RESUMO
The cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway is instrumental to antitumor immunity, yet the underlying molecular and cellular mechanisms are complex and still unfolding. A new paradigm suggests that cancer cells' cGAS-synthesized cGAMP can be transferred to tumor-infiltrating immune cells, eliciting STING-dependent IFN-ß response for antitumor immunity. Nevertheless, how the tumor microenvironment may shape this process remains unclear. In this study, we found that extracellular ATP, an immune regulatory molecule widely present in the tumor microenvironment, can potentiate cGAMP transfer, thereby boosting the STING signaling and IFN-ß response in murine macrophages and fibroblasts. Notably, genetic ablation or chemical inhibition of murine volume-regulation anion channel LRRC8/volume-regulated anion channel (VRAC), a recently identified cGAMP transporter, abolished ATP-potentiated cGAMP transfer and STING-dependent IFN-ß response, revealing a crucial role of LRRC8/VRAC in the cross-talk of extracellular ATP and cGAMP. Mechanistically, ATP activation of the P2X family receptors triggered Ca2+ influx and K+ efflux, promoting reactive oxygen species production. Moreover, ATP-evoked K+ efflux alleviated the phosphorylation of VRAC's obligate subunit LRRC8A/SWELL1 on S174. Mutagenesis studies indicated that the phosphorylation of S174 on LRRC8A could act as a checkpoint for VRAC in the steady state and a rheostat of ATP responsiveness. In an MC38-transplanted tumor model, systemically blocking CD39 and ENPP1, hydroxylases of extracellular ATP and cGAMP, respectively, elevated antitumor NK, NKT, and CD8+ T cell responses and restrained tumor growth in mice. Altogether, this study establishes a crucial role of ATP in facilitating LRRC8/VRAC transport cGAMP in the tumor microenvironment and provides new insight into harnessing cGAMP transfer for antitumor immunity.
Assuntos
Trifosfato de Adenosina , Proteínas de Membrana , Nucleotídeos Cíclicos , Microambiente Tumoral , Animais , Nucleotídeos Cíclicos/metabolismo , Camundongos , Trifosfato de Adenosina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Microambiente Tumoral/imunologia , Interferon beta/metabolismo , Interferon beta/imunologia , Camundongos Endogâmicos C57BL , Humanos , Transdução de Sinais/imunologia , Camundongos Knockout , Linhagem Celular Tumoral , Cátions/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Nucleotidiltransferases/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismoRESUMO
INTRODUCTION: Patients with allergic bronchopulmonary aspergillosis (ABPA) suffer from repeated exacerbations. The involvement of T-cell subsets remains unclear. METHODS: We enrolled ABPA patients, asthma patients and healthy controls. T-helper type 1 (Th1), 2 (Th2) and 17 (Th17) cells, regulatory T-cells (Treg) and interleukin (IL)-21+CD4+T-cells in total or sorted subsets of peripheral blood mononuclear cells and ABPA bronchoalveolar lavage fluid (BALF) were analysed using flow cytometry. RNA sequencing of subsets of CD4+T-cells was done in exacerbated ABPA patients and healthy controls. Antibodies of T-/B-cell co-cultures in vitro were measured. RESULTS: ABPA patients had increased Th2 cells, similar numbers of Treg cells and decreased circulating Th1 and Th17 cells. IL-5+IL-13+IL-21+CD4+T-cells were rarely detected in healthy controls, but significantly elevated in the blood of ABPA patients, especially the exacerbated ones. We found that IL-5+IL-13+IL-21+CD4+T-cells were mainly peripheral T-helper (Tph) cells (PD-1+CXCR5-), which also presented in the BALF of ABPA patients. The proportions of circulating Tph cells were similar among ABPA patients, asthma patients and healthy controls, while IL-5+IL-13+IL-21+ Tph cells significantly increased in ABPA patients. Transcriptome data showed that Tph cells of ABPA patients were Th2-skewed and exhibited signatures of follicular T-helper cells. When co-cultured in vitro, Tph cells of ABPA patients induced the differentiation of autologous B-cells into plasmablasts and significantly enhanced the production of IgE. CONCLUSION: We identified a distinctly elevated population of circulating Th2-skewed Tph cells that induced the production of IgE in ABPA patients. It may be a biomarker and therapeutic target for ABPA.
