RESUMO
Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK-cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.
Assuntos
Aspergilose , Aspergillus fumigatus , Antígeno CD56 , Células Matadoras Naturais , Humanos , Aspergillus fumigatus/imunologia , Células Matadoras Naturais/imunologia , Antígeno CD56/metabolismo , Antígeno CD56/imunologia , Aspergilose/imunologia , Aspergilose/microbiologia , Ativação Linfocitária/imunologia , Polissacarídeos/metabolismo , Polissacarídeos/imunologia , Parede Celular/imunologia , Parede Celular/metabolismoRESUMO
Small-cell lung cancer (SCLC) is a fatal disease with limited treatment options. Circulating tumor cells (CTCs) in liquid biopsy samples may serve as predictive and prognostic biomarkers; but the analysis of CTCs is still challenging. By using microfluidic or density gradient CTC enrichment in combination with immunofluorescent (IF) staining or qPCR of CTC-related transcripts, we achieved a 60.8% to 88.0% positivity in SCLC blood samples. Epithelial and neuroendocrine transcripts including the druggable target DLL3 were associated with shorter overall survival (OS), indicating the clinical value of these markers in terms of differential diagnosis and treatment decisions. High CTC counts and the presence of CTC duplets detected by IF staining were prognostic for OS, and thus may serve as indicators of disease progression or therapy failure. In patient samples with high CTC load detected by IF staining, a concordance of the transcripts positivity in circulating free plasma RNA and CTCs was observed. Our data emphasize the role of CTCs and CTC-related transcripts and underline the clinical value of liquid biopsy analysis in SCLC.
Assuntos
Neoplasias Pulmonares , Células Neoplásicas Circulantes , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Prognóstico , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Biomarcadores Tumorais/genética , Proteínas de Membrana , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Aspergillus fumigatus is an opportunistic fungal pathogen causing detrimental infections in immunocompromised individuals. Dendritic cells (DCs) are potent antigen-presenting cells and recognize the A. fumigatus cell wall component ß-1,3 glucan via Dectin-1, followed by DC maturation and cytokine release. Here, we demonstrate that human primary myeloid DCs (mDCs) interact with different morphotypes of A. fumigatus. Dectin-1 is expressed on mDCs and is down-regulated after contact with A. fumigatus, indicating that mDCs recognize A. fumigatus via this receptor. Blocking of Dectin-1, followed by stimulation with depleted zymosan diminished the up-regulation of the T-cell co-stimulatory molecules CD40, CD80, HLA-DR and CCR7 on mDCs and led to decreased release of the cytokines TNF-α, IL-8, IL-1ß and IL-10.
Assuntos
Aspergillus fumigatus/imunologia , Células Dendríticas/imunologia , Regulação para Baixo/imunologia , Interações Hospedeiro-Patógeno/imunologia , Lectinas Tipo C/imunologia , Células Cultivadas , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Citometria de Fluxo , Humanos , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/efeitos dos fármacos , Lectinas Tipo C/metabolismo , Zimosan/análogos & derivados , Zimosan/farmacologiaRESUMO
High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections.
Assuntos
Aspergillus fumigatus/genética , Citomegalovirus/genética , Interações Hospedeiro-Patógeno/genética , Aspergillus fumigatus/patogenicidade , Linhagem Celular , Coinfecção , Citocinas/metabolismo , Citomegalovirus/patogenicidade , Células Dendríticas/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunidade Inata , Ativação Linfocitária , Monócitos/metabolismo , NF-kappa B/metabolismo , RNA-Seq/métodos , Análise de Sequência de RNA/métodos , Transdução de Sinais , Transcriptoma/genéticaRESUMO
Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1ß, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56brightCD16dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1ß, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/fisiologia , Células Matadoras Naturais/imunologia , Actinas/metabolismo , Corticosteroides/farmacologia , Adulto , Idoso , Antígeno CD56/metabolismo , Movimento Celular , Células Cultivadas , Quimiocinas/metabolismo , Feminino , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other.
