RESUMO
Graft-versus-host disease (GvHD) is a frequent life-threatening complication following allogeneic HSC transplantation (HSCT). IL-10 is a regulatory cytokine with important roles during GvHD, yet its relevant sources, and mode of action, remain incompletely defined in this disease. Using IL-10-deficient donor or host mice (BALB/c or C57BL/6, respectively) in a MHC-mismatched model for acute GvHD, we found a strongly aggravated course of the disease with increased mortality when either donor or host cells could not produce this cytokine. A lack of IL-10 resulted in increased allogeneic T-cell responses and enhanced activation of host DCs in spleen and MLNs. Remarkably, IL-10 was prominently produced by host- and donor-derived CD5(int) CD1d(int) TIM-1(int) B cells in this disease, and consistent with this, allogeneic HSCT resulted in exacerbated GvHD when mice lacking IL-10 expression in B cells were used as donor or host, compared with controls. Taken together, this study demonstrates that host and donor B cell-derived IL-10 provides a unique mechanism of suppression of acute GvHD, and suggests that DCs are the targets of this B cell-mediated suppressive effect. These findings open novel therapeutic possibilities based on the use of B cells to increase the feasibility of allogeneic HSCT.
Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Interleucina-10/imunologia , Linfócitos T/imunologia , Aloenxertos , Animais , Linfócitos B/patologia , Células Dendríticas/patologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Linfócitos T/patologiaRESUMO
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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.
RESUMO
Polymorphonuclear neutrophils (PMN) are important for the control of invasive aspergillosis (IA), a major threat to immunocompromised individuals. For clearance of Aspergillus fumigatus infections, PMN employ their potent oxidative and non-oxidative mechanisms. To clarify the relative contribution of these mechanisms, we analyzed p47(phox-/-), gp91(phox-/-) and elastase (ELA) deficient mice (ELANE) after intratracheal infection with A. fumigatus. Infected p47(phox-/-) and gp91(phox-/-) mice died within 4 days and had a significant higher fungal burden in the lungs compared to wild-type controls. Interestingly, the survival of ELANE mice after infection was unimpaired suggesting that ELA is not essential here. Nevertheless, A. fumigatus clearance was delayed in ELANE mice indicating a partial contribution of ELA to fungal immunity. Comparing p47(phox-/-), gp91(phox-/-) or ELANE mice for PMN activation and recruitment to the lungs, we were unable to detect significant differences in vitro or in vivo among mutant or wild-type strains suggesting intact PMN functionality of basic effector mechanisms. Fungal killing in vitro by ELA deficient PMN was comparably reduced as in p47(phox-/-) and gp91(phox-/-) deficient PMN corroborating the importance of oxidative and non-oxidative PMN mechanisms for the control of fungal outgrowth. Taken together, this suggests that intact oxidative as well as non-oxidative PMN effector functions are highly relevant for the control of A. fumigatus infections in vitro and in vivo. While ELA contributes to clearance of A. fumigatus, the oxidative functions are essential for survival.
Assuntos
Aspergillus fumigatus/fisiologia , Aspergilose Pulmonar Invasiva/imunologia , Elastase de Leucócito/metabolismo , Pulmão/patologia , Neutrófilos/imunologia , Animais , Antígenos de Fungos/imunologia , Movimento Celular/genética , Células Cultivadas , Humanos , Imunidade Celular/genética , Elastase de Leucócito/genética , Pulmão/microbiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Neutrófilos/microbiologia , Estresse Oxidativo/genéticaRESUMO
Triggering receptor expressed on myeloid cells 1 (TREM-1) is an important mediator of innate inflammatory responses in microbial infections and sepsis. TREM-1 ligation on neutrophils (PMN) or monocytes results in the production of proinflammatory cytokines. Engagement of TREM-1 induces the activation of MAP kinases as well as rapid Ca(2+) mobilization. However, a detailed understanding of TREM-1 signaling pathways is currently lacking. We evaluated the TREM-1 signaling hierarchy in monocytic cells and found that the acute myeloid leukemia cell line MUTZ-3 expresses TREM-1 in a natural and functional manner. We compared essential signaling molecules of the TREM-1, TLR and NLR cascade in MUTZ-3 cells as well as primary monocytes or PMN by Western blot analysis. These studies confirmed the essential role of phosphatidyl inositide 3-kinase (PI3K) and p38MAPK in the TREM-1 as well as the TLR or NLR cascade of monocytic cells. Importantly, PI3K and p38MAPK signals in monocytic cells both control Ca(2+) mobilization and are directly connected in the TREM-1 signaling hierarchy, which contrasts previous results obtained in PMN. Taken together, our results indicate cell type-specific differences in the TREM-1 signaling cascade and contribute to an enhanced understanding of the regulation of innate inflammatory responses.
