RESUMO
Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.
Assuntos
Epigênese Genética , Interferon Tipo I , Coriomeningite Linfocítica , Vírus da Coriomeningite Linfocítica , Células B de Memória , Animais , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Camundongos , Vírus da Coriomeningite Linfocítica/imunologia , Células B de Memória/imunologia , Camundongos Endogâmicos C57BL , Receptor de Interferon alfa e beta/genética , Memória Imunológica/imunologia , Doença Crônica , Subpopulações de Linfócitos B/imunologia , Análise de Célula ÚnicaRESUMO
cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.
Assuntos
Imunidade Inata , Proteínas de Membrana , Camundongos , Animais , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Macrófagos/metabolismo , Nucleotidiltransferases/metabolismo , DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/genéticaRESUMO
Stimulator of Interferon Genes (STING) is a cytosolic sensor of cyclic dinucleotides (CDNs). The activation of dendritic cells (DC) via the STING pathway, and their subsequent production of type I interferon (IFN) is considered central to eradicating tumours in mouse models. However, this contribution of STING in preclinical murine studies has not translated into positive outcomes of STING agonists in phase I & II clinical trials. We therefore questioned whether a difference in human DC responses could be critical to the lack of STING agonist efficacy in human settings. This study sought to directly compare mouse and human plasmacytoid DCs and conventional DC subset responses upon STING activation. We found all mouse and human DC subsets were potently activated by STING stimulation. As expected, Type I IFNs were produced by both mouse and human plasmacytoid DCs. However, mouse and human plasmacytoid and conventional DCs all produced type III IFNs (i.e., IFN-λs) in response to STING activation. Of particular interest, all human DCs produced large amounts of IFN-λ1, not expressed in the mouse genome. Furthermore, we also found differential cell death responses upon STING activation, observing rapid ablation of mouse, but not human, plasmacytoid DCs. STING-induced cell death in murine plasmacytoid DCs occurred in a cell-intrinsic manner and involved intrinsic apoptosis. These data highlight discordance between STING IFN and cell death responses in mouse and human DCs and caution against extrapolating STING-mediated events in mouse models to equivalent human outcomes.
Assuntos
Interferon Tipo I , Animais , Morte Celular , Citosol/metabolismo , Células Dendríticas/metabolismo , Humanos , Interferon Tipo I/metabolismo , Proteínas de Membrana , Camundongos , Transdução de SinaisRESUMO
Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.
Assuntos
Células Dendríticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Feminino , Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismoRESUMO
Initiation of adaptive immunity to particulate antigens in lymph nodes largely depends on their presentation by migratory dendritic cells (DCs). DC subsets differ in their capacity to induce specific types of immunity, allowing subset-specific DC-targeting to influence vaccination and therapy outcomes. Faithful drug design, however, requires exact understanding of subset-specific versus global activation mechanisms. cDC1, the subset of DCs that excel in supporting immunity toward viruses, intracellular bacteria, and tumors, express uniquely high levels of the pattern recognition receptor TLR3. Using various murine genetic models, we show here that both, the cDC1 and cDC2 subsets of cDCs are activated and migrate equally well in response to TLR3 stimulation in a cell extrinsic and TNF-α dependent manner, but that cDC1 show a unique requirement for type I interferon signaling. Our findings reveal common and differing pathways regulating DC subset migration, offering important insights for the design of DC-based vaccination and therapy approaches.
