamycin reduced immunosuppression and susceptibility to secondary Candida infection. Thus, although rapamycin is typically an immunosuppressive medication, in the context of sepsis, rapamycin has the opposite effect. These results implicated an NKT cell/mTOR/IFN-γ axis in immunosuppression following endotoxemia or sepsis. In summary, in vivo iNKT cells activated mTORC1 in NK cells to produce IFN-γ, which worsened macrophage phagocytosis, clearance of secondary Candida infection, and mortality.

8.
Nat Genet ; 52(3): 247-253, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066938

RESUMO

Genetic studies have revealed that autoimmune susceptibility variants are over-represented in memory CD4+ T cell regulatory elements1-3. Understanding how genetic variation affects gene expression in different T cell physiological states is essential for deciphering genetic mechanisms of autoimmunity4,5. Here, we characterized the dynamics of genetic regulatory effects at eight time points during memory CD4+ T cell activation with high-depth RNA-seq in healthy individuals. We discovered widespread, dynamic allele-specific expression across the genome, where the balance of alleles changes over time. These genes were enriched fourfold within autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes. HLA-DQB1 alleles had one of three distinct transcriptional regulatory programs. Using CRISPR-Cas9 genomic editing we demonstrated that a promoter variant is causal for T cell-specific control of HLA-DQB1 expression. Our study shows that genetic variation in cis-regulatory elements affects gene expression in a manner dependent on lymphocyte activation status, contributing to the interindividual complexity of immune responses.


Assuntos
Autoimunidade/genética , Variação Genética , Antígenos HLA/genética , Cadeias beta de HLA-DQ/genética , Ativação Linfocitária/genética , Regiões Promotoras Genéticas/genética , Alelos , Linfócitos T CD4-Positivos , Sistemas CRISPR-Cas , Linhagem Celular , Regulação da Expressão Gênica , Loci Gênicos , Técnicas de Genotipagem , Antígenos HLA/metabolismo , Cadeias beta de HLA-DQ/metabolismo , Humanos , Imunidade Celular , Linfócitos T Reguladores
9.
Proc Natl Acad Sci U S A ; 117(10): 5532-5541, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32079724

RESUMO

The role of stromal fibroblasts in chronic inflammation is unfolding. In rheumatoid arthritis, leukocyte-derived cytokines TNF and IL-17A work together, activating fibroblasts to become a dominant source of the hallmark cytokine IL-6. However, IL-17A alone has minimal effect on fibroblasts. To identify key mediators of the synergistic response to TNF and IL-17A in human synovial fibroblasts, we performed time series, dose-response, and gene-silencing transcriptomics experiments. Here we show that in combination with TNF, IL-17A selectively induces a specific set of genes mediated by factors including cut-like homeobox 1 (CUX1) and IκBζ (NFKBIZ). In the promoters of CXCL1, CXCL2, and CXCL3, we found a putative CUX1-NF-κB binding motif not found elsewhere in the genome. CUX1 and NF-κB p65 mediate transcription of these genes independent of LIFR, STAT3, STAT4, and ELF3. Transcription of NFKBIZ, encoding the atypical IκB factor IκBζ, is IL-17A dose-dependent, and IκBζ only mediates the transcriptional response to TNF and IL-17A, but not to TNF alone. In fibroblasts, IL-17A response depends on CUX1 and IκBζ to engage the NF-κB complex to produce chemoattractants for neutrophil and monocyte recruitment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Inflamação/metabolismo , Interleucina-17/fisiologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Artrite Reumatoide/genética , Células Cultivadas , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Quimiocinas CXC/genética , Fatores Quimiotáticos/genética , Fibroblastos/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Inflamação/genética , Interleucina-17/farmacologia , Interleucina-6/genética , Metaloproteinase 3 da Matriz/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Líquido Sinovial , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/genética , Transcriptoma/efeitos da radiação , Fator de Necrose Tumoral alfa/farmacologia
10.
JCI Insight ; 4(20)2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31536480

RESUMO

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.


Assuntos
Linfócitos B/imunologia , Interleucinas/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Proto-Oncogênicas c-maf/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Idoso , Antígeno CD11c/metabolismo , Sistemas CRISPR-Cas/genética , Estudos de Casos e Controles , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Comunicação Celular/imunologia , Técnicas de Cultura de Células , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Interleucinas/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/sangue , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-maf/genética , RNA-Seq , Receptores CXCR5/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo
12.
EMBO J ; 38(14): e101260, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304630

RESUMO

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.


Assuntos
Perfilação da Expressão Gênica/métodos , Switching de Imunoglobulina , Imunoglobulina G/genética , Células T Matadoras Naturais/metabolismo , Nódulos Linfáticos Agregados/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Interleucina-4/genética , Camundongos , Células T Matadoras Naturais/citologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Técnicas de Transferência Nuclear , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteínas com Domínio T/genética , Vacinação
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.
Nature ; 570(7760): 246-251, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31142839

RESUMO

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.