Assuntos
Sinalização do Cálcio , Leucemia Mieloide Aguda/imunologia , Glicoproteínas de Membrana/metabolismo , Monócitos/imunologia , Neutrófilos/imunologia , Receptores Imunológicos/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Especificidade de Órgãos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Toll-Like/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Arginine depletion via myeloid cell arginase is critically involved in suppression of the adaptive immune system during cancer or chronic inflammation. On the other hand, arginine depletion is being developed as a novel anti-tumor metabolic strategy to deprive arginine-auxotrophic cancer cells of this amino acid. In human immune cells, arginase is mainly expressed constitutively in PMNs. We therefore purified human primary PMNs from healthy donors and analyzed PMN function as the main innate effector cell and arginase producer in the context of arginine deficiency. We demonstrate that human PMN viability, activation-induced IL-8 synthesis, chemotaxis, phagocytosis, generation of ROS, and fungicidal activity are not impaired by the absence of arginine in vitro. Also, profound pharmacological arginine depletion in vivo via ADI-PEG20 did not inhibit PMN functions in a mouse model of pulmonary invasive aspergillosis; PMN invasion into the lung, activation, and successful PMN-dependent clearance of Aspergillus fumigatus and survival of mice were not impaired. These novel findings add to a better understanding of immunity during inflammation-associated arginine depletion and are also important for the development of therapeutic arginine depletion as anti-metabolic tumor therapy.
Assuntos
Arginina/fisiologia , Neutrófilos/imunologia , Animais , Antineoplásicos/farmacologia , Apoptose , Arginase/sangue , Arginase/fisiologia , Arginina/análise , Arginina/farmacologia , Aspergillus fumigatus/imunologia , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Quimiotaxia de Leucócito , Citrulina/análise , Humanos , Hidrolases/farmacologia , Imunidade Inata , Interleucina-8/biossíntese , Interleucina-8/genética , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Fagocitose , Polietilenoglicóis/farmacologia , Cultura Primária de Células , Aspergilose Pulmonar/imunologia , Aspergilose Pulmonar/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão RespiratóriaRESUMO
Immunologic approaches to combat cancer aim at the induction of tumor-reactive immune responses to achieve long-term protection. In this context, we recently developed a transcutaneous immunization (TCI) method using the Toll-like receptor (TLR) 7 agonist imiquimod and a peptide epitope. Application onto intact skin induces potent cytotoxic T lymphocyte (CTL) responses and protection against transplanted tumors. The purpose of this study was to explore the effects of UV irradiation on imiquimod-based TCI. Here we show that skin exposure to low-dose UV light before TCI with imiquimod strongly boosts specific CTL responses leading to memory formation and enhanced tumor protection. Toward the mechanisms, we show that the activation of bone-marrow-derived dermal dendritic cells (DCs), but not Langerin-expressing DCs, is responsible for enhanced CTL activation. We describe an optimized TCI method that mediates enhanced CTL and antitumor responses by a DC- and TLR-dependent mechanism. These data may provide the basis for the future development of advanced vaccination protocols against tumors and persistent virus infections.
Assuntos
Neoplasias Cutâneas/prevenção & controle , Pele , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos da radiação , Raios Ultravioleta , Aminoquinolinas/imunologia , Aminoquinolinas/farmacologia , Animais , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Imiquimode , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Tolerância Imunológica/efeitos da radiação , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Memória Imunológica/efeitos da radiação , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/imunologia , Células de Langerhans/efeitos da radiação , Glicoproteínas de Membrana/agonistas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pele/citologia , Pele/imunologia , Pele/efeitos da radiação , Neoplasias Cutâneas/imunologia , Receptor 7 Toll-Like/agonistasRESUMO
BACKGROUND: The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (T(reg)) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses. METHODOLOGY/PRINCIPAL FINDINGS: TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3(+) regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10(-/-)) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of T(reg) in IL-10(-/-) mice and the use of B cell deficient JHT(-/-) mice, we can exclude T(reg) and B cells as source of IL-10 in the setting of TCI. CONCLUSION/SIGNIFICANCE: We identify T(reg) and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting T(reg) function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors.