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1.
PLoS Pathog ; 16(6): e1008621, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32544188

RESUMO

During tuberculosis, lung myeloid cells have two opposing roles: they are an intracellular niche occupied by Mycobacterium tuberculosis, and they restrict bacterial replication. Lung myeloid cells from mice infected with yellow-fluorescent protein expressing M. tuberculosis were analyzed by flow cytometry and transcriptional profiling to identify the cell types infected and their response to infection. CD14, CD38, and Abca1 were expressed more highly by infected alveolar macrophages and CD11cHi monocyte-derived cells compared to uninfected cells. CD14, CD38, and Abca1 "triple positive" (TP) cells had not only the highest infection rates and bacterial loads, but also a strong interferon-γ signature and nitric oxide synthetase-2 production indicating recognition by T cells. Despite evidence of T cell recognition and appropriate activation, these TP macrophages are a cellular compartment occupied by M. tuberculosis long-term. Defining the niche where M. tuberculosis resists elimination promises to provide insight into why inducing sterilizing immunity is a formidable challenge.


Assuntos
Antígenos CD11/imunologia , Macrófagos Alveolares , Monócitos , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/imunologia , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/imunologia , Animais , Antígenos CD11/genética , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/microbiologia , Monócitos/patologia , Mycobacterium tuberculosis/genética , Linfócitos T/imunologia , Linfócitos T/microbiologia , Linfócitos T/patologia , Tuberculose/genética , Tuberculose/patologia
2.
PLoS Pathog ; 16(5): e1008553, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453761

RESUMO

IRGM and its mouse orthologue Irgm1 are dynamin-like proteins that regulate vesicular remodeling, intracellular microbial killing, and pathogen immunity. IRGM dysfunction is linked to inflammatory bowel disease (IBD), and while it is thought that defective intracellular killing of microbes underscores IBD susceptibility, studies have yet to address how IRGM/Irgm1 regulates immunity to microbes relevant to intestinal inflammation. Here we find that loss of Irgm1 confers marked susceptibility to Citrobacter rodentium, a noninvasive intestinal pathogen that models inflammatory responses to intestinal bacteria. Irgm1-deficient mice fail to control C. rodentium outgrowth in the intestine, leading to systemic pathogen spread and host mortality. Surprisingly, susceptibility due to loss of Irgm1 function was not linked to defective intracellular killing of C. rodentium or exaggerated inflammation, but was instead linked to failure to remodel specific colon lamina propria (C-LP) myeloid cells that expand in response to C. rodentium infection and are essential for C. rodentium immunity. Defective immune remodeling was most striking in C-LP monocytes, which were successfully recruited to the infected C-LP, but subsequently underwent apoptosis. Apoptotic susceptibility was induced by C. rodentium infection and was specific to this setting of pathogen infection, and was not apparent in other settings of intestinal inflammation. These studies reveal a novel role for Irgm1 in host defense and suggest that deficiencies in survival and remodeling of C-LP myeloid cells that control inflammatory intestinal bacteria may underpin IBD pathogenesis linked to IRGM dysfunction.


Assuntos
Citrobacter rodentium/imunologia , Colo/imunologia , Infecções por Enterobacteriaceae/imunologia , Proteínas de Ligação ao GTP/deficiência , Doenças Inflamatórias Intestinais/imunologia , Monócitos/imunologia , Animais , Colo/microbiologia , Colo/patologia , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/patologia , Proteínas de Ligação ao GTP/imunologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/patologia , Camundongos , Camundongos Knockout , Monócitos/microbiologia , Monócitos/patologia , Membrana Mucosa/imunologia , Membrana Mucosa/microbiologia , Membrana Mucosa/patologia
3.
Nat Commun ; 11(1): 2331, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393780

RESUMO

Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-ß1-transporting vesicles in response to the pathogenic fungus Candida albicans. Soluble ß-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-ß1-transporting vesicles. CR3-dependence is demonstrated using CR3-deficient (CD11b knockout) monocytes generated by CRISPR-CAS9 genome editing and isolated from CR3-deficient (CD11b knockout) mice. These vesicles reduce the pro-inflammatory response in human M1-macrophages as well as in whole blood. Binding of the vesicle-transported TGF-ß1 to the TGF-ß receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-ß1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-ß1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway.


Assuntos
Imunomodulação , Antígeno de Macrófago 1/metabolismo , Monócitos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Apoptose , Candida albicans/metabolismo , Candida albicans/ultraestrutura , Regulação para Baixo , Difusão Dinâmica da Luz , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Monócitos/microbiologia , Monócitos/ultraestrutura , Transporte Proteico , Solubilidade , Transcrição Genética , Regulação para Cima , beta-Glucanas/metabolismo
4.
Immunity ; 52(4): 591-605.e6, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32294405

RESUMO

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.


Assuntos
Endorribonucleases/metabolismo , Monócitos/imunologia , Neutrófilos/imunologia , RNA Bacteriano/metabolismo , RNA de Protozoário/metabolismo , Receptor 8 Toll-Like/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Endorribonucleases/imunologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Escherichia coli/química , Escherichia coli/imunologia , Edição de Genes/métodos , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/imunologia , Monócitos/microbiologia , Monócitos/parasitologia , Neutrófilos/microbiologia , Neutrófilos/parasitologia , Plasmodium falciparum/química , Plasmodium falciparum/imunologia , Cultura Primária de Células , Estabilidade de RNA , RNA Bacteriano/imunologia , RNA de Protozoário/imunologia , Serratia marcescens/química , Serratia marcescens/imunologia , Staphylococcus aureus/química , Staphylococcus aureus/imunologia , Streptococcus/química , Streptococcus/imunologia , Células THP-1 , Receptor 8 Toll-Like/imunologia
5.
mBio ; 11(2)2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317318

RESUMO

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C-DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.IMPORTANCE Ehrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.


Assuntos
Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Basigina/metabolismo , Ehrlichia chaffeensis/fisiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas c-vav/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Animais , Basigina/genética , Humanos , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
6.
Mediators Inflamm ; 2020: 4185273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32089643

RESUMO

Periodontitis is a prevalent chronic inflammatory disease triggered by a synergistic and dysbiotic microbiota present in the oral biofilm. This in vitro study is aimed at evaluating the regulation of cyclooxygenase (COX)2 expression and production by the periodontopathogen Filifactor alocis in human gingival fibroblastic (HGF-1) and monocytic (THP-1) cells and also at investigating the underlying cellular pathway mechanisms. HGF-1 and THP-1 cells were exposed either to F. alocis or to the proinflammatory cytokine tumor necrosis factor alpha (TNFα) for 1 and 2 d to examine the COX2 expression by qPCR. COX2 protein levels were evaluated by ELISA in F. alocis-stimulated cells. Both types of cells were also stimulated with a blocking toll-like receptor (TLR)2 antibody or specific inhibitors against MAPKs. F. alocis significantly (p < 0.05) increased COX2 at both transcriptional and protein levels in both HGF-1 and THP-1 cells. Moreover, the stimulatory effect of F. alocis on COX2 was more pronounced in HGF-1 cells in comparison to THP-1 cells. F. alocis upregulated the COX2 expression in a dose-dependent manner in both type cells at 1 d. TNFα also significantly (p < 0.05) increased the COX2 expression in both cells. After preincubation of HGF-1 and THP-1 cells either with a neutralizing anti-TLR2 antibody or with specific MAPK inhibitors, the F. alocis-upregulated COX2 expression was significantly (p < 0.05) suppressed at 1 d. Our in vitro study provides original evidence that F. alocis stimulates COX2 production in fibroblastic and monocytic cells through TLR2 and MAPK mechanisms, suggesting a role of this periodontopathogen in the etiopathogenesis of periodontitis.