Assuntos
Aspergilose Broncopulmonar Alérgica , Linfócitos B , Líquido da Lavagem Broncoalveolar , Células Th2 , Humanos , Masculino , Feminino , Aspergilose Broncopulmonar Alérgica/imunologia , Adulto , Células Th2/imunologia , Pessoa de Meia-Idade , Estudos de Casos e Controles , Linfócitos B/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T Reguladores/imunologia , Asma/imunologia , Células Th17/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Dimerization of C-type lectin receptors (CLRs) or Toll-like receptors (TLRs) can alter their ligand binding ability, thereby modulating immune responses. However, the possibilities and roles of dimerization between CLRs and TLRs remain unclear. Here we show that C-type lectin receptor-2d (CLEC2D) forms homodimers, as well as heterodimers with TLR2. Quantitative ligand binding assays reveal that both CLEC2D homodimers and CLEC2D/TLR2 heterodimers have a higher binding ability to fungi-derived ß-glucans than TLR2 homodimers. Moreover, homo- or hetero-dimeric CLEC2D mediates ß-glucan-induced ubiquitination and degradation of MyD88 to inhibit the activation of transcription factor IRF5 and subsequent IL-12 production. Clec2d-deficient female mice are resistant to infection with Candida albicans, a human fungal pathogen, owing to the increase of IL-12 production and subsequent generation of IFN-γ-producing NK cells. Together, these data indicate that CLEC2D forms homodimers or heterodimers with TLR2, which negatively regulate antifungal immunity through suppression of IRF5-mediated IL-12 production. These homo- and hetero-dimers of CLEC2D and TLR2 provide an example of receptor dimerization to regulate host innate immunity against microbial infections.
Assuntos
Receptor 2 Toll-Like , beta-Glucanas , Animais , Feminino , Humanos , Camundongos , Antifúngicos , beta-Glucanas/metabolismo , Imunidade Inata , Fatores Reguladores de Interferon/metabolismo , Interleucina-12/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligantes , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/metabolismoRESUMO
During viral infections, stimulator of interferon genes (STING) exerts a positive protective immune response. Chen et al. now shed light on the distinct role of STING in fungal infections. STING translocates to the phagosome to negatively regulate the immune response against Candida albicans infection through the inhibition of Src-involved Syk phosphorylation.
RESUMO
Candida albicans deploys a variety of mechanisms such as morphological switch and elicitor release to promote virulence. However, the intricate interactions between the fungus and the host remain poorly understood, and a comprehensive inventory of fungal virulence factors has yet to be established. In this study, we identified a C. albicans secretory effector protein Sce1, whose induction and secretion are associated with vagina-simulative conditions and chlamydospore formation. Sequence alignment showed that Sce1 belongs to a Pir family in C. albicans, which is conserved across several fungi and primarily characterized as a ß-glucan binding protein in the Saccharomyces cerevisiae. Mechanically, Sce1 is primarily localized to the cell wall in a cleaved form as an alkali-labile ß-1,3-glucan binding protein and plays a role in masking ß-glucan in acidic environments and chlamydospores, a feature that might underline C. albicans' ability to evade host immunity. Further, a cleaved short form of Sce1 protein could be released into extracellular compartments and presented in bone marrow-derived macrophages infected with chlamydospores. This cleaved short form of Sce1 also demonstrated a unique ability to trigger the caspases-8/9-dependent apoptosis in various host cells. Correspondingly, genetic deletion of SCE1 led to dampened vaginal colonization of C. albicans and diminished fungal virulence during systemic infection. The discovery of Sce1 as a versatile virulence effector that executes at various compartments sheds light on the fungus-host interactions and C. albicans pathogenesis.
RESUMO
Programmed death ligand 1 (PD-L1) has been shown to be inducibly expressed on neutrophils to suppress host immunity during polymicrobial sepsis, virus and parasite infections. However, the role of PD-L1 on neutrophil-mediated antifungal immunity remains wholly unknown. Here, we show that the expression of PD-L1 on murine and human neutrophils was upregulated upon the engagement of C-type lectin receptor Dectin-1 with its ligand ß-glucans, exposed on fungal pathogen Candida albicans yeast. Moreover, ß-glucan stimulation induced PD-L1 translocation into nucleus to regulate the production of chemokines CXCL1 and CXCL2, which control neutrophil mobilization. Importantly, C. albicans infection-induced expression of PD-L1 leads to neutrophil accumulation in bone marrow, through mediating their autocrine secretion of CXCL1/2. Furthermore, neutrophil-specific deficiency of PD-L1 impaired CXCL1/2 secretion, which promoted neutrophil migration from bone marrow into the peripheral circulation, thereby conferring host resistance to C. albicans infection. Finally, either PD-L1 blockade or pharmacological inhibition of PD-L1 expression significantly increased neutrophil release from bone marrow to enhance host antifungal immunity. Our data together indicate that activation of Dectin-1/PD-L1 cascade by ß-glucans inhibits neutrophil release from bone marrow reserve, contributing to the negative regulation of antifungal innate immunity, which functions as a potent immunotherapeutic target against life-threatening fungi infections.
