RESUMO
Antibodies to DNA and chromatin drive autoimmunity in systemic lupus erythematosus (SLE). Null mutations and hypomorphic variants of the secreted deoxyribonuclease DNASE1L3 are linked to familial and sporadic SLE, respectively. We report that DNASE1L3-deficient mice rapidly develop autoantibodies to DNA and chromatin, followed by an SLE-like disease. Circulating DNASE1L3 is produced by dendritic cells and macrophages, and its levels inversely correlate with anti-DNA antibody response. DNASE1L3 is uniquely capable of digesting chromatin in microparticles released from apoptotic cells. Accordingly, DNASE1L3-deficient mice and human patients have elevated DNA levels in plasma, particularly in circulating microparticles. Murine and human autoantibody clones and serum antibodies from human SLE patients bind to DNASE1L3-sensitive chromatin on the surface of microparticles. Thus, extracellular microparticle-associated chromatin is a potential self-antigen normally digested by circulating DNASE1L3. The loss of this tolerance mechanism can contribute to SLE, and its restoration may represent a therapeutic opportunity in the disease.
Assuntos
Autoanticorpos/imunologia , Micropartículas Derivadas de Células/química , Cromatina/imunologia , DNA/imunologia , Endodesoxirribonucleases/genética , Lúpus Eritematoso Sistêmico/imunologia , Animais , Micropartículas Derivadas de Células/metabolismo , Modelos Animais de Doenças , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Humanos , Células Jurkat , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Plasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a "CC" end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.
Assuntos
DNA/genética , Endodesoxirribonucleases/genética , Lúpus Eritematoso Sistêmico/genética , Mutação , Animais , Estudos de Casos e Controles , DNA/sangue , Fragmentação do DNA , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Terapia Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Transgênicos , Especificidade por Substrato , Transdução GenéticaRESUMO
Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3-/- mice carrying Dnase1l3+/- fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3 Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity.
Assuntos
Ácidos Nucleicos Livres/sangue , Fragmentação do DNA , DNA/sangue , Endodesoxirribonucleases/metabolismo , Motivos de Nucleotídeos , Animais , Ácidos Nucleicos Livres/genética , DNA/genética , Endodesoxirribonucleases/genética , Feminino , Feto/metabolismo , Deleção de Genes , Camundongos , Camundongos Knockout , GravidezRESUMO
mAbs specific for surface proteins on APCs can serve as Ag-delivery vehicles that enhance immunogenicity. The practical use of such constructs is limited by the challenge of expressing and modifying full-sized mAbs. We generated single-domain Ab fragments (VHHs) specific for class II MHC (MHCII), CD11b, and CD36. VHH sequences were modified by inclusion of a C-terminal sortase motif to allow site-specific conjugation with various Ag payloads. We tested T cell activation using VHHs that target distinct APC populations; anti-MHCII adducts elicited strong activation of CD4+ T cells, whereas anti-CD11b showed CD8+ T cell activation superior to targeting via MHCII and CD36. Differences in Ag presentation among constructs were unrelated to dendritic cell subtype or routing to acidic compartments. When coupled to antigenic payloads, anti-MHCII VHH primed Ab responses against GFP, ubiquitin, an OVA peptide, and the α-helix of influenza hemagglutinin's stem; the last afforded protection against influenza infection. The versatility of the VHH scaffold and sortase-mediated covalent attachment of Ags suggests their broader application to generate desirable immune responses.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Células Dendríticas/fisiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Anticorpos de Domínio Único/metabolismo , Animais , Apresentação de Antígeno , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Camelídeos Americanos , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Influenza Humana/prevenção & controle , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/prevenção & controle , Anticorpos de Domínio Único/imunologiaRESUMO
The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.
Assuntos
Candida albicans , Candidíase/imunologia , Interações Hospedeiro-Patógeno/imunologia , Fagocitose/fisiologia , Esfingolipídeos/biossíntese , Animais , Candida albicans/imunologia , Linhagem Celular , Cromatografia em Camada Fina , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Espectrometria de Massas , CamundongosRESUMO
Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R, and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq-mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.
Assuntos
Receptores Histamínicos/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Técnicas Biossensoriais , Sinalização do Cálcio , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with poor prognosis and resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We previously showed that KEAP1 mutant tumors consume glutamine to support the metabolic rewiring associated with NRF2-dependent antioxidant production. Here, using preclinical patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the glutamine antagonist prodrug DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumors by inhibiting glutamine-dependent nucleotide synthesis and promoting antitumor T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we demonstrate that DRP-104 reverses T cell exhaustion, decreases Tregs, and enhances the function of CD4 and CD8 T cells, culminating in an improved response to anti-PD1 therapy. Our preclinical findings provide compelling evidence that DRP-104, currently in clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer.
Assuntos
Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Glutamina/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Inibidores Enzimáticos/uso terapêutico , MutaçãoRESUMO
Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.
