RESUMO
Rationale: Chemoradiation (CRT) is commonly used as an adjuvant or neoadjuvant treatment for colorectal cancer (CRC) patients. However, resistant cells manage to survive and propagate after CRT, increasing the risk of recurrence. Thus, better understanding the mechanism of resistant cancer cells is required to achieve better clinical outcomes. Methods: Here, we explored gene expression profiling of CRC patient tumors to identify therapy resistance genes and discovered that protein tyrosine phosphatase receptor type C (PTPRC), which encodes CD45, was increased in remnant tumor tissues after CRT and correlated with metastasis. Through multiple validations using patient tumors and CRC cell lines, we found for the first time the increase of CD45 expression in CRC (EpCAM+) epithelial cells surviving after CRT. Thus, we investigated the biological role and downstream events of CD45 were explored in human CRC cells and CRC mouse models. Results: Increased CD45 expression in cancer cells in pretreated primary tumors accounts for poor regression and recurrence-free survival in CRT-treated patients. High CD45 expression promotes CRC cell survival upon 5-fluorouracil or radiation treatment, while CD45 depletion sensitizes CRC cells to CRT. Intriguingly, CD45 is preferentially expressed in cancer stem-like cells (CSCs), as determined by spheroid culture and the expression of CSC markers, and is required for the distinct functions of CSCs, such as cancer initiation, repopulation, and metastasis. Mechanistically, CD45 phosphatase activity promotes Wnt transcriptional activity by stabilizing the ß-catenin protein, which collectively enhances stemness and the therapy-resistant phenotype. Conclusions: Our results highlight a novel function of CD45 as a mediator of CRT resistance and provide a potential therapy strategy for CRC therapy.
Assuntos
Neoplasias Colorretais/metabolismo , Antígenos Comuns de Leucócito/genética , Via de Sinalização Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/fisiopatologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/fisiologia , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Antígenos Comuns de Leucócito/metabolismo , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Estudos Retrospectivos , Células-Tronco/metabolismo , Transcriptoma/genéticaRESUMO
Neurexins (Nrxns) and LAR-RPTPs (leukocyte common antigen-related protein tyrosine phosphatases) are presynaptic adhesion proteins responsible for organizing presynaptic machineries through interactions with nonoverlapping extracellular ligands. Here, we report that two members of the LAR-RPTP family, PTPσ and PTPδ, are required for the presynaptogenic activity of Nrxns. Intriguingly, Nrxn1 and PTPσ require distinct sets of intracellular proteins for the assembly of specific presynaptic terminals. In addition, Nrxn1α showed robust heparan sulfate (HS)-dependent, high-affinity interactions with Ig domains of PTPσ that were regulated by the splicing status of PTPσ. Furthermore, Nrxn1α WT, but not a Nrxn1α mutant lacking HS moieties (Nrxn1α ΔHS), inhibited postsynapse-inducing activity of PTPσ at excitatory, but not inhibitory, synapses. Similarly, cis expression of Nrxn1α WT, but not Nrxn1α ΔHS, suppressed the PTPσ-mediated maintenance of excitatory postsynaptic specializations in mouse cultured hippocampal neurons. Lastly, genetics analyses using male or female Drosophila Dlar and Dnrx mutant larvae identified epistatic interactions that control synapse formation and synaptic transmission at neuromuscular junctions. Our results suggest a novel synaptogenesis model whereby different presynaptic adhesion molecules combine with distinct regulatory codes to orchestrate specific synaptic adhesion pathways.SIGNIFICANCE STATEMENT We provide evidence supporting the physical interactions of neurexins with leukocyte common-antigen related receptor tyrosine phosphatases (LAR-RPTPs). The availability of heparan sulfates and alternative splicing of LAR-RPTPs regulate the binding affinity of these interactions. A set of intracellular presynaptic proteins is involved in common for Nrxn- and LAR-RPTP-mediated presynaptic assembly. PTPσ triggers glutamatergic and GABAergic postsynaptic differentiation in an alternative splicing-dependent manner, whereas Nrxn1α induces GABAergic postsynaptic differentiation in an alternative splicing-independent manner. Strikingly, Nrxn1α inhibits the glutamatergic postsynapse-inducing activity of PTPσ, suggesting that PTPσ and Nrxn1α might control recruitment of a different pool of postsynaptic machinery. Drosophila orthologs of Nrxns and LAR-RPTPs mediate epistatic interactions in controlling synapse structure and strength at neuromuscular junctions, underscoring the physiological significance in vivo.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Antígenos Comuns de Leucócito/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Potenciais Pós-Sinápticos Excitadores/fisiologia , Espaço Extracelular/metabolismo , Feminino , Células HEK293 , Humanos , Larva , Masculino , Camundongos , Conformação Molecular , Moléculas de Adesão de Célula Nervosa/metabolismo , Gravidez , Terminações Pré-Sinápticas/metabolismo , Ratos , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genética , Transmissão Sináptica/fisiologiaRESUMO
Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found 2 peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on T cell immunoglobulin and mucin domain containing 4 (Tim-4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FAK family-interacting protein of 200 kDa resulted in Tim-4+ TAM loss via ROS-mediated apoptosis and elevated T cell immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated macrophages positive for complement receptor of the immunoglobulin superfamily (CRIg) were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat patients with ovarian cancer with peritoneal metastasis.