Assuntos
Neutrófilos , beta-Glucanas , Animais , Camundongos , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Antifúngicos/metabolismo , Medula Óssea , Candida albicans/fisiologia , beta-Glucanas/farmacologia , beta-Glucanas/metabolismoRESUMO
Cryptococcosis is a potentially lethal disease that is primarily caused by the fungus Cryptococcus neoformans, treatment options for cryptococcosis are limited. Here, we show glucuronoxylomannan, the major polysaccharide component of C. neoformans, induces the recruitment of neutrophilic myeloid-derived suppressor cells in mice and patients with cryptococcosis. Depletion of neutrophilic myeloid-derived suppressor cells enhances host defense against C. neoformans infection. We identify C-type lectin receptor-2d recognizes glucuronoxylomannan to potentiate the immunosuppressive activity of neutrophilic myeloid-derived suppressor cells by initiating p38-mediated production of the enzyme arginase-1, which inhibits T-cell mediated antifungal responses. Notably, pharmacological inhibition of arginase-1 expression by a specific inhibitor of p38, SB202190, or an orally available receptor tyrosine kinase inhibitor, vandetanib, significantly enhances T-cell mediated antifungal responses against cryptococcosis. These data reveal a crucial suppressive role of neutrophilic myeloid-derived suppressor cells during cryptococcosis and highlight a promising immunotherapeutic application by inhibiting arginase-1 production to combat infectious diseases.
Assuntos
Criptococose , Cryptococcus neoformans , Células Supressoras Mieloides , Animais , Antifúngicos , Arginase , Criptococose/microbiologia , Criptococose/terapia , Fatores Imunológicos , Imunoterapia , Camundongos , Linfócitos TRESUMO
Intestinal fungi are critical for modulating host immune homeostasis and underlying mechanisms remain unclear. We show that dendritic cell (DC)-specific deficiency of casitas B-lineage lymphoma (c-Cbl) renders mice susceptible to dextran sodium sulfate (DSS)-induced colitis. Mechanistically, we identify that c-Cbl functions downstream of Dectin-2 and Dectin-3 to mediate the ubiquitination and degradation of noncanonical nuclear factor κB subunit RelB. Thus, c-Cbl deficiency in DCs promotes α-mannan-induced activation of RelB, which suppresses p65-mediated transcription of an anti-inflammatory cytokine gene, il10, thereby aggravating DSS-induced colitis. Moreover, suppressing fungal growth with fluconazole or inhibition of RelB activation in vivo attenuates colitis in mice with DC-specific deletion of c-Cbl. We also demonstrate an interaction between c-Cbl and c-Abl tyrosine kinase and find that treatment with DPH, a c-Abl agonist, synergistically increases fungi-induced c-Cbl activation to restrict colitis. Together, these findings unravel a previously unidentified fungi-induced c-Cbl/RelB axis that sustains intestinal homeostasis and protects against intestinal inflammation.
