Asunto(s)
Inmunoterapia , Neoplasias , Humanos , Neoplasias/inmunología , Neoplasias/terapia , Inmunoterapia/métodos , Enfermedad Crónica , Animales , Escape del Tumor , Evasión InmuneAsunto(s)
Neoplasias , Linfocitos T , Humanos , Neoplasias/genética , Neoplasias/terapia , InmunoterapiaRESUMEN
BACKGROUND: Tumor microenvironment-associated T cell senescence is a key limiting factor for durable effective cancer immunotherapy. A few studies have demonstrated the critical role of the tumor suppressor TP53-derived p53 isoforms in cellular senescence process of non-immune cells. However, their role in lymphocytes, in particular tumor-antigen (TA) specific T cells remain largely unexplored. METHODS: Human T cells from peripheral blood were retrovirally engineered to coexpress a TA-specific T cell receptor and the Δ133p53α-isoform, and characterized for their cellular phenotype, metabolic profile and effector functions. RESULTS: Phenotypic analysis of Δ133p53α-modified T cells revealed a marked reduction of the T-cell inhibitory molecules (ie, CD160 and TIGIT), a lower frequency of senescent-like CD57+ and CD160+ CD8+ T cell populations, and an increased number of less differentiated CD28+ T cells. Consistently, we demonstrated changes in the cellular metabolic program toward a quiescent T cell state. On a functional level, Δ133p53α-expressing T cells acquired a long-term proliferative capacity, showed superior cytokine secretion and enhanced tumor-specific killing in vitro and in mouse tumor model. Finally, we demonstrated the capacity of Δ133p53α to restore the antitumor response of senescent T cells isolated from multiple myeloma patients. CONCLUSION: This study uncovered a broad effect of Δ133p53α isoform in regulating T lymphocyte function. Enhancing fitness and effector functions of senescent T cells by modulation of p53 isoforms could be exploited for future translational research to improve cancer immunotherapy and immunosenescence-related diseases.
Asunto(s)
Inmunoterapia/métodos , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Microambiente TumoralRESUMEN
Elimination of suppressive T cells may enable and enhance cancer immunotherapy. Here, we demonstrate that the cell membrane protein SLAMF7 was highly expressed on immunosuppressive CD8+CD28-CD57+ Tregs in multiple myeloma (MM). SLAMF7 expression associated with T cell exhaustion surface markers and exhaustion-related transcription factor signatures. T cells from patients with a high frequency of SLAMF7+CD8+ T cells exhibited decreased immunoreactivity towards the MART-1aa26-35*A27L antigen. A monoclonal anti-SLAMF7 antibody (elotuzumab) specifically depleted SLAMF7+CD8+ T cells in vitro and in vivo via macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). Anti-SLAMF7 treatment of MM patients depleted suppressive T cells in peripheral blood. These data highlight SLAMF7 as a marker for suppressive CD8+ Treg and suggest that anti-SLAMF7 antibodies can be used to boost anti-tumoral immune responses in cancer patients.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Depleción Linfocítica/métodos , Mieloma Múltiple/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Adulto , Anciano , Animales , Antineoplásicos/uso terapéutico , Apoptosis , Proliferación Celular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Mieloma Múltiple/patología , Mieloma Múltiple/terapia , Pronóstico , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Myeloid cell arginase-mediated arginine depletion with consecutive inhibition of T cell functions is a key component of tumor immune escape. Both, granulocytic myeloid-derived suppressor cells (G-MDSC) and conventional mature human polymorphonuclear neutrophil granulocytes (PMN) express high levels of arginase 1 and can act as suppressor cells of adaptive anti-cancer immunity. Here we demonstrate that pharmacological inhibition of PMN-derived arginase 1 not only prevents the suppression of T cell functions but rather leads to a strong hyperactivation of T cells. Human PMN were incubated in cell culture medium in the absence or presence of an arginase inhibitor. T cells from healthy donors were then activated either polyclonally or in an antigen-specific manner in the supernatants of the PMN cultures at different PMN-T cell ratios. T cell proliferation was completely suppressed in these supernatants in the absence of an arginase inhibitor. Arginase inhibition led to a strong hyperinduction of T cell proliferation, which exceeded control activation conditions up to 25-fold. The hyperinduction was correlated with higher PMN-T cell ratios and was only apparent when PMN arginase activity was blocked sufficiently. The T cell stimulatory factor was liberated very early by PMN and was present in the < 3 kDa fraction of the PMN supernatants. Increased T cell production of specific proinflammatory cytokines by PMN supernatant in the presence of arginase inhibitor was apparent. Upon arginase inhibition, downregulation of important T cell membrane activation and costimulation proteins was completely prevented or de novo induction accelerated. Antigen-specific T cell cytotoxicity against tumor cells was enhanced by PMN supernatant itself and could be further increased by PMN arginase blockade. Finally, we analyzed anergic T cells from multiple myeloma patients and noticed a complete reversal of anergy and the induction of strong proliferation upon T cell activation in PMN supernatants by arginase inhibition. In summary, we discovered a potent PMN-mediated hyperactivation of human T cells, which is apparent only when PMN arginase-mediated arginine depletion is concurrently inhibited. Our findings are clearly relevant for the analysis and prevention of human tumor immune escape in conjunction with the application of arginase inhibitors already being developed clinically.