Assuntos
Autofagia , Macrófagos Peritoneais/patologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Neoplasias Ovarianas/patologia , Estresse Oxidativo , Neoplasias Peritoneais/secundário , Adaptação Fisiológica , Animais , Proteínas Relacionadas à Autofagia/fisiologia , Feminino , Humanos , Antígenos Comuns de Leucócito/fisiologia , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/metabolismo , Receptores CCR2/fisiologiaRESUMO
Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1ß secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1ß levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1ß secretion after PVL exposure and controls S. aureus lung burdens.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Exotoxinas/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/citologia , Leucocidinas/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Receptor da Anafilatoxina C5a/efeitos dos fármacos , Staphylococcus aureus , Animais , Antígeno CD11b/imunologia , Sistemas CRISPR-Cas , Diferenciação Celular , Células Cultivadas , Exotoxinas/deficiência , Técnicas de Introdução de Genes , Humanos , Interleucina-1beta/metabolismo , Antígenos Comuns de Leucócito/fisiologia , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Fragmentos de Peptídeos/imunologia , Pneumonia Estafilocócica/imunologia , Subunidades Proteicas , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/fisiologia , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/fisiologiaRESUMO
BACKGROUND: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. HYPOTHESIS: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. STUDY DESIGN: Controlled laboratory study. METHODS: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques-BM aspiration and BM collection-after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. RESULTS: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45-CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56- cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. CONCLUSION: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. CLINICAL RELEVANCE: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.
Assuntos
Cartilagem/fisiologia , Regeneração/fisiologia , Coleta de Tecidos e Órgãos/métodos , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/fisiologia , Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Cartilagem/lesões , Doenças das Cartilagens , Contagem de Células , Diferenciação Celular , Condrogênese/fisiologia , Feminino , Citometria de Fluxo , Humanos , Ílio/cirurgia , Antígenos Comuns de Leucócito/fisiologia , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Osteogênese/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Células-Tronco/fisiologiaRESUMO
KEY POINTS: Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling. ABSTRACT: Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross-sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration.
Assuntos
Fibroblastos/fisiologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/lesões , Músculo Esquelético/fisiologia , Mioblastos/fisiologia , Regeneração/fisiologia , Adulto , Antígenos CD/fisiologia , Antígenos de Diferenciação Mielomonocítica/fisiologia , Células Cultivadas , Técnicas de Cocultura , Estimulação Elétrica , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Antígenos Comuns de Leucócito/fisiologia , Macrófagos/fisiologia , Masculino , Contração Muscular , Proteína 2 Semelhante ao Fator 7 de Transcrição/fisiologiaRESUMO
The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Epitopos , Sinapses Imunológicas/fisiologia , Mieloma Múltiplo/tratamento farmacológico , Receptores Fc/imunologia , Linfócitos T/imunologia , Animais , Citocinas/metabolismo , Humanos , Antígenos Comuns de Leucócito/fisiologia , Ativação Linfocitária , Macaca fascicularis , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Receptor de Morte Celular Programada 1/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Fc/análiseRESUMO
BACKGROUND: The developmental origin of the c-kit expressing progenitor cell pool in the adult heart has remained elusive. Recently, it has been discovered that the injured heart is enriched with c-kit(+) cells, which also express the hematopoietic marker CD45. METHODS AND RESULTS: In this study, we characterize the phenotype and transcriptome of the c-kit+/CD45+/CD11b+/Flk-1+/Sca-1±(B-type) cell population, originating from the left atrial appendage. These cells are defined as cardiac macrophage progenitors. We also demonstrate that the CD45+ progenitor cell population activates heart development, neural crest and pluripotency-associated pathways in vitro, in conjunction with CD45 down-regulation, and acquire a c-kit+/CD45-/CD11b-/Flk-1-/Sca-1+ (A-type) phenotype through cell fusion and asymmetric division. This putative spontaneous reprogramming evolves into a highly proliferative, partially myogenic phenotype (C-type). CONCLUSIONS: Our data suggests that A-type cells and cardiac macrophage precursor cells (B-type) have a common lineage origin, possibly resolving some current conundrums in the field of cardiac regeneration.