Assuntos
Biofilmes , Endodesoxirribonucleases , Sepse , Infecções Estafilocócicas , Animais , Camundongos , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Desoxirribonucleases/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Armadilhas Extracelulares/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/metabolismoRESUMO
Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We have previously shown that KEAP1 mutant tumors have increased glutamine consumption to support the metabolic rewiring associated with NRF2 activation. Here, using patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the novel glutamine antagonist DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumor growth by inhibiting glutamine-dependent nucleotide synthesis and promoting anti-tumor CD4 and CD8 T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we discover that DRP-104 reverses T cell exhaustion and enhances the function of CD4 and CD8 T cells culminating in an improved response to anti-PD1 therapy. Our pre-clinical findings provide compelling evidence that DRP-104, currently in phase 1 clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer. Furthermore, we demonstrate that by combining DRP-104 with checkpoint inhibition, we can achieve suppression of tumor intrinsic metabolism and augmentation of anti-tumor T cell responses.
RESUMO
Lung cancer treatment has benefited greatly through advancements in immunotherapies. However, immunotherapy often fails in patients with specific mutations like KEAP1, which are frequently found in lung adenocarcinoma. We established an antigenic lung cancer model and used it to explore how Keap1 mutations remodel the tumor immune microenvironment. Using single-cell technology and depletion studies, we demonstrate that Keap1-mutant tumors diminish dendritic cell and T cell responses driving immunotherapy resistance. This observation was corroborated in patient samples. CRISPR-Cas9-mediated gene targeting revealed that hyperactivation of the NRF2 antioxidant pathway is responsible for diminished immune responses in Keap1-mutant tumors. Importantly, we demonstrate that combining glutaminase inhibition with immune checkpoint blockade can reverse immunosuppression, making Keap1-mutant tumors susceptible to immunotherapy. Our study provides new insight into the role of KEAP1 mutations in immune evasion, paving the way for novel immune-based therapeutic strategies for KEAP1-mutant cancers.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Evasão da Resposta Imune , Linhagem Celular Tumoral , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/terapia , Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/tratamento farmacológico , Mutação/genética , Imunoterapia , Microambiente TumoralRESUMO
Tumor mutations can influence the surrounding microenvironment leading to suppression of anti-tumor immune responses and thereby contributing to tumor progression and failure of cancer therapies. Here we use genetically engineered lung cancer mouse models and patient samples to dissect how LKB1 mutations accelerate tumor growth by reshaping the immune microenvironment. Comprehensive immune profiling of LKB1 -mutant vs wildtype tumors revealed dramatic changes in myeloid cells, specifically enrichment of Arg1 + interstitial macrophages and SiglecF Hi neutrophils. We discovered a novel mechanism whereby autocrine LIF signaling in Lkb1 -mutant tumors drives tumorigenesis by reprogramming myeloid cells in the immune microenvironment. Inhibiting LIF signaling in Lkb1 -mutant tumors, via gene targeting or with a neutralizing antibody, resulted in a striking reduction in Arg1 + interstitial macrophages and SiglecF Hi neutrophils, expansion of antigen specific T cells, and inhibition of tumor progression. Thus, targeting LIF signaling provides a new therapeutic approach to reverse the immunosuppressive microenvironment of LKB1 -mutant tumors.
RESUMO
Phagocytosis by alveolar macrophages is the obligate first step in Mycobacterium tuberculosis (Mtb) infection, yet the mechanism underlying this process is incompletely understood. Here, we show that Mtb invasion relies on an intact sphingolipid biosynthetic pathway. Inhibition or knockout of early sphingolipid biosynthetic enzymes greatly reduces Mtb uptake across multiple phagocytic cell types without affecting other forms of endocytosis. While the phagocytic receptor dectin-1 undergoes normal clustering at the pathogen contact sites, sphingolipid biosynthetic mutant cells fail to segregate the regulatory phosphatase CD45 from the clustered receptors. Blocking sphingolipid production also impairs downstream activation of Rho GTPases, actin dynamics, and phosphoinositide turnover at the nascent phagocytic cup. Moreover, we found that production of sphingomyelin, not glycosphingolipids, is essential for Mtb uptake. Collectively, our data support a critical role of sphingomyelin biosynthesis in an early stage of Mtb infection and provide novel insights into the mechanism underlying phagocytic entry of this pathogen.IMPORTANCEMycobacterium tuberculosis (Mtb) invades alveolar macrophages through phagocytosis to establish infection and cause disease. The molecular mechanisms underlying Mtb entry are still poorly understood. Here, we report that an intact sphingolipid biosynthetic pathway is essential for the uptake of Mtb by phagocytes. Disrupting sphingolipid production affects the segregation of the regulatory phosphatase CD45 from the nascent phagosome, a critical step in the progression of phagocytosis. We also show that blocking sphingolipid biosynthesis impairs activation of small GTPases and phosphoinositide turnover at the host-pathogen contact sites. Moreover, production of sphingomyelin, not glycosphingolipids, is critical for the phagocytic uptake of Mtb These data demonstrate a vital role for sphingomyelin biosynthesis in an early step of Mtb infection, defining a potential target for antimycobacterial therapeutics.
Assuntos
Interações Hospedeiro-Patógeno , Macrófagos Alveolares/microbiologia , Mycobacterium tuberculosis/fisiologia , Fagocitose/fisiologia , Esfingomielinas/biossíntese , Animais , Vias Biossintéticas , Células Cultivadas , Humanos , Macrófagos Alveolares/imunologia , Camundongos , Mycobacterium tuberculosis/imunologia , Células RAW 264.7 , Transdução de Sinais , Células THP-1RESUMO
Antibodies to double-stranded DNA (dsDNA) are prevalent in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis, yet the nature and regulation of antigenic cell-free DNA (cfDNA) are poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity. We report that >50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation, which was associated with neutralizing autoantibodies to DNASE1L3. These patients had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens included DNA-associated proteins such as HMGB1. Our results reveal autoantibody-mediated impairment of DNASE1L3 activity as a common nongenetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE.
Assuntos
Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , DNA/imunologia , Endodesoxirribonucleases/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Adulto , Animais , Anticorpos Antinucleares/sangue , Autoanticorpos/sangue , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/imunologia , Criança , Endodesoxirribonucleases/sangue , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Células HEK293 , Proteína HMGB1/imunologia , Proteína HMGB1/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Nefrite Lúpica/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Índice de Gravidade de DoençaRESUMO
Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSCs) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady-state. Labeled cells, comprising primarily long-term HSCs and some short-term HSCs, produced megakaryocytic lineage progeny within 1 wk in a process that required only two to three cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 wk and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic, and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 wk of tracing. These results show that continuous differentiation of HSCs rapidly produces major hematopoietic lineages and cell types and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid, and lymphoid differentiation.
Assuntos
Células-Tronco Adultas/imunologia , Diferenciação Celular/imunologia , Divisão Celular/imunologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Adultas/citologia , Animais , Células Dendríticas/citologia , Células Dendríticas/imunologia , Granulócitos/citologia , Granulócitos/imunologia , Células-Tronco Hematopoéticas/citologia , Cinética , Megacariócitos/citologia , Megacariócitos/imunologia , Camundongos , Camundongos Transgênicos , Monócitos/citologia , Monócitos/imunologiaRESUMO
Inflammatory bowel disease (IBD) is a chronic inflammatory condition caused by an aberrant immune response to microbial components of the gastrointestinal tract. Plasmacytoid dendritic cells (pDCs) are innate immune cells specialized in the production of type I interferons and were recently implicated in the pathogenesis of autoimmune disorders such as lupus and scleroderma. While pDCs were shown to infiltrate intestinal mucosa of IBD patients and proposed to participate in intestinal inflammation, their net contribution to the disease remains unclear. We addressed this question by targeting the pDC-specific transcription factor TCF4 (E2-2) in experimental IBD caused by deficiency of Wiskott-Aldrich syndrome protein (WASP) or of interleukin-10 (IL-10). Monoallelic Tcf4 deletion, which was previously shown to abrogate experimental lupus, did not affect autoimmunity manifestations or colitis in WASP-deficient animals. Furthermore, conditional biallelic Tcf4 targeting resulted in a near-complete pDC ablation, yet had no effect on the development of colitis in IL-10-deficient mice. Our results suggest that, in contrast to other inflammatory and autoimmune diseases, pDCs do not play a major role in the pathogenesis of intestinal inflammation during IBD.
Assuntos
Colite/imunologia , Células Dendríticas/imunologia , Doenças Inflamatórias Intestinais/imunologia , Nefrite Lúpica/imunologia , Fator de Transcrição 4/metabolismo , Animais , Colite/genética , Humanos , Imunidade Inata , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Fator de Transcrição 4/genética , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
Droplet-based single-cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While this approach offers the exciting promise to deconvolute cellular heterogeneity in diseased tissues, the lack of cost-effective and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we developed a 3D-printed, low-cost droplet microfluidic control instrument and deploy it in a clinical environment to perform single-cell transcriptome profiling of disaggregated synovial tissue from five rheumatoid arthritis patients. We sequence 20,387 single cells revealing 13 transcriptomically distinct clusters. These encompass an unsupervised draft atlas of the autoimmune infiltrate that contribute to disease biology. Additionally, we identify previously uncharacterized fibroblast subpopulations and discern their spatial location within the synovium. We envision that this instrument will have broad utility in both research and clinical settings, enabling low-cost and routine application of microfluidic techniques.
Assuntos
Artrite Reumatoide/genética , Microfluídica/métodos , RNA/genética , Análise de Célula Única/métodos , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Microfluídica/economia , Microfluídica/instrumentação , RNA/metabolismo , Análise de Célula Única/economia , Análise de Célula Única/instrumentação , Membrana Sinovial/citologia , Membrana Sinovial/metabolismoRESUMO
The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system - such as neutrophils, dendritic cells, and macrophages - retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4. Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 µm, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.