Assuntos
Colite , NF-kappa B , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Animais , Colite/induzido quimicamente , Fungos/metabolismo , Inflamação , Camundongos , NF-kappa B/metabolismo , Ubiquitina-Proteína LigasesRESUMO
The recently identified anion channel LRRC8 volume-regulated anion channels (VRACs) are heteromeric hexamers constituted with the obligate LRRC8A subunit paired with at least one of the accessory LRRC8B to LRRC8E subunits. In addition to transport chloride, taurine, and glutamate, LRRC8 VRACs also transport the anticancer agent cisplatin and STING agonists 2'3'-cyclic GMP-AMP (cGAMP) and cyclic dinucleotides; hence, they are implicated in a variety of physiological and pathological processes, such as cell swelling, stroke, cancer, and viral infection. Although the subunit composition largely determines VRAC substrate specificity, the opening of various VRAC pores under physiological and pathological settings remains enigmatic. In this study, we demonstrated that VRACs comprising LRRC8A and LRRC8E (LRRC8A/E-containing VRACs), specialized in cGAMP transport, can be opened by a protein component present in serum under resting condition. Serum depletion ablated the tonic activity of LRRC8A/E-containing VRACs, decreasing cGAMP transport in various human and murine cells. Also, heating or proteinase K treatment abolished the ability of serum to activate VRAC. Genetic analyses revealed a crucial role for cGAMP synthase (cGAS) in serum/TNF-promoted VRAC activation. Notably, the presence of cGAS on the plasma membrane, rather than its DNA-binding or enzymatic activity, enabled VRAC activation. Moreover, phospholipid PIP2 seemed to be instrumental in the membrane localization of cGAS and its association with VRACs. Corroborating a role for LRRC8A/D-containing VRACs in cisplatin transport, serum and TNF markedly potentiated cisplatin uptake and killing of cancer cells derived from human or mouse. Together, these observations provide new insights into the complex regulation of VRAC activation and suggest a novel approach to enhance the efficacy of cGAMP and cisplatin in treating infection and cancer.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Inflamação/tratamento farmacológico , Proteínas de Membrana/imunologia , Nucleotídeos Cíclicos/farmacologia , Animais , Antineoplásicos/imunologia , Cisplatino/imunologia , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nucleotídeos Cíclicos/imunologiaRESUMO
Aluminum-containing adjuvants have been used for nearly 100 years to enhance immune responses in billions of doses of vaccines. To date, only a few adjuvants have been approved for use in humans, among which aluminum-containing adjuvants are the only ones widely used. However, the medical need for potent and safe adjuvants is currently continuously increasing, especially those triggering cellular immune responses for cytotoxic T lymphocyte activation, which are urgently needed for the development of efficient virus and cancer vaccines. Manganese is an essential micronutrient required for diverse biological activities, but its functions in immunity remain undefined. We previously reported that Mn2+ is important in the host defense against cytosolic dsDNA by facilitating cGAS-STING activation and that Mn2+ alone directly activates cGAS independent of dsDNA, leading to an unconventional catalytic synthesis of 2'3'-cGAMP. Herein, we found that Mn2+ strongly promoted immune responses by facilitating antigen uptake, presentation, and germinal center formation via both cGAS-STING and NLRP3 activation. Accordingly, a colloidal manganese salt (Mn jelly, MnJ) was formulated to act not only as an immune potentiator but also as a delivery system to stimulate humoral and cellular immune responses, inducing antibody production and CD4+/CD8+ T-cell proliferation and activation by either intramuscular or intranasal immunization. When administered intranasally, MnJ also worked as a mucosal adjuvant, inducing high levels of secretory IgA. MnJ showed good adjuvant effects for all tested antigens, including T cell-dependent and T cell-independent antigens, such as bacterial capsular polysaccharides, thus indicating that it is a promising adjuvant candidate.
Assuntos
Adjuvantes Imunológicos/farmacologia , Manganês/farmacologia , Sais/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antivirais/farmacologia , Vacinas Anticâncer/imunologia , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Interleucina-1/biossíntese , Interleucina-18/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotidiltransferases/metabolismo , Subunidades Proteicas/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The morphological switch between yeast and hyphae of Candida albicans is essential for its interaction with the host defense system. However, the lack of understanding of host-pathogen interactions during C. albicans infection greatly hampers the development of effective immunotherapies. Here, we found that priming with the C. albicans FLO8-deficient (flo8) mutant, locked in yeast form, protected mice from subsequent lethal C. albicans infection. Deficiency of Dectin-2, a fungus-derived α-mannan recognition receptor, completely blocked flo8 mutant-induced protection. Mechanistically, the flo8 mutant-induced Dectin-2/CARD9-mediated IL-10 production in DCs and macrophages to block thymus atrophy by inhibiting the C. albicans-induced apoptosis of thymic T cells, which facilitated the continuous output of naive T cells from the thymus to the spleen. Continuous recruitment of naive T cells to the spleen enhanced Th1-biased antifungal immune responses. Consequently, depletion of CD4+ T cells or blockade of IL-10 receptor function using specific antibodies in mice completely blocked the protective effects of flo8 mutant priming against C. albicans infection. Moreover, mannans exposed on the surface of the flo8 mutant were responsible for eliciting protective immunity by inhibiting the C. albicans-induced apoptosis of thymic T cells to sustain the number of naive T cells in the spleen. Importantly, priming with the flo8 mutant extensively protected mice from polymicrobial infection caused by cecal ligation and puncture (CLP) by enhancing Th1-biased immune responses. Together, our findings imply that targeting FLO8 in C. albicans elicits protective immune responses against polymicrobial infections and that mannans extracted from the flo8 mutant are potential immunotherapeutic candidate(s) for controlling infectious diseases.