Assuntos
Apêndice Atrial/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Antígenos Comuns de Leucócito/fisiologia , Macrófagos/fisiologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/fisiologia , Animais , Apêndice Atrial/citologia , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosAssuntos
Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Antígenos Comuns de Leucócito/fisiologia , Terapia de Alvo Molecular , Quinoxalinas/uso terapêutico , Antineoplásicos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Antígenos Comuns de Leucócito/antagonistas & inibidores , Antígenos Comuns de Leucócito/imunologia , Quinoxalinas/farmacologia , Transdução de Sinais/imunologiaRESUMO
The continuous recirculation of mature lymphocytes and their entry into the peripheral lymph nodes are crucial for the development of an immune response to foreign antigens. Occasionally, the entry and the subsequent response of T lymphocytes in these sites lead to severe inflammation and pathological conditions. Here, we characterized the tetraspanin molecule, CD151, as a regulator of T cell motility in health and in models of inflammatory bowel disease. CD151 formed a cell surface complex with VLA-4 and LFA-1 integrins, and its activation led to enhanced migration of T cells. Picomolar levels of CCL2 that were previously shown to inhibit T-cell migration to lymph nodes suppressed CD151 expression and dissociated CD151-integrin complexes in T lymphocytes, resulting in attenuated migration toward T-cell attractant chemokines. To directly inhibit CD151 function, a truncated CD151 peptide fragment mimicking of the CD151 extracellular loop was designed. CD151 extracellular loop inhibited T-cell migration in vitro and in vivo and attenuated the development of dextrane sulfate sodium-induced colitis. Thus, CD151 is a key orchestrator of T cell motility; interference with its proper function results in attenuated progression of inflammatory bowel disease.
Assuntos
Movimento Celular/imunologia , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Inflamação/imunologia , Linfócitos T/imunologia , Tetraspanina 24/fisiologia , Animais , Estudos de Casos e Controles , Movimento Celular/fisiologia , Seguimentos , Proteínas de Homeodomínio/fisiologia , Humanos , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , Receptores CCR2/fisiologiaRESUMO
CD45 is a receptor-like tyrosine phosphatase that positively regulates BCR signaling by dephosphorylating the inhibitory tyrosine of the Src family kinases. We showed previously that a single point mutation, E613R, introduced into the cytoplasmic membrane-proximal "wedge" domain of CD45 is sufficient to drive a lupus-like autoimmune disease on a susceptible genetic background. To clarify the molecular mechanism of this disease, we took advantage of a unique allelic series of mice in which the expression of CD45 is varied across a broad range. Although both E613R B cells and those with supraphysiologic CD45 expression exhibited hyperresponsive BCR signaling, they did so by opposite regulation of the Src family kinase Lyn. We demonstrated that the E613R allele of CD45 does not function as a hyper- or hypomorphic allele but rather alters the substrate specificity of CD45 for Lyn. Despite similarly enhancing BCR signaling, only B cells with supraphysiologic CD45 expression became anergic, whereas only mice harboring the E613R mutation developed frank autoimmunity on a susceptible genetic background. We showed that selective impairment of a Lyn-dependent negative-regulatory circuit in E613R B cells drove autoimmunity in E613R mice. This demonstrates that relaxing negative regulation of BCR signaling, rather than enhancing positive regulation, is critical for driving autoimmunity in this system.