Assuntos
Células Dendríticas/imunologia , Intestinos/imunologia , Receptor 3 Toll-Like/metabolismo , Animais , Vacinas Anticâncer , Movimento Celular , Células Cultivadas , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Receptor 3 Toll-Like/imunologiaRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) presents an increasing threat to public health, with antimicrobial resistance on the rise and infections endemic in the hospital setting. Despite a global research effort to understand and combat antimicrobial resistance, less work has focused on understanding the nuances in the immunopathogenesis of clinical strains. In particular, there is a surprising gap of knowledge in the literature pertaining to how clinical strains are recognized by dendritic cells (DCs). Here, we show that the activation of DCs is compromised in response to MRSA strains resistant to the last-line antibiotic daptomycin. We found a significant reduction in the secretion of proinflammatory cytokines including tumor necrosis factor-α, interleukin-6, regulated upon activation, normal T cell expressed, and secreted and macrophage inflammatory protein-1ß, as well as decreased expression of CD80 by DCs responding to daptomycin-resistant MRSA. We further demonstrate that this phenotype is coincident with the acquisition of specific point mutations in the cardiolipin synthase gene cls2, and, partly, in the bifunctional lysylphosphatidylglycerol flippase/synthetase gene mprF, which are genes that are often mutated in clinical daptomycin-resistant strains. Therefore, throughout infection and antibiotic therapy, MRSA has the capacity to not only develop further antibiotic resistance, but also develop resistance to immunological recognition by DCs, because of single amino acid point mutations occurring under the selective pressures of both host immunity and antibiotic therapy. Understanding the diversity of clinical MRSA isolates and the nuances in their immune recognition will have important implications for future therapeutics and the treatment of these infections.
Assuntos
Daptomicina , Células Dendríticas/imunologia , Farmacorresistência Bacteriana/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Animais , Antígeno B7-1/imunologia , Citocinas/imunologia , Regulação da Expressão Gênica , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , CamundongosRESUMO
The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.
Assuntos
Células Dendríticas/fisiologia , Endossomos/metabolismo , Exonucleases/metabolismo , Hepatite/genética , Macrófagos/fisiologia , Glicoproteínas de Membrana/metabolismo , Fosfolipase D/metabolismo , Doença de Alzheimer/genética , Animais , Artrite Reumatoide/genética , DNA de Cadeia Simples/imunologia , Exonucleases/genética , Estudo de Associação Genômica Ampla , Humanos , Interferon gama/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipase D/genética , Escleroderma Sistêmico/genética , Transdução de Sinais , Receptor Toll-Like 9/metabolismoRESUMO
Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.
Assuntos
Imunidade Inata , Proteínas de Membrana/metabolismo , Transporte de RNA , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/imunologia , Linhagem Celular , Citoplasma , Proteína DEAD-box 58/metabolismo , Modelos Animais de Doenças , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/imunologia , Endossomos/metabolismo , Feminino , Expressão Gênica , Técnicas de Inativação de Genes , Herpes Simples/genética , Herpes Simples/imunologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas de Transporte de Nucleotídeos , Ligação Proteica , Transporte Proteico , RNA Viral/genética , RNA Viral/metabolismo , Transdução de Sinais , Receptor 3 Toll-Like/metabolismoRESUMO
Plasmacytoid dendritic cells (pDCs) play an important role in immunity to certain pathogens and immunopathology in some autoimmune diseases. They are thought to have a longer lifespan than conventional DCs (cDCs), largely based on a slower rate of BrdU labeling by splenic pDCs. Here we demonstrated that pDC expansion and therefore BrdU labeling by pDCs occurs in bone marrow (BM). The rate of labeling was similar between BM pDCs and spleen cDCs. Therefore, slower BrdU labeling of spleen pDCs likely reflects the "migration time" (â¼2 days) for BrdU labeled pDCs to traffic to the spleen, not necessarily reflecting longer life span. Tracking the decay of differentiated DCs showed that splenic pDCs and cDCs decayed at a similar rate. We suggest that spleen pDCs have a shorter in vivo lifespan than estimated utilizing some of the previous approaches. Nevertheless, pDC lifespan varies between mouse strains. pDCs from lupus-prone NZB mice survived longer than C57BL/6 pDCs. We also demonstrated that activation either positively or negatively impacted on the survival of pDCs via different cell-death mechanisms. Thus, pDCs are also short-lived. However, the pDC lifespan is regulated by genetic and environmental factors that may have pathological consequence.