Assuntos
Clostridiales/metabolismo , Ciclo-Oxigenase 2/metabolismo , Fibroblastos/microbiologia , Monócitos/microbiologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo
7.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32041787

RESUMO

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.


Assuntos
Antígenos CD18/metabolismo , Integrinas/metabolismo , Fagócitos/metabolismo , Fagócitos/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , Células CHO , Linhagem Celular , Cricetulus , Camundongos , Monócitos/metabolismo , Monócitos/microbiologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Ligação Proteica/fisiologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Talina/metabolismo , Células Th1
8.
Biochem Biophys Res Commun ; 522(1): 26-32, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31735338

RESUMO

Early secreted antigenic target 6-kDa protein (ESAT6) is an essential virulence factor of Mycobacterium tuberculosis (MTb). However, ESAT6 helped fighting MTb infection according to vaccine studies. It's unclear whether ESAT6 confers protection via enhancing the innate immunity of macrophages, which are the first-line defense against MTb. We profiled the global transcriptional changes and characterized the innate immunity of THP-1 macrophages treated with ESAT6. We found ESAT6 promoted the phagocytosis ability, enhanced reactive oxygen species (ROS) generation and accelerated glucose metabolism in macrophages. Meanwhile, ESAT6 induced a distinctive phenotype of macrophages with a concurrence of pro-inflammatory and anti-inflammatory cytokines and chemokines. ESAT6 increased the expression of HIF1α mRNA and protein. Interfering HIF1α with siRNA defected the capacity of phagocytosis and ROS generation as well as glucose metabolism. Thus, ESAT6 enhanced the protective innate immunity of macrophages partially via HIF1α. This study provided clues for developing therapies against tuberculosis by targeting ESAT6.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Imunidade Inata , Macrófagos/imunologia , Glucose/metabolismo , Humanos , Inflamação , Monócitos/microbiologia , Mycobacterium tuberculosis/metabolismo , Fagocitose , Fenótipo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células THP-1
9.
Infect Genet Evol ; 77: 104077, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669366

RESUMO

Macrophages play a major role in the control and elimination of invading Mycobacterium tuberculosis (Mtb). Long intergenic noncoding RNA erythroid prosurvival (lincRNA-EPS) plays an important role in regulating various biologic processes in macrophages, including inflammatory responses, cell apoptosis, and autophagy. Whereas the effect of lincRNA-EPS in regulating the immune response of macrophages to Mtb is little studied. This study aimed to explore lincRNA-EPS expression in monocytes from patients with active pulmonary tuberculosis (PTB) and from healthy individuals. We also sought to investigate the effect of lincRNA-EPS on Bacillus Calmette-Guérin (BCG)-infected macrophages apoptosis and autophagy. Our study found that lincRNA-EPS expression was down-regulated in the monocytes from patients with active PTB compared with healthy individuals, accompanied by significant attenuated monocyte apoptosis and enhanced autophagy. In vitro, knockdown of lincRNA-EPS inhibited apoptosis and promoted autophagy in BCG-infected RAW264.7 macrophages. Moreover, we revealed that lincRNA-EPS regulated apoptosis and autophagy of BCG-infected RAW264.7 macrophages via JNK/MAPK signaling pathway. In conclusion, our findings demonstrated that knockdown of lincRNA-EPS inhibits apoptosis and enhances autophagy by activating the JNK/MAPK signaling pathway in BCG-infected RAW264.7 macrophages. Suggesting that lincRNA-EPS could serve as a new potential therapeutic target for PTB.