Assuntos
Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Tolerância Imunológica , Antígenos Comuns de Leucócito/fisiologia , Proteínas Tirosina Fosfatases Semelhantes a Receptores/fisiologia , Alelos , Animais , Subpopulações de Linfócitos B/citologia , Variação Genética/imunologia , Tolerância Imunológica/genética , Antígenos Comuns de Leucócito/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores/deficiência , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genética , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
OBJECTIVE: To identify the optimum intra-articular multipotent stromal cell (MSC) tissue source in the canine stifle. STUDY DESIGN: Experimental. SAMPLE POPULATION: Infrapatellar adipose tissue, synovium lining the joint capsule, and synovium surrounding the cranial cruciate ligament (CrCL) from normal stifles of 6 dogs. METHODS: Nucleated cell density for each tissue was determined, and cell doublings (CD) and doubling times (DT) were quantified for expansion rates. Adipogenic, osteogenic, and chondrogenic differentiation was confirmed with light microscopy. Fibroblastic, adipogenic, and osteogenic colony forming unit frequencies were determined for multipotentiality. Tissue-specific target gene expression was assessed, and percentages of CD29(+) , CD34(+) , CD44(+) , CD45(+) , and CD90(+) cells quantified. RESULTS: Adipose tissue had the highest MSC density (ASC). The CD decreased with increasing passages for all cell types, and ASC values tended to be higher. Multipotentiality decreased with passage, but remained highest in ASC. Tissue-specific target gene expression was higher in induced versus noninduced cells, and ASCs had the highest upregulation across passages. Most cells were CD29(+) , CD44(+) , CD90(+) , and percentages decreased with passage. Within cell types, there were more CD29(+) ASC in early passages and more CD44(+) and CD90(+) ASC in later passages. CONCLUSIONS: ASC had the highest in vitro expansion rates, CFU frequencies, tissue-specific target gene expression, and percentages of MSC immunophenotypes.
Assuntos
Tecido Adiposo/citologia , Antígenos CD/fisiologia , Células-Tronco Multipotentes/fisiologia , Células Estromais/citologia , Animais , Ligamento Cruzado Anterior/citologia , Antígenos CD/imunologia , Antígenos CD34/fisiologia , Contagem de Células/veterinária , Divisão Celular/fisiologia , Cães , Feminino , Receptores de Hialuronatos/fisiologia , Imunofenotipagem/veterinária , Integrina beta1/fisiologia , Antígenos Comuns de Leucócito/fisiologia , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/imunologia , Joelho de Quadrúpedes/citologia , Células Estromais/imunologia , Células Estromais/fisiologia , Antígenos Thy-1/fisiologiaRESUMO
Immunological and neural synapses share properties such as the synaptic cleft, adhesion molecules, stability, and polarity. However, the mismatch in scale has limited the utility of these comparisons. The discovery of phosphatase micro-exclusion from signaling elements in immunological synapses and innate phagocytic synapses define a common functional unit at a common sub-micron scale across synapse types. Bundling of information from multiple antigen receptor microclusters by an immunological synapse has parallels to bundling of multiple synaptic inputs into a single axonal output by neurons, allowing integration and coincidence detection. Bonafide neuroimmune synapses control the inflammatory reflex. A better understanding of the shared mechanisms between immunological and neural synapses could aid in the development of new therapeutic modalities for immunological, neurological, and neuroimmunological disorders alike.
Assuntos
Sinapses Imunológicas/fisiologia , Sinapses/fisiologia , Animais , Evolução Biológica , Células Dendríticas/imunologia , Humanos , Capeamento Imunológico , Inflamação/imunologia , Inflamação/fisiopatologia , Antígenos Comuns de Leucócito/fisiologia , Cooperação Linfocítica , Modelos Biológicos , Modelos Imunológicos , Neuroimunomodulação/fisiologia , Neurônios/fisiologia , Fagocitose , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores Imunológicos/imunologia , Transdução de Sinais/fisiologia , Nervo Vago/fisiologiaRESUMO
ß-Defensins are known for their antimicrobial activity and belong to the molecular barrier of the innate immune system against invading pathogens. In addition, it has been shown that some members of the ß-defensin superfamily have the capacity to promote local innate inflammatory and systemic adaptive immune responses, mediated in part by the interaction with CCR6. We found that mouse ß-defensin 14 (mBD14, Defb14), a newly identified member of the mouse ß-defensin superfamily, is expressed in mouse fibrosarcoma tumor tissue. Tumor cells overexpressing mBD14 demonstrated enhanced solid tumor growth in syngeneic C57BL/6 mice concomitant with increased vascularization of these tumors. Furthermore, mBD14-overexpressing tumors demonstrated increased expression of proangiogenic MIP-2 (CXCL2) ex vivo. In contrast, vascular endothelial growth factor expression was not affected. Cellular analysis of tumor-infiltrating leukocytes revealed a significant increase of CCR6(+) B220(+) lymphocytes in solid tumors derived from mBD14-overexpressing tumor cells. Enhanced tumor growth of mBD14-overexpressing fibrosarcomas was abolished in CCR6-deficient mice, which was paralleled by decreased infiltration of CCR6(+) B220(+) lymphocytes, indicating the requirement of CCR6 expression on host cells. Previously, the interaction of activated, LTαß(+), lymphocytes with lymphotoxin ß-receptor-expressing fibrosarcoma tumor cells has been identified as a new CXCL2-dependent proangiogenic pathway. Coexpression of a soluble lymphotoxin ß-receptor:Ig fusion protein, an inhibitor of CXCL2-dependent angiogenesis, in mBD14-overexpressing fibrosarcoma tumor cells abolished enhanced solid tumor growth. Thus, we conclude that mBD14 expression by tumor-infiltrating host cells results in the chemoattraction of CCR6(+) B220(+) lymphocytes, which in turn initiates a proangiogenic pathway leading to enhanced angiogenesis and organized tumor tissue development.