Asunto(s)
Arginasa/metabolismo , Activación de Linfocitos/inmunología , Neutrófilos/enzimología , Linfocitos T/inmunología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Mieloma Múltiple/inmunología , Neutrófilos/inmunología , Escape del Tumor/inmunologíaRESUMEN
Interfering with tumor metabolism by specifically restricting the availability of extracellular nutrients is a rapidly emerging field of cancer research. A variety of tumor entities depend on the uptake of the amino acid arginine since they have lost the ability to synthesize it endogenously, that is they do not express the rate limiting enzyme for arginine synthesis, argininosuccinate synthase (ASS). Arginine transport through the plasma membrane of mammalian cells is mediated by eight different transporters that belong to two solute carrier (SLC) families. In the present study we found that the proliferation of primary as well as immortalized chronic lymphocytic leukemia (CLL) cells depends on the availability of extracellular arginine and that primary CLL cells do not express ASS and are therefore arginine-auxotrophic. The cationic amino acid transporter-1 (CAT-1) was the only arginine importer expressed in CLL cells. Lentiviral-mediated downregulation of the CAT-1 transporter in HG3 CLL cells significantly reduced arginine uptake, abolished cell proliferation and impaired cell viability. In a murine CLL xenograft model, tumor growth was significantly suppressed upon induced downregulation of CAT-1 in the CLL cells. Our results suggest that inhibition of CAT-1 is a promising new therapeutic approach for CLL.
RESUMEN
Genetic engineering of T cells with a T cell receptor (TCR) targeting tumor antigen is a promising strategy for cancer immunotherapy. Inefficient expression of the introduced TCR due to TCR mispairing may limit the efficacy and adversely affect the safety of TCR gene therapy. Here, we evaluated the safety and therapeutic efficiency of an optimized single-chain TCR (scTCR) specific for an HLA-A2.1-restricted (non-mutated) p53(264-272) peptide in adoptive T cell transfer (ACT) models using our unique transgenic mice expressing human p53 and HLA-A2.1 that closely mimic the human setting. Specifically, we showed that adoptive transfer of optimized scTCR-redirected T cells does not induce on-target and off-target autoimmunity. Furthermore, ACT resulted in full tumor protection and led to a long-lived effective, antigen-specific memory T cell response in syngeneic and xenograft models. Taken together, the study demonstrated that our scTCR specific for the broadly expressed tumor-associated antigen p53(264-272) can eradicate p53+A2.1+ tumor cells without inducing off-target or self-directed toxicities in mouse models of ACT. These data strongly support the improved safety and therapeutic efficacy of high-affinity p53scTCR for TCR-based immunotherapy of p53-associated malignancies.