Assuntos
Regulação para Baixo , Macrófagos/microbiologia , Mycobacterium bovis/patogenicidade , RNA Longo não Codificante/genética , Tuberculose Pulmonar/genética , Adulto , Animais , Apoptose , Autofagia , Estudos de Casos e Controles , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Macrófagos/química , Macrófagos/citologia , Masculino , Camundongos , Modelos Biológicos , Monócitos/química , Monócitos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Células RAW 264.7 , Tuberculose Pulmonar/microbiologia
10.
Microbes Infect ; 22(3): 137-143, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31770592

RESUMO

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermally dimorphic fungi of the genus Paracoccidioides that affects predominantly 30-60-year-old male rural workers. The main clinical forms of the disease are acute/subacute, chronic (CF); almost all CF patients develop pulmonary fibrosis, and they also exhibit emphysema due to smoke. An important cytokine in this context, IL-1ß, different from the others, is produced by an intracellular multimolecular complex called inflammasome that is activated by pathogens and/or host signs of damage. Inflammasome has been recognized for its contribution to chronic inflammatory diseases, from that, we hypothesized that this activation could be involved in paracoccidioidomycosis, contributing to chronic inflammation. While inflammasome activation has been demonstrated in experimental models of Paracoccidioides brasiliensis infection, no information is available in patients, leading us to investigate the participation of NLRP3-inflammasome machinery in CF/PCM patients from a Brazilian endemic area. Our findings showed increased priming in mRNA levels of NLRP3 inflammasome genes by monocytes of PCM patients in vitro than healthy controls. Similar intracellular protein expression of NLRP3, CASP-1, ASC, and IL-1ß were also observed in freshly isolated monocytes of PCM patients and smoker controls. Increased expression of NLRP3 and ASC was observed in monocytes from PCM patients under hypoxia in comparison with smoker controls. For the first time, we showed that primed monocytes of CF-PCM patients were associated with enhanced expression of components of NLRP3-inflammasome due to smoke. Also, hypoxemia boosted this machinery. These findings reinforce the systemic low-grade inflammation activation observed in PCM during and after treatment.


Assuntos
Monócitos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Paracoccidioidomicose/imunologia , Fumar , Hipóxia Celular , Humanos , Infecções Fúngicas Invasivas/imunologia , Infecções Fúngicas Invasivas/microbiologia , Pneumopatias Fúngicas/microbiologia , Monócitos/microbiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Paracoccidioides , Paracoccidioidomicose/microbiologia , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/microbiologia
11.
Microb Pathog ; 139: 103910, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31809795

RESUMO

The life cycle of Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), appears to involve interactions with spleen and head kidney macrophages. To develop an in vitro model for studying this, F. psychrophilum was incubated with a rainbow trout splenic monocyte/macrophage-like cell line (RTS11) and fundamental macrophage functions evaluated. The animal cell basal medium, L15, supplemented with bovine serum (FBS) supports RTS11 maintenance, and surprisingly, L15 with 2% FBS (L15/FBS) also supported F. psychrophilum growth. L15/FBS in which the bacteria had been grown is referred to as F. psychrophilum conditioned medium (FpCM). Adding FpCM to RTS11 cultures caused a small, yet significant, percentage of cells to die, many cells to become more diffuse, and phagocytosis to be temporarily reduced. FpCM also significantly stimulated transcript expression for pro-inflammatory cytokines (IL-1ß, TNFα and IL-6) and the anti-inflammatory cytokine (IL-10) after one day of exposure but this upregulation rapidly declined over time. Adding live F. psychrophilum to RTS11 cultures also altered the cellular morphology and stimulated cytokine expression more profoundly than FpCM. Additionally, the phagocytic activity of RTS11 was also significantly impaired by live F. psychrophilum, but not to the same extent as when exposed to FpCM. Adding heat-killed bacteria to RTS11 cultures elicited few changes. These bacteria/RTS11 co-cultures should be useful for gaining a deeper understanding of the pathogenesis of F. psychrophilum and may aid in the development of effective measures to prevent infection and spread of this troublesome disease.


Assuntos
Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/veterinária , Flavobacterium/fisiologia , Macrófagos/microbiologia , Monócitos/microbiologia , Oncorhynchus mykiss/microbiologia , Animais , Linhagem Celular , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/patogenicidade , Interleucina-10/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Oncorhynchus mykiss/imunologia , Baço/imunologia , Baço/microbiologia , Virulência
12.
Immunology ; 159(4): 393-403, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31880316

RESUMO

Signalling lymphocyte activation molecule family member 9 (SLAMF9) is an orphan receptor of the CD2/SLAM family of leucocyte surface proteins. Examination of SLAMF9 expression and function indicates that SLAMF9 promotes inflammation by specialized subsets of antigen-presenting cells. Within healthy liver and circulating mouse peripheral blood mononuclear cells, SLAMF9 is expressed on CD11b+ , Ly6C- , CD11clow , F4/80low , MHC-II+ , CX3 CR1+ mononuclear phagocytes as well as plasmacytoid dendritic cells. In addition, SLAMF9 can be found on peritoneal B1 cells and small (F4/80low ), but not large (F4/80high ), peritoneal macrophages. Upon systemic challenge with Salmonella enterica Typhimurium, Slamf9-/- mice were impaired in their ability to clear the infection from the liver. In humans, SLAMF9 is up-regulated upon differentiation of monocytes into macrophages, and lipopolysaccharide stimulation of PMA-differentiated, SLAMF9 knockdown THP-1 cells showed an essential role of SLAMF9 in production of granulocyte-macrophage colony-stimulating factor, tumour necrosis factor-α, and interleukin-1ß. Taken together, these data implicate SLAMF9 in the initiation of inflammation and clearance of bacterial infection.


Assuntos
Células Dendríticas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Fígado/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/microbiologia , Diferenciação Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/microbiologia , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interações Hospedeiro-Patógeno/genética , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Lipopolissacarídeos/farmacologia , Fígado/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/microbiologia , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária/deficiência , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Células THP-1 , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
13.
Dev Comp Immunol ; 103: 103511, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31580833

RESUMO

ß-defensin is a cationic host defense peptide actively participating in host innate immune response against pathogens. In teleost fish, ß-defensin exhibits a diversity in genotypes and functions. Herein, a ß-defensin homolog (PaBD) was identified from ayu, Plecoglossus altivelis, showing multiple tissues' upregulation against Vibrio anguillarum challenge. In vivo experiments revealed that intraperitoneal injection of chemically synthesized mature PaBD (mPaBD) increased the survival rate of V. anguillarum-infected ayu, accompanied by reduced bacterial load and decreased tissue mRNA levels of tumor necrosis factor α (PaTNF-α) and interleukin 1ß (PaIL-1ß). However, in vitro, mPaBD showed weak bactericidal activity against V. anguillarum. Interestingly, mPaBD enhanced phagocytosis, intracellular bacterial killing, and respiratory burst of ayu monocytes/macrophages (MO/MΦ). Moreover, it inhibited mRNA levels of PaIL-1ß and PaTNF-α in MO/MФ upon V. anguillarum infection. In conclusion, PaBD protects ayu against V. anguillarum challenge not only through its direct antibacterial ability, but also through its immunomodulation in MO/MΦ.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Osmeriformes/imunologia , Vibrioses/veterinária , Vibrio/fisiologia , beta-Defensinas/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Carga Bacteriana/efeitos dos fármacos , Citocinas/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Proteínas de Peixes/administração & dosagem , Proteínas de Peixes/genética , Imunomodulação , Macrófagos/imunologia , Macrófagos/microbiologia , Monócitos/imunologia , Monócitos/microbiologia , Osmeriformes/classificação , Osmeriformes/genética , Fagocitose , Filogenia , Explosão Respiratória , Alinhamento de Sequência , Taxa de Sobrevida , Distribuição Tecidual , Vibrio/efeitos dos fármacos , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrioses/prevenção & controle , beta-Defensinas/administração & dosagem , beta-Defensinas/genética
14.
J Immunol Res ; 2019: 1462098, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815150

