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1.
Immunobiology ; 225(1): 151867, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31761474

RESUMO

Neutrophil extracellular traps (NETs) are formed by polymorphonuclear neutrophils (PMN) and contribute to the innate host defense by binding and killing bacterial and fungal pathogens. Because NET formation depends on histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), we used PAD4 gene deficient (Pad4-/-) mice in a mouse model of invasive pulmonary aspergillosis (IPA) to address the contribution of NETs to the innate host defense in vivo. After the induction (24 h) of IPA by i.t. infection with Aspergillus fumigatus conidia, Pad4-/- mice revealed lower fungal burden in the lungs, accompanied by less acute lung injury, TNFα and citH3 compared to wildtype controls. These findings suggest that release of NETs contributes to tissue damage and limits control of fungal outgrowth. Thus inhibition of NETosis might be a useful strategy to maintain neutrophil function and avoid lung damage in patients suffering from IPA, especially in those suffering from preexisting pulmonary disease.


Assuntos
Aspergillus fumigatus/fisiologia , Armadilhas Extracelulares/metabolismo , Aspergilose Pulmonar Invasiva/metabolismo , Pulmão/metabolismo , Neutrófilos/imunologia , Animais , Apoptose , Citrulinação/genética , Modelos Animais de Doenças , Humanos , Imunidade Inata , Aspergilose Pulmonar Invasiva/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína-Arginina Desiminase do Tipo 4/genética
2.
Eur J Immunol ; 49(11): 2083-2094, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31393597

RESUMO

Transcutaneous immunization (TCI) is a novel vaccination strategy that utilizes skin-associated lymphatic tissue to induce immune responses. Employing T-cell epitopes and the TLR7 agonist imiquimod onto intact skin mounts strong primary, but limited memory CTL responses. To overcome this limitation, we developed a novel imiquimod-containing vaccination platform (IMI-Sol) rendering superior primary CD8+ and CD4+ T-cell responses. However, it has been unclear whether IMI-Sol per se is restricted in terms of memory formation and tumor protection. In our present work, we demonstrate that the combined administration of IMI-Sol and CD40 ligation unleashes fullblown specific T-cell responses in the priming and memory phase, strongly enhancing antitumor protection in mice. Interestingly, these effects were entirely CD4+ T cell independent, bypassing the necessity of helper T cells. Moreover, blockade of CD70 in vivo abrogated the boosting effect of CD40 ligation, indicating that the adjuvant effect of CD40 in TCI is mediated via CD70 on professional APCs. Furthermore, this work highlights the so far underappreciated importance of the CD70/CD27 interaction as a promising adjuvant target in TCI. Summing up, we demonstrate that the novel formulation IMI-Sol represents a powerful vaccination platform when applied in combination with sufficient adjuvant thereby overcoming current limitations of TCI.


Assuntos
Ligante CD27/imunologia , Ligante de CD40/administração & dosagem , Imiquimode/administração & dosagem , Melanoma Experimental/terapia , Neoplasias Cutâneas/terapia , Linfócitos T Citotóxicos/efeitos dos fármacos , Administração Cutânea , Aloenxertos , Animais , Ligante CD27/genética , Citotoxicidade Imunológica/efeitos dos fármacos , Expressão Gênica , Rejeição de Enxerto , Imunização/métodos , Memória Imunológica/efeitos dos fármacos , Imunoterapia/métodos , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagem , Pele/efeitos dos fármacos , Pele/imunologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia
3.
Artigo em Inglês | MEDLINE | ID: mdl-30910895