Asunto(s)
Autoinmunidad/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Terapia Genética , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Ratones , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Tumor-mediated immunosuppression via regulatory T-cells is a key player among the various immune-escape mechanisms in multiple myeloma. We analyzed the generation, distribution, function and immunophenotype of CD8+CD28- regulatory T-cells in patients with multiple myeloma. Functionality of CD8+CD28- T-cells was assessed by immunological assays using ex vivo generated antigen-specific T-cells from patients with plasma cell dyscrasias and healthy donors. Detailed analysis of distribution, immunophenotype and cytotoxic potential of CD8+CD28- T-cells was performed by flow cytometry and ELISA. We found that the amount of CD8+CD28- T-cells was directly correlated with the suppression of antigen-specific T-cell responses in patients with plasma cell dyscrasia. Analyzing the CD8+CD28- T-cells in detail, increased numbers of these cells were observed in the bone marrow (i.e., tumor microenvironment) of patients with plasma cell dyscrasia. Furthermore, we identified the expression of lymphocyte function-associated antigen 1 (LFA-1) as a marker of immunosuppression and defined the CD8+CD28-CD57+LFA-1high population as the relevant immunosuppressive compartment. These regulatory T-cells act as immunosuppressors via soluble factors and incubation with IL-10 augmented their immunosuppressive capacity. The immunosuppressive regulatory network of IL-10 and the CD8+CD28-CD57+LFA-1high regulatory T-cells show unique characteristics and contribute to the tumor immune escape mechanism in patients with multiple myeloma.
Asunto(s)
Antígenos CD28/inmunología , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Interleucina-10/farmacología , Mieloma Múltiple/inmunología , Paraproteinemias/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Estudios de Casos y Controles , Células Cultivadas , Humanos , Activación de Linfocitos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Paraproteinemias/metabolismo , Paraproteinemias/patología , Subgrupos de Linfocitos T/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/efectos de los fármacosAsunto(s)
Neoplasias Hematológicas/terapia , Inmunoterapia/métodos , Leucemia Mieloide/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Enfermedad Aguda , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Neoplasias Hematológicas/inmunología , Humanos , Inmunoconjugados/inmunología , Inmunoconjugados/uso terapéutico , Leucemia Mieloide/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Resultado del TratamientoRESUMEN
Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/ß T-cell receptors (TCRα/ß). However, potential mispairing of introduced TCRα/ß-chains with endogenous ß/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vß-fragment to the TCR Cß-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/ß-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vß-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vß. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/ß-positive T-cells.
Asunto(s)
Complejo CD3/inmunología , Leucemia de Células T/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Biomarcadores de Tumor , Complejo CD3/genética , Membrana Celular , Proliferación Celular , Humanos , Inmunoterapia Adoptiva , Leucemia de Células T/genética , Leucemia de Células T/patología , Ratones , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transducción de Señal , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
Cervical cancer, the second leading cause of cancer deaths in women, is the consequence of high-risk human papillomavirus (HPV) infections. Toward the development of therapeutic vaccines that can induce both innate and adaptive mucosal immune responses, we analyzed intravaginal (ivag) vaccine delivery of live attenuated Salmonella enterica serovar Typhimurium expressing HPV16L1 as a model antigen. Innate immune responses were examined in cervicovaginal tissues by determining gene expression patterns by microarray analysis using nylon membranes imprinted with cDNA fragments coding for inflammation-associated genes. At 24 h, a wide range of genes, including those for chemokines and Th1- and Th2-type cytokine and chemokine receptors were up-regulated in mice ivag immunized with Salmonella compared to control mice. However, the majority of transcripts returned to their steady-state levels 1 week after immunization, suggesting a transient inflammatory response. Indeed, cervicovaginal histology of immunized mice showed a massive, but transient, infiltration of macrophages and neutrophils, while T cells were still increased after 7 days. Ivag immunization also induced humoral and antitumor immune responses, i.e., serum and vaginal anti-HPV16VLP antibody titers similar to those induced by oral immunization, and significant protection in tumor protection experiments using HPV16-expressing C3 tumor cells. These results show that ivag immunization with live attenuated Salmonella expressing HPV16 antigens modulates the local mucosal gene expression pattern into a transient proinflammatory profile, elicits strong systemic and mucosal immunity against HPV16, and confers protection against HPV16 tumor cells subcutaneously implanted in mice. Examination of the efficacy with which ivag HPV16E7E6 Salmonella induces regression of tumors located in cervicovaginal tissue is warranted.