RESUMO

Diabetes mellitus, a metabolic disease characterized by hyperglycemia and poor glucose control, is a risk factor for Mycobacterium tuberculosis (M. tuberculosis) infection and the development of active tuberculosis. To evaluate whether M. tuberculosis infection susceptibility is associated with an intrinsic factor in monocytes from type 2 diabetes (T2D) patients or it is associated with hyperglycemia per se, we analyzed TLR-2 and TLR-4 expression by flow cytometry and the cytokines IL-1ß, IL-6, IL-8, IL-10, and TNF-α by cytometric bead array assays, either stimulated with TLR-2 and TLR-4 ligands or infected with M. tuberculosis in the whole blood from T2D patients (n = 43) and healthy subjects (n = 26) or in CD14+ monocytes from healthy subjects cultured in high glucose (HG) (30 mM). The intracellular growth of M. tuberculosis was evaluated by CFU counts at 0, 1, and 3 days in both monocytes from T2D patients and monocytes from healthy subjects cultured in HG. We did not find significant differences in TLR expression, cytokine production, or growth of M. tuberculosis in monocytes from T2D patients compared with those in monocytes from healthy subjects. Despite these results, in vitro assays of monocytes cultured with 30 mM glucose led to significantly increased TLR-2 and TLR-4 basal expression compared to those of monocytes cultured with 11 mM glucose (P < 0.05). Conversely, the production of IL-6 by TLR-2 ligand stimulation, of IL-1ß, IL-6, and IL-8 by TLR-4 ligand stimulation, and of IL-8 by M. tuberculosis infection significantly decreased in monocytes cultured in HG (P < 0.05). Additionally, the intracellular survival of M. tuberculosis increased in monocytes in HG after day 3 of culture (P < 0.05). In conclusion, HG decreased IL-8 production and the intracellular growth control of M. tuberculosis by monocytes, supporting the hypothesis that hyperglycemia plays an important role in the impaired immune responses to M. tuberculosis in patients with T2D.


Assuntos
Diabetes Mellitus Tipo 2/imunologia , Glucose/farmacologia , Hiperglicemia/imunologia , Monócitos/imunologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose Pulmonar/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/microbiologia , Hiperglicemia/patologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Lipoproteínas/farmacologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/microbiologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Cultura Primária de Células , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
15.
PLoS One ; 14(12): e0226778, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877174

RESUMO

Francisella tularensis, a category-A bioterrorism agent causes tularemia. F. tularensis suppresses the immune response of host cells and intracellularly proliferates. However, the detailed mechanisms of immune suppression and intracellular growth are largely unknown. Here we developed a transposon mutant library to identify novel pathogenic factors of F. tularensis. Among 750 transposon mutants of F. tularensis subsp. novicida (F. novicida), 11 were isolated as less cytotoxic strains, and the genes responsible for cytotoxicity were identified. Among them, the function of slt, which encodes soluble lytic transglycosylase (SLT) was investigated in detail. An slt deletion mutant (Δslt) was less toxic to the human monocyte cell line THP-1 vs the wild-type strain. Although the wild-type strain proliferated in THP-1 cells, the number of intracellular Δslt mutant decreased in comparison. The Δslt mutant escaped from phagosomes during the early stages of infection, but the mutant was detected within the autophagosome, followed by degradation in lysosomes. Moreover, the Δslt mutant induced host cells to produce high levels of cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß, compared with the wild-type strain. These results suggest that the SLT of F. novicida is required for immune suppression and escape from autophagy to allow its survival in host cells.


Assuntos
Proteínas de Bactérias/imunologia , Francisella tularensis/imunologia , Glicosiltransferases/imunologia , Tularemia/imunologia , Animais , Linhagem Celular , Francisella tularensis/crescimento & desenvolvimento , Humanos , Evasão da Resposta Imune , Lisossomos/imunologia , Lisossomos/microbiologia , Camundongos , Monócitos/imunologia , Monócitos/microbiologia , Fagossomos/imunologia , Fagossomos/microbiologia , Tularemia/microbiologia
16.
Pol J Microbiol ; 68(4): 439-447, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880888

RESUMO

Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes - Typhimurium, Enteritidis, and Choleraesuis - in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy - significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes ­ Typhimurium, Enteritidis, and Choleraesuis ­ in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy ­ significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.