Assuntos
Fibrossarcoma/imunologia , Fibrossarcoma/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Receptores CCR6/fisiologia , beta-Defensinas/fisiologia , Animais , Feminino , Fibrossarcoma/metabolismo , Antígenos Comuns de Leucócito/biossíntese , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Receptores CCR6/biossíntese , Células Tumorais Cultivadas , beta-Defensinas/biossínteseRESUMO
T cell development depends on continuous recruitment of progenitors from bone marrow (BM) to the thymus via peripheral blood. However, both phenotype and functional characteristics of physiological T cell precursors remain ill-defined. Here, we characterized a putative CD135(+)CD27(+) T cell progenitor population, which lacked expression of CD127, CD90, and high levels of CD117 and was therefore termed triple negative precursor (TNP). TNPs were present in both BM and blood and displayed robust T lineage potential, but virtually no myeloid or B lineage potential, in vitro. However, TNPs did not efficiently generate T lineage progeny after intravenous or intrathymic transfer, suggesting that a physiological thymic microenvironment does not optimally support T cell differentiation from TNPs. Thus, we propose that physiological T cell precursors are confined to populations expressing either CD127, CD90, or high levels of CD117 in addition to CD135 and CD27 and that TNPs may have other physiological functions.
Assuntos
Medula Óssea/imunologia , Linhagem da Célula , Células-Tronco Hematopoéticas/imunologia , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Linfócitos T/imunologia , Antígenos Thy-1/metabolismo , Animais , Medula Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
Natural killer T (NKT) cells represent an important regulatory T cell subset that develops in the thymus and contains immature (NK1.1(lo)) and mature (NK1.1(hi)) cell subsets. Here we show in mice that an inherited mutation in heterogeneous ribonucleoprotein L-like protein (hnRNPLL(thunder)), that shortens the survival of conventional T cells, has no discernible effect on NKT cell development, homeostasis or effector function. Thus, Hnrpll deficiency effectively increases the NKTâ¶T cell ratio in the periphery. However, Hnrpll mutation disrupts CD45RA, RB and RC exon silencing of the Ptprc mRNA in both NKT and conventional T cells, and leads to a comparably dramatic shift to high molecular weight CD45 isoforms. In addition, Hnrpll mutation has a cell intrinsic effect on the expression of the developmentally regulated cell surface marker NK1.1 on NKT cells in the thymus and periphery but does not affect cell numbers. Therefore our results highlight both overlapping and divergent roles for hnRNPLL between conventional T cells and NKT cells. In both cell subsets it is required as a trans-acting factor to regulate alternative splicing of the Ptprc mRNA, but it is only required for survival of conventional T cells.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Células Matadoras Naturais/imunologia , Antígenos Comuns de Leucócito/fisiologia , Linfócitos T/imunologia , Animais , Éxons , CamundongosRESUMO
STUDY OBJECTIVES: Sleep loss triggers changes in inflammatory signaling pathways in the brain and periphery. The mechanisms that underlie these changes are ill-defined. The Toll-like receptor 4 (TLR4) activates inflammatory signaling cascades in response to endogenous and pathogen-associated ligands known to be elevated in association with sleep loss. TLR4 is therefore a possible mediator of some of the inflammation-related effects of sleep loss. Here we describe the baseline electroencephalographic sleep phenotype and the biochemical and electroencephalographic responses to sleep loss in TLR4-deficient mice. DESIGN, MEASUREMENTS AND RESULTS: TLR4-deficient mice and wild type controls were subjected to electroencephalographic and electromyographic recordings during spontaneous sleep/wake cycles and during and after sleep restriction sessions of 3, 6, and 24-h duration, during which sleep was disrupted by an automated sleep restriction system. Relative to wild type control mice, TLR4-deficient mice exhibited an increase in the duration of the primary daily waking bout occurring at dark onset in a light/dark cycle. The amount of time spent in non-rapid eye movement sleep by TLR4-deficient mice was reduced in proportion to increased wakefulness in the hours immediately after dark onset. Subsequent to sleep restriction, EEG measures of increased sleep drive were attenuated in TLR4-deficient mice relative to wild-type mice. TLR4 was enriched 10-fold in brain cells positive for the cell surface marker CD11b (cells of the monocyte lineage) relative to CD11b-negative cells in wild type mouse brains. To assess whether this population was affected selectively by TLR4 knockout, flow cytometry was used to count F4/80- and CD45-positive cells in the brains of sleep deprived and time of day control mice. While wild-type mice exhibited a significant reduction in the number of CD11b-positive cells in the brain after 24-h sleep restriction, TLR4-deficient mice did not. CONCLUSION: These data demonstrate that innate immune signaling pathways active in the monocyte lineage, including presumably microglia, detect and mediate in part the cerebral reaction to sleep loss.