Asunto(s)
Administración Intravaginal , Proteínas de la Cápside/inmunología , Papillomavirus Humano 16/inmunología , Proteínas Oncogénicas Virales/inmunología , Vacunas contra Papillomavirus/inmunología , Salmonella typhimurium/genética , Animales , Anticuerpos Antineoplásicos , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/genética , Cuello del Útero/inmunología , Citocinas/biosíntesis , Citocinas/genética , Femenino , Perfilación de la Expresión Génica , Papillomavirus Humano 16/genética , Macrófagos/inmunología , Ratones , Neoplasias/inmunología , Neoplasias/prevención & control , Neutrófilos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas Virales/genética , Vacunas contra Papillomavirus/administración & dosificación , Vacunas contra Papillomavirus/genética , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/genética , Salmonella typhimurium/crecimiento & desarrollo , Linfocitos T/inmunología , Factores de Tiempo , Vagina/inmunologíaRESUMEN
BACKGROUND: Streptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation. METHODS: 103 CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2-/-, CD14-/- and CD14-/-/TLR2-/- mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF. RESULTS: TLR2-/- mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2-/- than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14-/- and TLR2-/-/CD14-/- mice, but only 79% of TLR2-/- mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2-/- and CD14-/- mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2-/- mice were rescued. CONCLUSION: During pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Antibacterianos/farmacología , Ácidos Hidroxámicos/farmacología , Meningitis Neumocócica/tratamiento farmacológico , Infecciones Neumocócicas/tratamiento farmacológico , Receptor Toll-Like 2/inmunología , Proteína ADAM17 , Animales , Ceftriaxona/farmacología , Quimioterapia Adyuvante , Femenino , Receptores de Lipopolisacáridos/inmunología , Masculino , Meningitis Neumocócica/enzimología , Meningitis Neumocócica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones Neumocócicas/enzimología , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/crecimiento & desarrollo , Receptor Toll-Like 2/deficiencia , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Phagocytosis and killing of Streptococcus pneumoniae was compared in blood-derived wild-type (WT) and Toll-like receptor 2 (TLR2)-deficient (TLR2-/-) polymorphonuclear leukocytes (PMN). Phagocytosis of green fluorescent protein-transformed pneumococci was delayed in TLR2-/- PMN. These cells exhibited also a lower oxidative bactericidal activity against S. pneumoniae than WT PMN, suggesting that TLR2 modulates bacterial clearance in PMN.
Asunto(s)
Granulocitos/microbiología , Fagocitosis , Streptococcus pneumoniae/inmunología , Receptor Toll-Like 2/deficiencia , Animales , Células Cultivadas , Granulocitos/química , Granulocitos/inmunología , Proteínas Fluorescentes Verdes/análisis , Ratones , Ratones Noqueados , Oxidación-Reducción , Fagocitosis/genética , Receptor Toll-Like 2/genéticaRESUMEN
The phagocyte pattern recognition receptor Toll-like receptor 2 (TLR2) and the multi-receptor adaptor MyD88 contribute to the reduction of bacterial load in infections with intra- and extra-cellular Gram-positive bacteria. Their mechanism of antibacterial action is mostly unresolved but evident in vivo by an increased pathogen burden in infected TLR2-/- and MyD88-/- compared to C57BL/6 wild type (wt) mice. We had previously observed higher bacterial numbers in brains of TLR2-/- than of wt mice with meningitis. Here we study bacteria-phagocyte interaction by comparing S. pneumoniae distribution and localization in wt and TLR2-/- brain by confocal microscopy using a green fluorescent protein-transformed encapsulated S. pneumoniae (C5017). Colony-forming units were similarly distributed in TLR2-/- and wt mice and exclusively localized in meninges and ventricles. Bacteria were more abundant in ventricles, in and around TLR2-/- than wt GLT1v+ plexus choroideus epithelial cells. S. pneumoniae were also found in and around Gr-1+ granulocytes, but never in F4/80+ macrophages, Iba1+ microglia, GFAP+ astrocytes, Meca-31+ endothelial cells or Neun+ neurons of either mouse strain. The results indicate that TLR2 does not change bacterial distribution, but may contribute to antibacterial defense by modulating S. pneumoniae adherence and uptake in plexus epithelia.