Assuntos
Aneurisma Aórtico/microbiologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Animais , Aneurisma Aórtico/genética , Aneurisma Aórtico/imunologia , Linhagem Celular , Humanos , Interferon-alfa/genética , Interferon-alfa/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Antígeno Ki-1/genética , Antígeno Ki-1/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Monócitos/imunologia , Monócitos/microbiologia , Plasmídeos/genética , Plasmídeos/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Sorogrupo , Virulência
17.
Sci Rep ; 9(1): 15779, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673005

RESUMO

The gingival epithelium is a physical and immunological barrier to the microbiota of the oral cavity, which interact through soluble mediators with the immune cells that patrol the tissue at the gingival epithelium. We sought to develop a three-dimensional gingivae-biofilm interface model using a commercially available gingival epithelium to study the tissue inflammatory response to oral biofilms associated with "health", "gingivitis" and "periodontitis". These biofilms were developed by sequential addition of microorganisms to mimic the formation of supra- and sub-gingival plaque in vivo. Secondly, to mimic the interactions between gingival epithelium and immune cells in vivo, we integrated peripheral blood mononuclear cells and CD14+ monocytes into our three-dimensional model and were able to assess the inflammatory response in the immune cells cultured with and without gingival epithelium. We describe a differential inflammatory response in immune cells cultured with epithelial tissue, and more so following incubation with epithelium stimulated by "gingivitis-associated" biofilm. These results suggest that gingival epithelium-derived soluble mediators may control the inflammatory status of immune cells in vitro, and therefore targeting of the epithelial response may offer novel therapies. This multi-cellular interface model, both of microbial and host origin, offers a robust in vitro platform to investigate host-pathogens at the epithelial surface.


Assuntos
Bactérias/imunologia , Fenômenos Fisiológicos Bacterianos , Biofilmes/crescimento & desenvolvimento , Gengiva , Mediadores da Inflamação/imunologia , Monócitos , Mucosa Bucal , Técnicas de Cocultura , Gengiva/imunologia , Gengiva/microbiologia , Gengiva/patologia , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Monócitos/imunologia , Monócitos/microbiologia , Monócitos/patologia , Mucosa Bucal/imunologia , Mucosa Bucal/microbiologia , Mucosa Bucal/patologia
18.
Sci Rep ; 9(1): 16868, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727989

RESUMO

Septic arthritis is one of the most aggressive joint diseases. Although caused predominantly by S. aureus, Gram-negative bacteria, Pseudomonas aeruginosa among them, account for a significant percentage of the causal agents of septic arthritis. However, septic arthritis caused by P. aeruginosa has not been studied thus far, due to lack of an animal model. NMRI mice were inoculated with different doses of P. aeruginosa. The clinical course of septic arthritis and radiological changes of joints were examined. Furthermore, the host molecular and cellular mechanisms involved in P. aeruginosa-induced septic arthritis were investigated. Inoculation of mice with P. aeruginosa caused septic arthritis in a dose-dependent manner. Neutrophil depletion led to higher mortality and more severe joint destruction (p < 0.01). In contrast, monocyte depletion resulted in higher mortality (p < 0.05) but similar arthritis severity compared to controls. Mice depleted of CD4+ T-cells inoculated with P. aeruginosa displayed less severe bone damage (p < 0.05). For the first time, a mouse model for P. aeruginosa septic arthritis is presented. Our data demonstrate that neutrophils play a protective role in P. aeruginosa septic arthritis. Monocytes/macrophages, on the other hand, are only essential in preventing P. aeruginosa-induced mortality. Finally, CD4+ T-cells are pathogenic in P. aeruginosa septic arthritis.