Assuntos
Candidíase , Sepse , Animais , Proteínas Adaptadoras de Sinalização CARD , Candida albicans/fisiologia , Hifas , Mananas/farmacologia , CamundongosRESUMO
Background: Pseudomonas aeruginosa (PA) is one of the most common Gram-negative bacteria causing hospital-acquired pulmonary infection, with high drug resistance and mortality. Therefore, it is urgent to introduce new non-antibiotic treatment strategies. Mesenchymal stem cells (MSCs), as important members of the stem cell family, were demonstrated to alleviate pathological damage in acute lung injury. However, the potential mechanism how MSC alleviate acute lung infection caused by PA remains unclear. Objective: The purpose of this study was to investigate the effects of Adipose-derived mesenchymal stem cells (ASCs) on acute pulmonary infections and the possible mechanisms how ASCs reduce pulmonary inflammation induced by PA. Methods: The therapeutic and mechanistic effects of ASCs on PA pulmonary infection were evaluated respectively in a murine model as well as in an in vitro model stimulated by PA and co-cultured with ASCs. Results: 1. ASCs treatment significantly reduced the bacterial load, inflammation of lung tissue and histopathological damage by PA. 2. PA infection mainly activated Nod-like receptor containing a caspase activating and recruitment domain 4 (NLRC4) inflammasome in the lung of mice. ASCs attenuated acute lung infection in mice by inhibiting NLRC4 inflammasome activation. 3. NLRC4-/- mice showed a significant improvement in survival rate and lung bacterial load after PA infection. 4. ASCs mainly increased expression and secretion of STC-1 in response to PA-stimulated NLRC4 inflammasome activation. Conclusions: PA infection attenuated macrophage phagocytosis through activation of NLRC4 inflammasome in macrophages, which eventually led to pulmonary inflammatory damage in mouse; ASCs reduced the activation of NLRC4 inflammasome in macrophages induced by PA infection, thereby increasing the phagocytic ability of macrophages, and ultimately improving lung tissue damage in mouse; ASCs may inhibit NLRC4 inflammasome through the secretion of STC-1.
Assuntos
Inflamassomos , Células-Tronco Mesenquimais , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio , Inflamassomos/metabolismo , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pseudomonas aeruginosa/metabolismoRESUMO
INTRODUCTION: The characteristics of Allergic Bronchopulmonary Aspergillosis (ABPA) based on its radiological classification is still unclear. OBJECTIVES: To investigate the clinical significances of ABPA patients with central bronchiectasis (ABPA-CB) by different radiological classifications of mucus plugs. METHODS: ABPA-CB patients from a pulmonary hospital between 2008 and 2015 were retrospectively included and analysed. According to the chest imaging in their first visit to physician, the ABPA-CB patients were divided into two groups based on the presence of high-attenuation mucus (HAM) or low-attenuation mucus (LAM). The primary endpoint was ABPA relapse within 1 year since the glucocorticoid withdrawal. The relationship between the imaging findings and the clinical prognosis was illuminated. RESULTS: A total of 125 ABPA patients were analysed in this study. Compared to the LAM group, the HAM group presented higher blood eosinophil cells counts, higher rates of Aspergillus detection isolated in sputum and expectoration of brownish-black mucus plugs, more affected lobes and segments, poorer pulmonary function and higher rate of relapse. CONCLUSIONS: The clinical characteristics and prognosis of ABPA-CB patients are closely related to its radiological phenotype of mucus plugs in the central bronchiectasis. Clinicians should promote a diversity of personalized treatments for different patients with different radiological characteristics.
Assuntos
Aspergillus/isolamento & purificação , Bronquiectasia/etiologia , Broncoscopia/métodos , Muco/microbiologia , Aspergilose Pulmonar/complicações , Tomografia Computadorizada por Raios X/métodos , Adulto , Bronquiectasia/classificação , Bronquiectasia/diagnóstico , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Aspergilose Pulmonar/diagnóstico , Aspergilose Pulmonar/microbiologia , Estudos RetrospectivosRESUMO
The incidence of Aspergillus fumigatus infection and the rate of resistance to antifungal drugs have sharply increased in recent years. LL37 has been reported as a host defense peptide with broad-spectrum antibacterial activities. However, the role of LL37 during A. fumigatus infection remains unclear. Here, we examined the interaction between LL37 and A. fumigatus and found that synthetic LL37 could directly bind to the surface of A. fumigatus, disrupting the integrity of the cell wall in vitro. LL37 inhibited mycelial growth in a concentration-dependent manner, rather than fungicidal effect even at high concentration (e.g., 20 µM). Interestingly, low concentrations of LL37 (e.g., 4 µM) significantly attenuated mycelial adhesion and prevented the invasion and destruction of epithelial cells. Following LL37 treatment, the levels of proinflammatory cytokines released by A. fumigatus-stimulated macrophages decreased significantly, accompanied by downregulation of M1 type markers. In a mouse model of pulmonary A. fumigatus infection, LL37-treated mice showed lower amounts of fungi load, moderate pathological damage, and reduced proinflammatory cytokines. Further, LL37 transgenic mice (LL37+/+) were examined to investigate the effects of endogenous LL37 in an A. fumigatus infection model and showed lower susceptibility to A. fumigatus infection in comparison with wild-type mice. In addition, LL37 also played a protective role in an immunosuppressed mouse model of A. fumigatus infection. Thus, LL37 inhibits A. fumigatus infection via directly binding to mycelia and reducing excessive inflammation. LL37 or its analogs may therefore constitute potential drug components for A. fumigatus infection.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Aspergilose/metabolismo , Aspergillus fumigatus/metabolismo , Inflamação/prevenção & controle , Animais , Antifúngicos , Células Cultivadas , Citocinas/metabolismo , Células Epiteliais/metabolismo , Feminino , Proteínas Fúngicas/metabolismo , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Virulência/fisiologiaRESUMO
Cryptococcus neoformans and Cryptococcus gattii cause life-threatening meningoencephalitis or lung diseases in immunocompetent individuals or immunocompromised ones. C. neoformans and C. gattii are subdivided into five serotypes based on their capsular glucuronoxylomannan (GXM). C. neoformans consists of serotypes A, D, and AD hybrid, and C. gattii consists of serotypes B and C. Given structural differences of GXM between C. neoformans and C. gattii, it remains unclear that how innate immune system recognizes GXM. Here, we report that C-type lectin receptor Dectin-3 (MCL encoded by Clec4d) is a direct receptor for GXMs from C. neoformans serotype AD (C.n-AD) and C. gattii serotype B (C.g-B). GXMs from C.n-AD and C.g-B activated NF-κB and ERK pathways to induce pro-inflammatory cytokine production, whereas it was completely abolished due to deficiency of Dectin-3 or caspase recruitment domain family member 9 (CARD9). Upon pulmonary C.n-AD and C.g-B infection, Dectin-3- and CARD9-deficient mice were highly susceptible and showed augmented lung injury due to impairment of alveolar macrophage accumulation and killing activities. Our study provides the first biological and genetic evidence demonstrating that Dectin-3 recognizes GXM of C.n-AD and C.g-B to initiate host defense against cryptococcosis.
Assuntos
Criptococose/imunologia , Cryptococcus gattii , Cryptococcus neoformans , Lectinas Tipo C/metabolismo , Polissacarídeos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Proteínas Adaptadoras de Sinalização CARD/deficiência , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Feminino , Mediadores da Inflamação/metabolismo , Pneumopatias/metabolismo , Pneumopatias/microbiologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Polissacarídeos/isolamento & purificaçãoRESUMO
The adaptor CARD9 functions downstream of C-type lectin receptors (CLRs) for the sensing of microbial infection, which leads to responses by the TH1 and TH17 subsets of helper T cells. The single-nucleotide polymorphism rs4077515 at CARD9 in the human genome, which results in the substitution S12N (CARD9S12N), is associated with several autoimmune diseases. However, the function of CARD9S12N has remained unknown. Here we generated CARD9S12N knock-in mice and found that CARD9S12N facilitated the induction of type 2 immune responses after engagement of CLRs. Mechanistically, CARD9S12N mediated CLR-induced activation of the non-canonical transcription factor NF-κB subunit RelB, which initiated production of the cytokine IL-5 in alveolar macrophages for the recruitment of eosinophils to drive TH2 cell-mediated allergic responses. We identified the homozygous CARD9 mutation encoding S12N in patients with allergic bronchopulmonary aspergillosis and revealed activation of RelB and production of IL-5 in peripheral blood mononuclear cells from these patients. Our study provides genetic and functional evidence demonstrating that CARD9S12N can turn alveolar macrophages into IL-5-producing cells and facilitates TH2 cell-mediated pathologic responses.