Asunto(s)
Encéfalo/microbiología , Meningitis Neumocócica/microbiología , Receptores Inmunológicos/inmunología , Streptococcus pneumoniae/aislamiento & purificación , Animales , Encéfalo/inmunología , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Meningitis Neumocócica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Receptores Inmunológicos/deficiencia , Streptococcus pneumoniae/inmunología , Receptor Toll-Like 2RESUMEN
TNF is a marker of disease activity in bacterial meningitis. To investigate TNF modulation by Toll-like receptor-2 (TLR2), we studied temporal and anatomical expression patterns of TLR2 and TNF in a pneumococcal meningitis model in wild type (wt) and TLR2(-/-) mice. We show by in situ hybridization that transcripts of TLR2 and of the comolecules CD14, MD-2, TLR1/6 strongly increased and colocalized with TNF in CD45-positive infiltrating cells in the ventricles, corpus callosum and the meninges. TNF gene and protein expression was stronger in TLR2(-/-) than wt brains and associated with increased IkappaB expression suggesting that TLR2 is controlling inflammation via TNF regulation.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Meningitis Neumocócica/metabolismo , Receptor Toll-Like 2/deficiencia , Factor de Necrosis Tumoral alfa/metabolismo , Análisis de Varianza , Animales , Northern Blotting/métodos , Western Blotting/métodos , Encéfalo/microbiología , Encéfalo/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Interleucina-10/genética , Interleucina-10/metabolismo , Leucocitos/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Listeria monocytogenes/patogenicidad , Antígeno 96 de los Linfocitos/metabolismo , Meningitis Neumocócica/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Streptococcus pneumoniae/patogenicidad , Factores de Tiempo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
CD14 is a myeloid receptor for bacterial cell membrane/wall components, for which we previously showed a strong induction in cerebrospinal fluid (CSF) during meningitis. Here, we studied CD14 function in murine Streptococcus pneumoniae meningitis by using wild-type (WT), CD14(-/-) mice, and WT mice pretreated with neutralizing anti-CD14 antibodies. Early polymorphonuclear leukocytes (PMN) immigration was more pronounced in CSF of CD14(-/-) than of WT mice. This was not a result of altered adherence molecule expression in blood and CSF PMN or brain endothelial cells. Macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine levels were similar in CSF in both strains, but MIP-2 was higher in infected brain and in brain-derived endothelial cells infected in vitro in CD14(-/-) than in WT mice. CD14(-/-) PMN demonstrated increased expression of CXC chemokine receptor 2 (CXCR2) after infection and stronger in vitro chemotaxis than WT PMN toward CSF from WT or CD14(-/-) mice and toward MIP-2. Excess PMN migration in CD14(-/-) mice did not result in improved bacterial clearing but in increased tumor necrosis factor in CSF, higher disease severity, and earlier death. Pretreatment with anti-CXCR2 reduced PMN infiltration into CSF and brain MIP-2 production and abolished earlier mortality in CD14(-/-) mice. In conclusion, CD14 plays a protective role in pneumococcal meningitis by slowing PMN migration via MIP-2 and CXCR2 modulation.
Asunto(s)
Receptores de Lipopolisacáridos/fisiología , Meningitis Neumocócica/inmunología , Monocinas/biosíntesis , Infiltración Neutrófila/inmunología , Neutrófilos/fisiología , Receptores de Interleucina-8B/biosíntesis , Animales , Anticuerpos Monoclonales/uso terapéutico , Encéfalo/metabolismo , Encéfalo/patología , Quimiocina CXCL2 , Receptores de Lipopolisacáridos/genética , Meningitis Neumocócica/líquido cefalorraquídeo , Meningitis Neumocócica/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocinas/líquido cefalorraquídeo , Activación Neutrófila/fisiología , Tasa de SupervivenciaRESUMEN
Murine Kupffer cells (KCs) are heterogeneous and survive only for a short time in vitro. Here, a clonal, murine KC line was generated from transgenic mice, expressing the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2K(b) promoter. Thirty-three degrees Celsius and 37 degrees C but not 39 degrees C have been permissive for growth of the clone; it required conditioned media from hepatocytes and endothelial cells for proliferation. In contrast to primary cells, the cells of the clone were uniform, survived detachment, and could therefore be analyzed by cytofluorimetry. The clone, as primary KCs, constitutively expressed nonspecific esterase, peroxidase, MOMA-2, BM8, scavenger receptor A, CD14, and Toll-like receptor 4 (TLR4); the antigen-presenting molecules CD40, CD80, and CD1d; and endocytosed dextran-fluorescein isothiocyanate. It lacked complement, Fc receptors, F4/80 marker, and the phagosomal coat protein tryptophan aspartate-containing coat protein (TACO). The clone exhibited CD14- and TLR4/MD2-independent, plasma-dependent lipopolysaccharide (LPS) binding, Escherichia coli and Streptococcus pneumoniae phagocytosis, and LPS- and interferon-gamma-induced NO production but no tumor necrosis factor alpha, interleukin (IL)-6, or IL-10 release. The large size, surface-marker expression, and capacity to clear gram-negative and -positive bacteria indicate that the clone was derived from the periportal, large KC subpopulation. The clone allows molecular studies of anti-infective and immune functions of KCs.