Assuntos
Artrite Infecciosa/patologia , Modelos Animais de Doenças , Articulações/patologia , Neutropenia/patologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/patogenicidade , Animais , Artrite Infecciosa/imunologia , Artrite Infecciosa/microbiologia , Artrite Infecciosa/mortalidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/patologia , Feminino , Humanos , Articulações/imunologia , Articulações/microbiologia , Contagem de Leucócitos , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos , Monócitos/imunologia , Monócitos/microbiologia , Monócitos/patologia , Neutropenia/imunologia , Neutropenia/microbiologia , Neutropenia/mortalidade , Neutrófilos/imunologia , Neutrófilos/microbiologia , Neutrófilos/patologia , Especificidade de Órgãos , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/imunologia , Índice de Gravidade de Doença , Análise de Sobrevida
19.
Life Sci Alliance ; 2(5)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31585982

RESUMO

The type II IFN (IFNγ) enhances antimicrobial activity yet also drives expression of genes that amplify inflammatory responses. Hence, excessive IFNγ stimulation can be pathogenic. Here, we describe a previously unappreciated mechanism whereby IFNγ itself dampens myeloid cell activation. Staining of monocytes from Listeria monocytogenes-infected mice provided evidence of type I IFN-independent reductions in IFNGR1. IFNγ was subsequently found to reduce surface IFNGR1 on cultured murine myeloid cells and human CD14+ peripheral blood mononuclear cells. IFNγ-driven reductions in IFNGR1 were not explained by ligand-induced receptor internalization. Rather, IFNγ reduced macrophage Ifngr1 transcription by altering chromatin structure at putative Ifngr1 enhancer sites. This is a distinct mechanism from that used by type I IFNs. Ligand-induced reductions in IFNGR1 altered myeloid cell sensitivity to IFNγ, blunting activation of STAT1 and 3. Our data, thus, reveal a mechanism by which IFNGR1 abundance and myeloid cell sensitivity to IFNγ can be modulated in the absence of type I IFNs. Multiple mechanisms, thus, exist to calibrate macrophage IFNGR1 abundance, likely permitting the fine tuning of macrophage activation and inflammation.


Assuntos
Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Listeria monocytogenes/imunologia , Células Mieloides/imunologia , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Animais , Antígenos CD4/metabolismo , Células Cultivadas , Cromatina/química , Cromatina/genética , Elementos Facilitadores Genéticos , Feminino , Humanos , Ligantes , Masculino , Camundongos , Monócitos/imunologia , Monócitos/microbiologia , Células Mieloides/citologia , Transcrição Genética
20.
Elife ; 82019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31637998

RESUMO

Monocyte counts are increased during human tuberculosis (TB) but it has not been determined whether Mycobacterium tuberculosis (Mtb) directly regulates myeloid commitment. We demonstrated that exposure to Mtb directs primary human CD34+ cells to differentiate into monocytes/macrophages. In vitro myeloid conversion did not require type I or type II IFN signaling. In contrast, Mtb enhanced IL-6 responses by CD34+ cell cultures and IL-6R neutralization inhibited myeloid differentiation and decreased mycobacterial growth in vitro. Integrated systems biology analysis of transcriptomic, proteomic and genomic data of large data sets of healthy controls and TB patients established the existence of a myeloid IL-6/IL6R/CEBP gene module associated with disease severity. Furthermore, genetic and functional analysis revealed the IL6/IL6R/CEBP gene module has undergone recent evolutionary selection, including Neanderthal introgression and human pathogen adaptation, connected to systemic monocyte counts. These results suggest Mtb co-opts an evolutionary recent IFN-IL6-CEBP feed-forward loop, increasing myeloid differentiation linked to severe TB in humans.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Interferons/metabolismo , Interleucina-6/metabolismo , Monócitos/metabolismo , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Antígenos CD34 , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Hidrolases , Interferons/genética , Interleucina-6/genética , Macrófagos/microbiologia , Monócitos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Células Mieloides/fisiologia , Proteômica , Receptores de Interleucina-6 , Índice de Gravidade de Doença , Transcriptoma , Tuberculose/metabolismo
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