Assuntos
Encéfalo/fisiologia , Monócitos/fisiologia , Privação do Sono/fisiopatologia , Receptor 4 Toll-Like/fisiologia , Animais , Antígenos de Diferenciação/fisiologia , Encéfalo/fisiopatologia , Eletroencefalografia , Eletromiografia , Citometria de Fluxo , Antígenos Comuns de Leucócito/fisiologia , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sono/fisiologia , Receptor 4 Toll-Like/deficiênciaRESUMO
Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors in vitro and their recovery in transplanted recipients in vivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Leucemia Promielocítica Aguda/patologia , Antígenos Comuns de Leucócito/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Animais , Anticorpos Monoclonais/farmacologia , Adesão Celular , Ensaio de Unidades Formadoras de Colônias , Sobrevivência de Enxerto , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células HL-60/transplante , Mobilização de Células-Tronco Hematopoéticas , Humanos , Antígenos Comuns de Leucócito/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Quimera por Radiação , Células Tumorais Cultivadas/transplanteRESUMO
BACKGROUND: Inflammatory bowel disease (IBD) consists of Crohn's disease (CD) and ulcerative colitis (UC), two widespread diseases of unknown, multifactorial etiology. Colitis pathology involves a pathological angiogenic response where increases in vascular density participate in colitis histopathology. Vascular endothelial growth factor-A (VEGF-A) is a potent angiogenesis stimulator known to be involved in pathological angiogenesis in several diseases including colitis. However, the pathogenic importance of specific VEGF-A isoforms during T-cell-mediated experimental colitis remains largely unknown. METHODS: The CD4⺠CD45RB(high) T-cell transfer model of experimental colitis was used for these studies. The VEGF lac-Z transgenic reporter mouse was used to examine specific cellular sources of VEGF-A production. The VEGF164 aptamer (Macugen), adenoviral VEGF164, and the VEGF Trap were used to evaluate pathological importance. RESULTS: VEGF lac-Z reporter mice experiments showed that both infiltrating T cells and local tissue cells produce VEGF-A in the colon during disease. Inhibition of VEGF164 using a highly selective RNA aptamer significantly attenuated CD4⺠CD45RB(high) T-cell-dependent experimental colitis by reducing pathological angiogenesis and inflammatory pathology. Conversely, broad-spectrum VEGF inhibition with VEGF Trap did not attenuate disease, nor did adenoviral VEGF164 overexpression significantly alter colitis pathology. CONCLUSIONS: VEGF164 is actively produced by multiple cell types during T-cell-mediated colitis. Importantly, specific inhibition of the VEGF164 isoform during T-cell-mediated colitis dose-dependently attenuated disease progression, while broad-scale inhibition of all VEGF-A isoforms was not therapeutically beneficial.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite/etiologia , Colite/metabolismo , Modelos Animais de Doenças , Linfócitos T Reguladores/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Aptâmeros de Nucleotídeos/farmacologia , Colite/patologia , Colo/imunologia , Colo/metabolismo , Colo/patologia , Ensaio de Imunoadsorção Enzimática , Proteínas de Homeodomínio , Humanos , Inflamação/prevenção & controle , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica/prevenção & controle , Isoformas de Proteínas , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.