Assuntos
Células da Medula Óssea/citologia , Células Dendríticas/citologia , Baço/citologia , Animais , Células da Medula Óssea/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Células Dendríticas/metabolismo , Camundongos , Baço/metabolismoRESUMO
Ras guanine nucleotide-releasing protein-4 (RasGRP4) is an evolutionarily conserved calcium-regulated, guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor. While an important intracellular signaling protein for CD117+ mast cells (MCs), its roles in other immune cells is less clear. In this study, we identified a subset of in vivo-differentiated splenic CD117+ dendritic cells (DCs) in wild-type (WT) C57BL/6 mice that unexpectedly contained RasGRP4 mRNA and protein. In regard to the biologic significance of these data to innate immunity, LPS-treated splenic CD117+ DCs from WT mice induced natural killer (NK) cells to produce much more interferon-γ (IFN-γ) than comparable DCs from RasGRP4-null mice. The ability of LPS-responsive MCs to cause NK cells to increase their expression of IFN-γ was also dependent on this intracellular signaling protein. The discovery that RasGRP4 is required for CD117+ MCs and DCs to optimally induce acute NK cell-dependent immune responses to LPS helps explain why this signaling protein has been conserved in evolution.
Assuntos
Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Lipopolissacarídeos/farmacologia , Mastócitos/imunologia , Proteínas Proto-Oncogênicas c-kit/imunologia , Fatores ras de Troca de Nucleotídeo Guanina/fisiologia , Animais , Técnicas de Cocultura , Interferon gama/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligante OX40 , Transdução de Sinais , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Fatores de Necrose Tumoral/metabolismoRESUMO
DCs often require stimulation from CD4(+) T cells to propagate CD8(+) T cell responses, but precisely how T cell help optimizes the priming capacity of DCs and why this appears to differ between varying types of CD8(+) T cell immunity remains unclear. We show that CD8(+) T cell priming upon HSV-1 skin infection depended on DCs receiving stimulation from both IFN-α/ß and CD4(+) T cells to provide IL-15. This was not an additive effect but resulted from CD4(+) T cells amplifying DC production of IL-15 in response to IFN-α/ß. We also observed that increased innate stimulation reversed the helper dependence of CD8(+) T cell priming and that the innate stimulus, rather than the CD4(+) T cells themselves, determined how "help'" was integrated into the priming response by DCs. These findings identify T cell help as a flexible means to amplify varying suboptimal innate signals in DCs.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Quimiocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Herpesvirus Humano 1/fisiologia , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Interferon beta/metabolismo , Interleucina-15/metabolismo , Interleucina-6/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Dermatopatias/patologia , Dermatopatias/virologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141(+) DC, CD1c(+) DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c(+) DC and CD141(+) DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c(+) DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141(+) DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c(+) DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8(+) T cells, CD141(+) DC are superior in their capacity to cross-present protein Ag to CD8(+) T cells following activation with polyinosinic-polycytidylic acid. CD141(+) DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141(+) and CD1c(+) DC and evaluate adjuvants and DC-targeting strategies in vivo.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos CD1/metabolismo , Antígenos de Superfície/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Glicoproteínas/metabolismo , Proteínas de Membrana/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Apresentação de Antígeno/imunologia , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/imunologia , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/metabolismo , Feminino , Humanos , Imunoglobulinas/metabolismo , Interferon gama/metabolismo , Interleucina-12/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Ativação Linfocitária/imunologia , Proteínas de Membrana/administração & dosagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Antígenos de Histocompatibilidade Menor , Poli I-C/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Quimiocinas/metabolismo , Trombomodulina , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5'-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Inosina/genética , Edição de RNA , RNA Viral/genética , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Adenosina/genética , Adenosina/imunologia , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Animais , Sequência de Bases , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/genética , Inosina/imunologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/imunologia , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/genéticaRESUMO