Assuntos
Aspergilose Broncopulmonar Alérgica/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Interleucina-5/biossíntese , Macrófagos Alveolares/imunologia , Células Th2/imunologia , Animais , Aspergilose Broncopulmonar Alérgica/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Humanos , Interleucina-5/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Polimorfismo de Nucleotídeo Único , Transdução de Sinais/imunologiaAssuntos
Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Dano ao DNA , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/metabolismo , Animais , Antineoplásicos/farmacologia , Proteína 10 de Linfoma CCL de Células B/deficiência , Proteínas Adaptadoras de Sinalização CARD/deficiência , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/deficiênciaRESUMO
Activation of various C-type lectin receptors (CLRs) initiates potent proinflammatory responses against various microbial infections. However, how activated CLRs are negatively regulated remains unknown. In this study, we report that activation of CLRs Dectin-2 and Dectin-3 by fungi infections triggers them for ubiquitination and degradation in a Syk-dependent manner. Furthermore, we found that E3 ubiquitin ligase Casitas B-lineage lymphoma protein b (Cbl-b) mediates the ubiquitination of these activated CLRs through associating with each other via adapter protein FcR-γ and tyrosine kinase Syk, and then the ubiquitinated CLRs are sorted into lysosomes for degradation by an endosomal sorting complex required for transport (ESCRT) system. Therefore, the deficiency of either Cbl-b or ESCRT subunits significantly decreases the degradation of activated CLRs, thereby resulting in the higher expression of proinflammatory cytokines and inflammation. Consistently, Cbl-b-deficient mice are more resistant to fungi infections compared with wild-type controls. Together, our study indicates that Cbl-b negatively regulates CLR-mediated antifungal innate immunity, which provides molecular insight for designing antifungal therapeutic agents.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Candida albicans/imunologia , Candidíase/imunologia , Lectinas Tipo C/imunologia , Proteínas Proto-Oncogênicas c-cbl/imunologia , Receptores Imunológicos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Candidíase/genética , Humanos , Imunidade Inata , Lectinas Tipo C/genética , Camundongos , Camundongos Knockout , Proteólise , Proteínas Proto-Oncogênicas c-cbl/genética , Receptores Imunológicos/genética , Quinase Syk/genética , Quinase Syk/imunologia , Ubiquitinação/genética , Ubiquitinação/imunologiaRESUMO
Multi-drug resistance of pathogenic microorganisms is becoming a serious threat, particularly to immunocompromised populations. The high mortality of systematic fungal infections necessitates novel antifungal drugs and therapies. Unfortunately, with traditional drug discovery approaches, only echinocandins was approved by FDA as a new class of antifungals in the past two decades. Drug efflux is one of the major contributors to multi-drug resistance, the modulator of drug efflux pumps is considered as one of the keys to conquer multi-drug resistance. In this study, we combined structure-based virtual screening and whole-cell based mechanism study, identified a natural product, beauvericin (BEA) as a drug efflux pump modulator, which can reverse the multi-drug resistant phenotype of Candida albicans by specifically blocking the ATP-binding cassette (ABC) transporters; meantime, BEA alone has fungicidal activity in vitro by elevating intracellular calcium and reactive oxygen species (ROS). It was further demonstrated by histopathological study that BEA synergizes with a sub-therapeutic dose of ketoconazole (KTC) and could cure the murine model of disseminated candidiasis. Toxicity evaluation of BEA, including acute toxicity test, Ames test, and hERG (human ether-à-go-go-related gene) test promised that BEA can be harnessed for treatment of candidiasis, especially the candidiasis caused by ABC overexpressed multi-drug resistant C. albicans.