Assuntos
Aspergillus fumigatus/imunologia , Aspergillus fumigatus/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Citocinas/metabolismo , Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interleucinas/metabolismo , Redes e Vias Metabólicas , Proteínas NLR/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , TranscriptomaRESUMO
Invasive aspergillosis (IA) is an infectious disease caused by the fungal pathogen Aspergillus fumigatus that mainly affects immunocompromised hosts. To investigate immune cell cross-talk during infection with A. fumigatus, we co-cultured natural killer (NK) cells and dendritic cells (DC) after stimulation with whole fungal structures, components of the fungal cell wall, fungal lysate or ligands for distinct fungal receptors. Both cell types showed activation after stimulation with fungal components and were able to transfer activation signals to the counterpart not stimulated cell type. Interestingly, DCs recognized a broader spectrum of fungal components and thereby initiated NK cell activation when those did not recognize fungal structures. These experiments highlighted the supportive function of DCs in NK cell activation. Furthermore, we focused on soluble DC mediated NK cell activation and showed that DCs stimulated with the TLR2/Dectin-1 ligand zymosan could maximally stimulate the expression of CD69 on NK cells. Thus, we investigated the influence of both receptors for zymosan, Dectin-1 and TLR2, which are highly expressed on DCs but show only minimal expression on NK cells. Specific focus was laid on the question whether Dectin-1 or TLR2 signaling in DCs is important for the secretion of soluble factors leading to NK cell activation. Our results show that Dectin-1 and TLR2 are negligible for NK cell activation. We conclude that besides Dectin-1 and TLR2 other receptors on DCs are able to compensate for the missing signal.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Comunicação Celular , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Antígenos de Fungos/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Lectinas Tipo C , Receptor 2 Toll-LikeRESUMO
Aspergillus fumigatus is a common airborne fungal pathogen of humans and a significant source of mortality in immunocompromised individuals. Here, we provide the most extensive cell wall proteome profiling to date of A. fumigatus resting conidia, the fungal morphotype pertinent to first contact with the host. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified proteins within the conidial cell wall by hydrogen-fluoride (HF)-pyridine extraction and proteins exposed on the surface using a trypsin-shaving approach. One protein, designated conidial cell wall protein A (CcpA), was identified by both methods and was found to be nearly as abundant as hydrophobic rodlet layer-forming protein RodA. CcpA, an amphiphilic protein, like RodA, peaks in expression during sporulation on resting conidia. Despite high cell wall abundance, the cell surface structure of ΔccpA resting conidia appeared normal. However, trypsin shaving of ΔccpA conidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen ΔccpA conidia led to higher activation of neutrophils and dendritic cells than was seen with wild-type conidia and caused significantly less damage to epithelial cells in vitro In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with ΔccpA conidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed to A. fumigatus conidia, suggesting a role for CcpA as a structural protein impacting conidial immunogenicity rather than possessing a protein-intrinsic immunosuppressive effect. Together, these data suggest that CcpA serves as a conidial stealth protein by altering the conidial surface structure to minimize innate immune recognition.IMPORTANCE The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of Aspergillus fumigatus, the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. A. fumigatus lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.
Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Proteoma/análise , Células A549 , Animais , Aspergilose/imunologia , Parede Celular/química , Cromatografia Líquida , Células Dendríticas/imunologia , Endocitose , Células Epiteliais/imunologia , Feminino , Proteínas Fúngicas/genética , Humanos , Hospedeiro Imunocomprometido , Proteínas de Membrana/genética , Camundongos , Ativação de Neutrófilo , Esporos Fúngicos/patogenicidade , Linfócitos T/imunologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.
Assuntos
Aspergillus fumigatus/patogenicidade , Antígeno CD56/metabolismo , Regulação para Baixo , Células Matadoras Naturais/microbiologia , Células Matadoras Naturais/ultraestrutura , Actinas/ultraestrutura , Aspergillus fumigatus/imunologia , Citoesqueleto/ultraestrutura , Citometria de Fluxo , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Microscopia Confocal , Microscopia Eletrônica de VarreduraRESUMO
The cytotoxicities of highly efficient salan-Ti(IV) complexes toward a range of cell lines, including drug-resistant cells, are reported along with preliminary mechanistic insights. Five salan-Ti(IV) complexes were investigated toward eight different human and murine cancer-derived cell lines, including colon, ovarian, lung, cervical, pancreatic, leukemic, skin, and breast. The salan complexes are more active toward the cells analyzed than cisplatin and the known titanium compound (bzac)(2) Ti(OiPr)(2) , and no cell line resistant to the salan complexes was identified. Moreover, the salan-Ti(IV) complexes are highly active toward both cisplatin-sensitive (A2780) and cisplatin-resistant (A2780CisR) human ovarian cancer cell lines. Similarly, the salan complexes are cytotoxic toward multi-drug-resistant (ABCB1-expressing) mouse lymphoma cell lines HU-1 and HU-2. Importantly, minimal or no activity was observed toward primary murine cells (bone marrow, heart, liver, kidney, spleen, and lung), supporting selectivity for cancer cells. Additionally, the salan complexes maintain high cytotoxicity for up to 24 h following exposure to cell culture medium, whereas reference complexes (bzac)(2) Ti(OiPr)(2) and Cp(2) TiCl(2) rapidly lose much of their activity upon exposure to medium, within ~1 h. The upregulation of p53 followed by cell-cycle arrest in G(1) phase is likely one mechanism of action of the salan complexes. Taken together, the results indicate that these compounds are selectively toxic to cancer cells and are able to circumvent two independent mechanisms of drug resistance, thus expanding the scope of their potential medicinal utility.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/química , Titânio/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Cisplatino/farmacologia , Complexos de Coordenação/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Titânio/químicaAssuntos
Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/terapia , Cardiologia , Consenso , Técnicas Eletrofisiológicas Cardíacas/normas , Garantia da Qualidade dos Cuidados de Saúde , Sociedades Médicas , Técnicas Eletrofisiológicas Cardíacas/instrumentação , Humanos , Pessoa de Meia-Idade , Melhoria de QualidadeRESUMO
Atrial fibrillation is the most common clinically significant dysrhythmia. It has great impact on quality of life especially related to complications such as stroke and heart failure as well as functional status and the cost of chronic treatment. Pharmacologic treatment strategies are now better defined and more efficacious. Side effects of drugs are better understood. Recent clinical studies have contributed recommended treatment guidelines. The identification of atrial ectopic foci in cardiac venous sites has defined targets for isolation ablations. Novel catheter ablation techniques are offering improved prognosis for patients with atrial fibrillation.