Interferon-producing plasmacytoid dendritic cells (pDC) are a specialized branch of the dendritic cell (DC) family, and their differentiation in mice is closely linked to that of conventional DC (cDC). Several different developmental pathways retain the potential to form pDC and are likely to contribute to the steady-state pDC population. A lymphoid pathway to DC development produces mainly pDC as a branch otherwise leading to B-cell development; such pDC may carry relics of a lymphoid past such as DJ rearrangements of immunoglobulin heavy chain (IgH) genes. The myeloid pathway to pDC and cDC is better known, but recent reassessment has revealed several substreams of development with separate DC-committed precursors. One substream has a lymphoid-like aspect, involving a precursor expressing RAG-1 and producing pDC with IgH gene rearrangements. Another more biased to cDC production produces pDC without such IgH gene rearrangements. Finally, there is the production of interferon-producing pDC-like cells that are not pDC but appear to be cDC precursors; these do not express key pDC markers such as CCR9. Initiation of the DC and then the pDC developmental program overrides any surface marker-expressed developmental bias to other myeloid or lymphoid lineages, resulting in an apparent convergent differentiation to the pDC form. A DC fate is sometimes imprinted early in development, upstream of identifiable myeloid, or lymphoid precursors. This suggests that DC, including pDC, represent a distinct hematopoietic lineage separate from conventional myeloid or lymphoid cells.
Assuntos
Células Dendríticas/citologia , Animais , Linhagem da Célula , Células Dendríticas/metabolismo , Hematopoese , HumanosRESUMO
Plasmacytoid dendritic cells (pDC) are the producers of type I IFNs in response to TLR9 ligands. However, we have found that when bone marrow is depleted of pDC, the IFN-α produced in response to TLR9 ligands is not fully removed. We assign the source of this non-pDC IFN-α as a newly described DC type. It displays the high IFN-α producing activity of pDC but to a more limited range of viruses. Unlike pDC, the novel DC display high T cell stimulation capacity. Moreover, unlike mouse pDC, they are matured with GM-CSF and are less prone to apoptosis upon activation stimuli, including viruses. We propose that these DC constitute a novel bone marrow inflammatory DC type, ideally geared to linking innate and adaptive immune responses in bone marrow via their potent IFN-α production and high T cell stimulatory capacity.
Assuntos
Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Interferon-alfa/imunologia , Linfócitos T/imunologia , Imunidade Adaptativa , Animais , Apoptose , Células da Medula Óssea/citologia , Células Dendríticas/citologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunidade Inata , Interferon-alfa/biossíntese , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poli I-C/farmacologia , Transdução de Sinais , Linfócitos T/citologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologiaRESUMO
Polyinosinic:polycytidylic acid (poly IC), a double-stranded RNA, is an effective adjuvant in vivo. IFN-λs (also termed IL-28/29) are potent immunomodulatory and antiviral cytokines. We demonstrate that poly IC injection in vivo induces large amounts of IFN-λ, which depended on hematopoietic cells and the presence of TLR3 (Toll-like receptor 3), IRF3 (IFN regulatory factor 3), IRF7, IFN-I receptor, Fms-related tyrosine kinase 3 ligand (FL), and IRF8 but not on MyD88 (myeloid differentiation factor 88), Rig-like helicases, or lymphocytes. Upon poly IC injection in vivo, the IFN-λ production by splenocytes segregated with cells phenotypically resembling CD8α(+) conventional dendritic cells (DCs [cDCs]). In vitro experiments revealed that CD8α(+) cDCs were the major producers of IFN-λ in response to poly IC, whereas both CD8α(+) cDCs and plasmacytoid DCs produced large amounts of IFN-λ in response to HSV-1 or parapoxvirus. The nature of the stimulus and the cytokine milieu determined whether CD8α(+) cDCs produced IFN-λ or IL-12p70. Human DCs expressing BDCA3 (CD141), which is considered to be the human counterpart of murine CD8α(+) DCs, also produced large amounts of IFN-λ upon poly IC stimulation. Thus, IFN-λ production in response to poly IC is a novel function of mouse CD8α(+) cDCs and their human equivalents.
Assuntos
Antígenos de Superfície/análise , Antígenos CD8/análise , Citocinas/biossíntese , Células Dendríticas/imunologia , Indutores de Interferon/farmacologia , Interleucinas/biossíntese , Poli I-C/farmacologia , Animais , Herpesvirus Humano 2 , Humanos , Fator Regulador 3 de Interferon/fisiologia , Fator Regulador 7 de Interferon/fisiologia , Fatores Reguladores de Interferon/fisiologia , Interferons , Interleucina-12/biossíntese , Camundongos , Parapoxvirus/imunologia , Trombomodulina , Receptor 3 Toll-Like/fisiologiaRESUMO
Dendritic cell (DC) populations consist of multiple subsets that are essential orchestrators of the immune system. Technological limitations have so far prevented systems-wide accurate proteome comparison of rare cell populations in vivo. Here, we used high-resolution mass spectrometry-based proteomics, combined with label-free quantitation algorithms, to determine the proteome of mouse splenic conventional and plasmacytoid DC subsets to a depth of 5,780 and 6,664 proteins, respectively. We found mutually exclusive expression of pattern recognition pathways not previously known to be different among conventional DC subsets. Our experiments assigned key viral recognition functions to be exclusively expressed in CD4(+) and double-negative DCs. The CD8alpha(+) DCs largely lack the receptors required to sense certain viruses in the cytoplasm. By avoiding activation via cytoplasmic receptors, including retinoic acid-inducible gene I, CD8alpha(+) DCs likely gain a window of opportunity to process and present viral antigens before activation-induced shutdown of antigen presentation pathways occurs.
Assuntos
RNA Helicases DEAD-box/biossíntese , Células Dendríticas/metabolismo , Proteômica/métodos , Infecções por Respirovirus/imunologia , Vírus Sendai/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Separação Celular , Células Cultivadas , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Citometria de Fluxo , Interações Hospedeiro-Patógeno , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteômica/instrumentação , Vírus Sendai/patogenicidadeRESUMO
The generation of dendritic cells (DCs) from monocytes and early progenitors in GM-CSF cultures has been the gold standard for in vitro generation of DCs for three decades. However, the most recent evidence suggests that these cultures represent the migratory and inflammatory DC subtypes and not the DC subtypes found in the steady state. By contrast a different culture method was described where mouse bone marrow is cultured with flt3 ligand for 9 days. Here, we describe this method in detail for the generation of the phenotypic, functional, and developmental equivalents of CD8(+), CD8(-), and plasmacytoid DCs. This includes growth and purification of recombinant flt3 ligand from Chinese hamster ovary cells, isolation of bone marrow cells, and phenotypic characterization of the subsets. This simple method allows generation of large numbers of DCs (60-100 million from one mouse) compared to splenic DC isolation (5 million per mouse).
Assuntos
Antígenos CD8/metabolismo , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Proteínas de Membrana/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células CHO , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Células Dendríticas/metabolismo , Citometria de Fluxo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mice lacking the ligand for Flt-3 (CD135) have a massive deficit of dendritic cells (DC) in all organs. This phenotype of FL (FL) knockout mice suggested that FL was the archetypal DC poietin in the steady state. However, FL knockout mice also have reduced numbers of common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) so it is possible that FL deficiency may limit the ability of other growth factors to drive DC development by limiting the pool of progenitor cells available. We found that DC development could be driven from BM cells of FL knockout mice using the myeloid growth factor M-CSF. The M-CSF-driven DC (MDC) developed independently of FL and resembled the DC types present in the spleen in the steady state.