RESUMO

There is a growing body of evidence for immunomodulatory side effects of antifungal agents on different immune cells, e.g., T cells. Therefore, the aim of our study was to clarify these interactions with regard to the effector functions of polymorphonuclear neutrophils (PMN). Human PMN were preincubated with fluconazole (FLC), voriconazole (VRC), posaconazole (POS), isavuconazole (ISA), caspofungin (CAS), micafungin (MFG), conventional amphotericin B (AMB), and liposomal amphotericin B (LAMB). PMN then were analyzed by flow cytometry for activation, degranulation, and phagocytosis and by dichlorofluorescein assay to detect reactive oxygen species (ROS). Additionally, interleukin-8 (IL-8) release was measured by enzyme-linked immunosorbent assay. POS led to enhanced activation, degranulation, and generation of ROS, whereas IL-8 release was reduced. In contrast, ISA-pretreated PMN showed decreased activation signaling, impaired degranulation, and lower generation of ROS. MFG caused enhanced expression of activation markers but impaired degranulation, phagocytosis, generation of ROS, and IL-8 release. CAS showed increased phagocytosis, whereas degranulation and generation of ROS were reduced. AMB led to activation of almost all effector functions besides impaired phagocytosis, whereas LAMB did not alter any effector functions. Independent from class, antifungal agents show variable influence on neutrophil effector functions in vitro Whether this is clinically relevant needs to be clarified.


Assuntos
Antifúngicos/farmacologia , Neutrófilos/metabolismo , Anfotericina B/farmacologia , Interleucina-8/metabolismo , Neutrófilos/efeitos dos fármacos , Nitrilas/farmacologia , Fagocitose/efeitos dos fármacos , Piridinas/farmacologia , Triazóis/farmacologia , Voriconazol/farmacologia
4.
Nat Immunol ; 19(12): 1319-1329, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30397348

RESUMO

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.


Assuntos
Adenocarcinoma/imunologia , Macrófagos/imunologia , Melanoma/imunologia , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologia , Acidose/imunologia , Adenocarcinoma/metabolismo , Animais , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Glicólise/imunologia , Humanos , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
5.
Sci Rep ; 8(1): 5558, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29615799

RESUMO

Triggering receptor expressed on myeloid cells (TREM)-1 on polymorphonuclear neutrophils (PMN) regulates innate immune activation in infectious and non-infectious conditions. TREM-1 ligation activates phosphatidyl-inositol 3 kinase (PI3K) triggering all neutrophil effector functions. As idelalisib is a PI3K inhibitor in clinical use for the treatment of non-Hodgkin lymphomas, we asked whether this inhibitor affects PMN functionalities. We analyzed PMNs from healthy donors or lymphoma patients for oxidative burst, phagocytosis, activation markers and IL-8 release upon TREM-1 or TLR ligation ex vivo. In addition, we performed western blot analyses to characterize the signaling events inhibited by idelalisib and other PI3K inhibitors. Upon TREM-1 ligation, the oxidative burst, degranulation, L-selectin shedding and cytokine release were all strongly reduced in the presence of idelalisib along impaired phosphorylation of P38, AKT and ERK by western blot analyses. In line with this, PMNs from patients receiving idelalisib also displayed an impaired TREM-1 mediated PMN activation ex vivo. In conclusion, PI3K inhibitors might cause a neutropenia-like susceptibility to infections in patients by leading to impaired PMN functionality. This should be considered when evaluating patients for infections treated with such inhibitors in daily clinical routine.


Assuntos
Anti-Inflamatórios/farmacologia , Neutrófilos/efeitos dos fármacos , Purinas/farmacologia , Quinazolinonas/farmacologia , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Anti-Inflamatórios/uso terapêutico , Contagem de Células , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-8/metabolismo , Neutrófilos/imunologia , Fagocitose/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Purinas/uso terapêutico , Quinazolinonas/uso terapêutico , Explosão Respiratória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
6.
Sci Rep ; 7(1): 7184, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28775254

RESUMO

Von Willebrand factor (VWF) is secreted as an acute phase protein during inflammation. ADAMTS-13 regulates the size and prothrombotic activity of VWF by it's specific proteolytic activity. To determine the relevance of this regulatory pathway for the innate inflammatory response by polymorphonuclear neutrophils (PMN), we employed a mouse model of invasive pulmonary aspergillosis (IPA) where PMN functionality is crucial for fungal clearance and survival. IPA was induced by intratracheal application of Aspergillus fumigatus (A. fumigatus) conidia in wildtype (129/Sv/Pas) or ADAMTS-13 deficient (Adamts13 -/-) mice. While neutropenic mice developed lethal IPA, all wildtype mice survived the infection. In contrast to wildtype or VWF deficient mice, Adamts13 -/- mice displayed more severe signs of disease with a lethal course in 24% with an increased fungal burden and signs of acute lung injury. Histology sections demonstrated a more pronounced perivascular leukocyte infiltration in support of a dysregulated inflammatory response in Adamts13 -/- mice. Importantly, we observed no general defect in the activation of neutrophil functions in response to conidia or hyphae in vitro. Therefore, we conclude that the proteolytic regulation of VWF by ADAMTS-13 or ADAMTS-13 by itself is an important mechanism to control PMN recruitment in acute inflammatory processes, such as fungal pneumonias.