Assuntos
Artrite Reumatoide/patologia , Fibroblastos/patologia , Animais , Osso e Ossos/patologia , Feminino , Fibroblastos/classificação , Fibroblastos/metabolismo , Gelatinases/metabolismo , Humanos , Inflamação/patologia , Articulações/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , RNA-Seq , Serina Endopeptidases/metabolismo , Análise de Célula Única , Membrana Sinovial/patologia , Antígenos Thy-1/metabolismo
15.
Nat Immunol ; 20(7): 928-942, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31061532

RESUMO

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Perfilação da Expressão Gênica , Membrana Sinovial/metabolismo , Transcriptoma , Artrite Reumatoide/patologia , Autoimunidade/genética , Biomarcadores , Biologia Computacional/métodos , Estudos Transversais , Citocinas/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Membrana Sinovial/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fluxo de Trabalho
16.
Sci Transl Med ; 11(491)2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068444

RESUMO

Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF+ inflammatory macrophages is enriched in RA tissues and is shaped by resident fibroblasts and the cytokine tumor necrosis factor (TNF). These macrophages promoted fibroblast invasiveness in an epidermal growth factor receptor-dependent manner, indicating that intercellular cross-talk in this inflamed setting reshapes both cell types and contributes to fibroblast-mediated joint destruction. In an ex vivo synovial tissue assay, most medications used to treat RA patients targeted HBEGF+ inflammatory macrophages; however, in some cases, medication redirected them into a state that is not expected to resolve inflammation. These data highlight how advances in our understanding of chronically inflamed human tissues and the effects of medications therein can be achieved by studies on local macrophage phenotypes and intercellular interactions.


Assuntos
Artrite Reumatoide/patologia , Fibroblastos/patologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Macrófagos/patologia , Polaridade Celular , Forma Celular , Humanos , Inflamação/patologia , Articulações/patologia , Análise de Célula Única , Membrana Sinovial/patologia
17.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1516-1524, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876808

RESUMO

Cadherins are homophilic cell-to-cell adhesion molecules that help cells respond to environmental changes. Newly formed cadherin junctions are associated with increased cell phosphorylation, but the pathways driving this signaling response are largely unknown. Since cadherins have no intrinsic signaling activity, this phosphorylation must occur through interactions with other signaling molecules. We previously reported that cadherin-11 engagement activates joint synovial fibroblasts, promoting inflammatory and degradative pathways important in rheumatoid arthritis (RA) pathogenesis. Our objective in this study was to discover interacting partners that mediate cadherin-11 signaling. Protein array screening showed that cadherin-11 extracellular binding domains linked to an Fc domain (cad11Fc) induced platelet-derived growth factor (PDGFR)-α phosphorylation in synovial fibroblasts and glioblastoma cells. PDGFRs are growth factor receptor tyrosine kinases that promote cell proliferation, survival, and migration in mesodermally derived cells. Increased PDGFR activity is implicated in RA pathology and associates with poor prognosis in several cancers, including sarcoma and glioblastoma. PDGFRα activation by cadherin-11 signaling promoted fibroblast proliferation, a signaling pathway independent from cadherin-11-stimulated IL-6 or matrix metalloproteinase (MMP)-3 release. PDGFRα phosphorylation mediated most of the cad11Fc-induced phosphatidyl-3-kinase (PI3K)/Akt activation, but only part of the mitogen-activated protein kinase (MAPK) response. PDGFRα-dependent signaling did not require cell cadherin-11 expression. Rather, cad11Fc immunoprecipitated PDGFRα, indicating a direct interaction between cadherin-11 and PDGFRα extracellular domains. This study is the first to report an interaction between cadherin-11 and PDGFRα and adds to our growing understanding that cadherin-growth factor receptor interactions help balance the interplay between tissue growth and adhesion.


Assuntos
Artrite Reumatoide/genética , Caderinas/genética , Fibroblastos/metabolismo , Osteoartrite/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Caderinas/metabolismo , Adesão Celular , Proliferação de Células , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Cápsula Articular/metabolismo , Cápsula Articular/patologia , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Cultura Primária de Células , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo
18.
Nat Immunol ; 20(3): 373, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30728493

