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1.
mBio ; 11(3)2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576678

RESUMO

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.


Assuntos
Betacoronavirus/imunologia , Antígeno CD47/metabolismo , Imunomodulação/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Células A549 , Imunidade Adaptativa/imunologia , Animais , Antígeno CD47/genética , Linhagem Celular Tumoral , Citocinas/imunologia , Feminino , Humanos , Imunidade Inata/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/imunologia , SARS-CoV-2 , Regulação para Cima/imunologia
2.
J Exp Med ; 216(7): 1615-1629, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31092533

RESUMO

Streptococcus pyogenes (Group A streptococcus; GAS) is a human pathogen causing diseases from uncomplicated tonsillitis to life-threatening invasive infections. GAS secretes EndoS, an endoglycosidase that specifically cleaves the conserved N-glycan on IgG antibodies. In vitro, removal of this glycan impairs IgG effector functions, but its relevance to GAS infection in vivo is unclear. Using targeted mass spectrometry, we characterized the effects of EndoS on host IgG glycosylation during the course of infections in humans. Substantial IgG glycan hydrolysis occurred at the site of infection and systemically in the severe cases. We demonstrated decreased resistance to phagocytic killing of GAS lacking EndoS in vitro and decreased virulence in a mouse model of invasive infection. This is the first described example of specific bacterial IgG glycan hydrolysis during infection and thereby verifies the hypothesis that EndoS modifies antibodies in vivo. This mechanisms of immune evasion could have implications for treatment of severe GAS infections and for future efforts at vaccine development.


Assuntos
Imunidade Adaptativa/imunologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Imunoglobulina G/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Feminino , Glicosilação , Humanos , Hidrólise , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Limite de Detecção , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/enzimologia , Tonsilite/imunologia , Tonsilite/microbiologia
3.
Nanotoxicology ; 10(6): 680-8, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26573343

RESUMO

Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.


Assuntos
Antituberculosos/uso terapêutico , Nanopartículas/química , Rifampina/uso terapêutico , Tioridazina/uso terapêutico , Tuberculose/tratamento farmacológico , Peixe-Zebra , Animais , Antituberculosos/química , Antituberculosos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Humanos , Ácido Láctico/química , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Rifampina/química , Rifampina/toxicidade , Tioridazina/química , Tioridazina/toxicidade , Tuberculose/microbiologia , Peixe-Zebra/microbiologia
4.
Infect Immun ; 82(9): 3790-801, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24958712

RESUMO

The polysaccharide capsule surrounding Streptococcus pneumoniae is essential for virulence. Recently, Streptococcus mitis, a human commensal and a close relative of S. pneumoniae, was also shown to have a capsule. In this study, the S. mitis type strain switched capsule by acquisition of the serotype 4 capsule locus of S. pneumoniae TIGR4, following induction of competence for natural transformation. Comparison of the wild type with the capsule-switching mutant and with a capsule deletion mutant showed that the capsule protected S. mitis against phagocytosis by RAW 264.7 macrophages. This effect was enhanced in the S. mitis strain expressing the S. pneumoniae capsule, which showed, in addition, increased resistance against early clearance in a mouse model of lung infection. Expression of both capsules also favored survival in human blood, and the effect was again more pronounced for the capsule-switching mutant. S. mitis survival in horse blood or in a mouse model of bacteremia was not significantly different between the wild type and the mutant strains. In all models, S. pneumoniae TIGR4 showed higher rates of survival than the S. mitis type strain or the capsule-switching mutant, except in the lung model, in which significant differences between S. pneumoniae TIGR4 and the capsule-switching mutant were not observed. Thus, we identified conditions that showed a protective function for the capsule in S. mitis. Under such conditions, S. mitis resistance to clearance could be enhanced by capsule switching to serotype 4, but it was enhanced to levels lower than those for the virulent strain S. pneumoniae TIGR4.


Assuntos
Cápsulas Bacterianas/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus mitis/imunologia , Animais , Bacteriemia/imunologia , Bacteriemia/microbiologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Cavalos/imunologia , Cavalos/microbiologia , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologia , Sorotipagem , Infecções Estreptocócicas/microbiologia , Streptococcus pneumoniae/imunologia , Virulência/imunologia
5.
J Cell Sci ; 126(Pt 14): 3043-54, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23687375

RESUMO

Nanoparticles (NPs) are increasingly used as biodegradable vehicles to selectively deliver therapeutic agents such as drugs or antigens to cells. The most widely used vehicle for this purpose is based on copolymers of lactic acid and glycolic acid (PLGA) and has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we describe fabrication of PLGA NPs containing either a high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was twofold: first to resolve the controversial issue of whether, after phagocytic uptake, PLGA NPs remain membrane-bound or whether they escape into the cytoplasm, as has been widely claimed. Second, we sought to make NPs that enclosed sufficient rifampicin to efficiently clear macrophages of infection with Mycobacterium bovis BCG. Using fluorescence microscopy and immuno-electron microscopy, in combination with markers for lysosomes, we show that BCG bacteria, as expected, localized to early phagosomes, but that at least 90% of PLGA particles were targeted to, and remained in, low pH, hydrolase-rich phago-lysosomes. Our data collectively argue that PLGA NPs remain membrane-enclosed in macrophages for at least 13 days and degrade slowly. Importantly, provided that the NPs are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9-12 days of treatment, as shown by estimates of the number of bacterial colonies in vitro.


Assuntos
Antibióticos Antituberculose/administração & dosagem , Portadores de Fármacos/química , Ácido Láctico , Macrófagos/microbiologia , Mycobacterium bovis/efeitos dos fármacos , Nanopartículas/química , Ácido Poliglicólico , Rifampina/administração & dosagem , Animais , Linhagem Celular , Membrana Celular/metabolismo , Contagem de Colônia Microbiana , Feminino , Masculino , Camundongos , Fagossomos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
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