7.
J Dermatol Sci ; 87(3): 260-267, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28823644

RESUMO

BACKGROUND: Transcutaneous immunization (TCI) is a non-invasive vaccination strategy targeting the skin-associated lymphoid tissue. Topical application of the TLR7 agonist imiquimod as adjuvant in combination with peptide antigens activates the innate immune system and mounts cytotoxic T lymphocyte (CTL) responses. OBJECTIVE: Based on the commercial 5% imiquimod-containing drug Aldara we aimed to develop an improved formulation with superior vaccination efficiencies. The primary target was the enhancement of mast cell activation as important key for the function of the innate immune system. METHODS: We investigated the effects of 9-phenanthrol (9-phe) on the activation of mast cells in vitro and in vivo. For TCI, we applied 0.2% 9-phe in Aldara or Aldara alone as adjuvants in combination with the MHC class I - restricted peptide SIINFEKL. To monitor vaccination, mast cell degranulation, migration of DC and frequencies of epitope-specific CTL was assessed. In a transgenic tumor model, the efficiencies of prophylactic immunization against a tumor antigen were also monitored. RESULTS: 9-phe induced degranulation of mast cells in vitro and upon topical application in vivo. A mixture of 0.2% 9-phe in Aldara showed superior results regarding the migration of DC and the expansion of antigen-specific CTL. Consequently, prophylactic immunization with 0.2% 9-phe in Aldara caused enhanced protection against tumor inoculation. CONCLUSION: Our data demonstrate that a simple modification of an adjuvant formulation can yield superior results in experimental vaccination protocols by boosting critical steps leading to the generation of an efficient CTL response.


Assuntos
Adjuvantes Imunológicos/farmacocinética , Degranulação Celular/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Melanoma/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Cutâneas/prevenção & controle , Linfócitos T Citotóxicos/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Adjuvantes Imunológicos/uso terapêutico , Administração Cutânea , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Animais , Cálcio/metabolismo , Movimento Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Imiquimode , Imunidade Inata/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenantrenos/farmacologia , Fenantrenos/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pele/citologia , Pele/efeitos dos fármacos , Pele/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinação/métodos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cell Immunol ; 308: 19-26, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27417453

RESUMO

Myelodysplastic syndrome (MDS) is a clonal stem cell disorder frequently associated with inefficient granulopoiesis showing dysplastic polymorphonuclear neutrophils (PMNs). To assess PMN functionality in MDS in a clinical routine setting, 30 MDS patients and ten healthy volunteers were analyzed for PMN and monocyte phenotype and function (degranulation, CD62L shedding, oxidative burst and phagocytosis) upon stimulation with lipopolysaccharide by multi-color flow cytometry (MCFC). Our data show a heterogeneous pattern for CD66, CD16 and CD64 expression on PMNs of MDS patients. CD62L shedding rate and CD66 degranulation were reduced. Interestingly, we detected correlations between the WHO adapted prognostic scoring system (WPSS) and CD16 expression on PMNs as well as the international prognostic scoring system (IPSS) and CD11b degranulation by MCFC, suggesting clinical relevance of MCFC based function testing. In conclusion, MCFC of myelodysplastic immunophenotypes and PMN functionality are applicable in clinical settings, but further prospective studies are needed to assess the practical clinical value of such analyses.


Assuntos
Monócitos/imunologia , Síndromes Mielodisplásicas/diagnóstico , Neutrófilos/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígeno CD11b/metabolismo , Degranulação Celular , Separação Celular , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Monitorização Imunológica , Monitorização Fisiológica , Síndromes Mielodisplásicas/imunologia , Prognóstico , Receptores de IgG/metabolismo
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