Asunto(s)
Línea Celular Transformada , Macrófagos del Hígado/citología , Animales , Antígenos Transformadores de Poliomavirus/genética , Técnicas de Cultivo de Célula/métodos , Células Clonales , Citocinas/biosíntesis , Endocitosis , Enzimas/metabolismo , Macrófagos del Hígado/química , Macrófagos del Hígado/inmunología , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/análisis , Ratones , Ratones Transgénicos , Mutación , Óxido Nítrico/biosíntesis , Fagocitosis , Fenotipo , Regiones Promotoras GenéticasRESUMEN
In this study we investigated whether induction of toll-like receptor 2 (TLR2) amplifies the effect of a cell wall component derived from gram-positive bacteria, namely peptidoglycan (PGN). Mice received a first systemic lipopolysaccharide (LPS) injection to pre-induce TLR2 in various regions of the brain, and 6 h later, a second administration of either LPS or PGN. The data show a robust transcriptional activation of TLR2, TNF-alpha and monocyte chemotactic protein-1 (MCP-1) in microglial cells of mice challenged twice with LPS, whereas PGN essentially abolished this response. TLR4 plays a critical role in this process, because C3H/HeJ mice no longer responded to LPS but exhibited a normal reaction to PGN. Conversely, a robust signal for genes encoding innate immune proteins was found in the brain of TLR2-deficient mice challenged with LPS. However, the second LPS bolus failed to trigger TNF-alpha and IL-12 in TLR2-deficient mice, while the same treatment caused a strong induction of these genes in the cerebral tissue of wild-type littermates. The present data provide evidence that cooperation exists between TLR4 and TLR2. While TLR4 is absolutely necessary to engage the innate immune response in the brain, TLR2 participates in the regulation of genes encoding TNF-alpha and IL-12 during severe endotoxemia. Such collaboration between TLR4 and TLR2 may be determinant for the transfer from the innate to the adaptive immunity within the CNS of infected animals.
Asunto(s)
Encéfalo/inmunología , Proteínas de Drosophila , Bacterias Gramnegativas/fisiología , Bacterias Grampositivas/fisiología , Glicoproteínas de Membrana/fisiología , Polisacáridos Bacterianos/farmacología , Receptores de Superficie Celular/fisiología , Adaptación Fisiológica , Animales , Encéfalo/anatomía & histología , Pared Celular/química , Sistema Nervioso Central/metabolismo , Corteza Cerebral/fisiología , Regulación de la Expresión Génica , Bacterias Gramnegativas/química , Bacterias Grampositivas/química , Hibridación in Situ , Lipopolisacáridos/farmacología , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Peptidoglicano/farmacología , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/genética , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Toll-like receptor-2 (TLR2) mediates host responses to gram-positive bacterial wall components. TLR2 function was investigated in a murine Streptococcus pneumoniae meningitis model in wild-type (wt) and TLR2-deficient (TLR2(-/-)) mice. TLR2(-/-) mice showed earlier time of death than wt mice (P<.02). Plasma interleukin-6 levels and bacterial numbers in blood and peripheral organs were similar for both strains. With ceftriaxone therapy, none of the wt but 27% of the TLR2(-/-) mice died (P<.04). Beyond 3 hours after infection, TLR2(-/-) mice had higher bacterial loads in brain than did wt mice, as assessed with luciferase-tagged S. pneumoniae by means of a Xenogen-CCD (charge-coupled device) camera. After 24 h, tumor necrosis factor activity was higher in cerebrospinal fluid of TLR2(-/-) than wt mice (P<.05) and was related to increased blood-brain barrier permeability (Evans blue staining, P<.02). In conclusion, the lack of TLR2 was associated with earlier death from meningitis, which was not due to sepsis but to reduced brain bacterial clearing, followed by increased intrathecal inflammation.