RESUMO

In the version of this article initially published, three authors (Hui-Fern Kuoy, Adam P. Uldrich and Dale. I. Godfrey) and their affiliations, acknowledgments and contributions were not included. The correct information is as follows:Ayano C. Kohlgruber1,2, Shani T. Gal-Oz3, Nelson M. LaMarche1,2, Moto Shimazaki1, Danielle Duquette4, Hui-Fern Koay5,6, Hung N. Nguyen1, Amir I. Mina4, Tyler Paras1, Ali Tavakkoli7, Ulrich von Andrian2,8, Adam P. Uldrich5,6, Dale I. Godfrey5,6, Alexander S. Banks4, Tal Shay3, Michael B. Brenner1,10* and Lydia Lynch1,4,9,10*1Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA. 2Division of Medical Sciences, Harvard Medical School, Boston, MA, USA. 3Department of Life Sciences, Ben-Gurion University of the Negev, Beersheba, Israel. 4Division of Endocrinology, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. 5Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Australia. 6ARC Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Australia. 7Department of General and Gastrointestinal Surgery, Brigham and Women's Hospital, Boston, MA, USA. 8Department of Microbiology and Immunology, Harvard Medical School, Boston, MA, USA. 9School of Biochemistry and Immunology, Trinity College, Dublin, Ireland. 10These authors jointly supervised this work: Michael B. Brenner, Lydia Lynch. *e-mail: mbrenner@research.bwh.harvard.edu; llynch@bwh.harvard.eduAcknowledgementsWe thank A.T. Chicoine, flow cytometry core manager at the Human Immunology Center at BWH, for flow cytometry sorting. We thank D. Sant'Angelo (Rutgers Cancer Institute) for providing Zbtb16-/- mice and R. O'Brien (National Jewish Health) for providing Vg4/6-/- mice. Supported by NIH grant R01 AI11304603 (to M.B.B.), ERC Starting Grant 679173 (to L.L.), the National Health and Medical Research Council of Australia (1013667), an Australian Research Council Future Fellowship (FT140100278 for A.P.U.) and a National Health and Medical Research Council of Australia Senior Principal Research Fellowship (1117766 for D.I.G.).Author contributionsA.C.K., L.L., and M.B.B. conceived and designed the experiments, and wrote the manuscript. A.C.K., N.M.L., L.L., H.N.N., M.S., T.P., and D.D. performed the experiments. S.T.G.-O. and T.S. performed the RNA-seq analysis. A.S.B. and A.I.M. provided advice and performed the CLAMS experiments. A.T. provided human bariatric patient samples. Parabiosis experiments were performed in the laboratory of U.v.A. H.-F.K., A.P.U. and D.I.G provided critical insight into the TCR chain usage of PLZF+ γδ T cells. M.B.B., N.M.L., and L.L. critically reviewed the manuscript.The errors have been corrected in the HTML and PDF version of the article.Correction to: Nature Immunology doi:10.1038/s41590-018-0094-2 (2018), published online 18 April 2018.

19.
Nat Commun ; 10(1): 687, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737409

RESUMO

How innate T cells (ITC), including invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, maintain a poised effector state has been unclear. Here we address this question using low-input and single-cell RNA-seq of human lymphocyte populations. Unbiased transcriptomic analyses uncover a continuous 'innateness gradient', with adaptive T cells at one end, followed by MAIT, iNKT, γδ T and natural killer cells at the other end. Single-cell RNA-seq reveals four broad states of innateness, and heterogeneity within canonical innate and adaptive populations. Transcriptional and functional data show that innateness is characterized by pre-formed mRNA encoding effector functions, but impaired proliferation marked by decreased baseline expression of ribosomal genes. Together, our data shed new light on the poised state of ITC, in which innateness is defined by a transcriptionally-orchestrated trade-off between rapid cell growth and rapid effector function.


Assuntos
Proliferação de Células/fisiologia , Linfócitos/metabolismo , Feminino , Ontologia Genética , Humanos , Imunidade Inata/fisiologia , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Células T Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/metabolismo
20.
Blood ; 133(6): 605-614, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30429159

RESUMO

More than 1 million apheresis platelet collections are performed annually in the United States. After 2 healthy plateletpheresis donors were incidentally found to have low CD4+ T-lymphocyte counts, we investigated whether plateletpheresis causes lymphopenia. We conducted a cross-sectional single-center study of platelet donors undergoing plateletpheresis with the Trima Accel, which removes leukocytes continuously with its leukoreduction system chamber. We recruited 3 groups of platelet donors based on the total number of plateletpheresis sessions in the prior 365 days: 1 or 2, 3 to 19, or 20 to 24. CD4+ T-lymphocyte counts were <200 cells per microliter in 0/20, 2/20, and 6/20 donors, respectively (P = .019), and CD8+ T-lymphocyte counts were low in 0/20, 4/20, and 11/20 donors, respectively (P < .001). The leukoreduction system chamber's lymphocyte-extraction efficiency was ∼15% to 20% for all groups. Immunophenotyping showed decreases in naive CD4+ T-lymphocyte and T helper 17 (Th17) cell percentages, increases in CD4+ and CD8+ effector memory, Th1, and regulatory T cell percentages, and stable naive CD8+ and Th2 percentages across groups. T-cell receptor repertoire analyses showed similar clonal diversity in all groups. Donor screening questionnaires supported the good health of the donors, who tested negative at each donation for multiple pathogens, including HIV. Frequent plateletpheresis utilizing a leukoreduction system chamber is associated with CD4+ and CD8+ T-cell lymphopenia in healthy platelet donors. The mechanism may be repeated extraction of these cells during plateletpheresis. The cytopenias do not appear to be harmful.


Assuntos
Doadores de Sangue/estatística & dados numéricos , Plaquetas/citologia , Linfopenia/etiologia , Plaquetoferese/efeitos adversos , Adulto , Idoso , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Prognóstico , Adulto Jovem
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