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1.
Front Cell Infect Microbiol ; 11: 676456, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34381738

RESUMO

Mycobacterium tuberculosis (Mtb) inhibits autophagy to promote its survival in host cells. However, the molecular mechanisms by which Mtb inhibits autophagy are poorly understood. Here, we report a previously unknown mechanism in which Mtb phosphoribosyltransferase (MtbPRT) inhibits autophagy in an mTOR, negative regulator of autophagy, independent manner by inducing histone hypermethylation (H3K9me2/3) at the Atg5 and Atg7 promoters by activating p38-MAPK- and EHMT2 methyltransferase-dependent signaling pathways. Additionally, we find that MtbPRT induces EZH2 methyltransferase-dependent H3K27me3 hypermethylation and reduces histone acetylation modifications (H3K9ac and H3K27ac) by upregulating histone deacetylase 3 to inhibit autophagy. In summary, this is the first demonstration that Mtb inhibits autophagy by inducing histone hypermethylation in autophagy-related genes to promote intracellular bacterial survival.


Assuntos
Histonas , Macrófagos/microbiologia , Mycobacterium tuberculosis , Pentosiltransferases/metabolismo , Autofagia , Histonas/metabolismo , Macrófagos/metabolismo , Metilação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Transdução de Sinais
2.
Methods Mol Biol ; 2341: 133-140, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264469

RESUMO

The use of cultured mammalian cells, whether immortalized cell lines or primary cells, is a well-known technique used as a substitute or prescreen for in vivo virulence potential of bacterial pathogens. This technique is also a way to examine host-pathogen interactions in a less complex environment compared to that found in whole animals. To this end, macrophage infection assays have become a key technique for studying the molecular mechanisms by which bacteria interact with the host. Herein, this chapter describes both how to produce macrophages from mouse bone marrow and the subsequent infection assays.


Assuntos
Técnicas de Cocultura/métodos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Animais , Linhagem Celular , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Células RAW 264.7
3.
FEBS Lett ; 595(16): 2160-2168, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34216493

RESUMO

The persistence of Helicobacter pylori in the human gastric mucosa implies that the immune response fails to clear the infection. We found that H. pylori compromises the antigen presentation ability of macrophages, because of the decline of the presenting molecules HLA-II. Here, we reveal that the main bacterial factor responsible for this effect is ADP-heptose, an intermediate metabolite in the biosynthetic pathway of lipopolysaccharide (LPS) that elicits a pro-inflammatory response in gastric epithelial cells. In macrophages, it upregulates the expression of miR146b which, in turn, would downmodulate CIITA, the master regulator for HLA-II genes. Hence, H. pylori, utilizing ADP-heptose, exploits a specific arm of macrophage response to establish its survival niche in the face of the immune defense elicited in the gastric mucosa.


Assuntos
Apresentação do Antígeno/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Helicobacter pylori/fisiologia , Heptoses/farmacologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Macrófagos/efeitos dos fármacos , Helicobacter pylori/metabolismo , Heptoses/química , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas Nucleares/metabolismo , Transativadores/metabolismo
4.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299184

RESUMO

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.


Assuntos
4-Butirolactona/análogos & derivados , Acinetobacter/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Homosserina/análogos & derivados , Macrófagos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , 4-Butirolactona/farmacologia , Acinetobacter/isolamento & purificação , Acinetobacter/patogenicidade , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Feminino , Homosserina/farmacologia , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Mitocôndrias/metabolismo , Percepção de Quorum , Espécies Reativas de Oxigênio/metabolismo , Fatores de Virulência/farmacologia
5.
Methods Mol Biol ; 2314: 167-182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235652

RESUMO

Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages, HoxB8 conditionally immortalized myeloid cells, Max Planck Institute alveolar macrophage-like cells, and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.


Assuntos
Macrófagos Alveolares/microbiologia , Macrófagos/microbiologia , Imagem Molecular/métodos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células Mieloides/microbiologia , Animais , Células Cultivadas , Humanos , Técnicas In Vitro , Macrófagos/patologia , Macrófagos Alveolares/patologia , Camundongos , Mycobacterium tuberculosis/patogenicidade , Células Mieloides/patologia
6.
Methods Mol Biol ; 2314: 261-271, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235657

RESUMO

Flow cytometry enables the measurement of tens of features on individual cells from complex mixtures. Flow cytometry enables high-throughput quantification of cell size, gene and protein expression. In the case of studies of host-pathogen interactions, this tool provides a facile way of identifying cells that have been successfully infected by a pathogen. Several recent technological advances have greatly improved throughput and the number of features that can be simultaneously monitored by this technique. Here, we describe common workflows to study Mycobacterium tuberculosis heterogeneity and host-M. tuberculosis interactions using flow cytometry and related technologies.


Assuntos
Citometria de Fluxo/métodos , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Macrófagos/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium tuberculosis/patogenicidade , Humanos , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/patologia
7.
Methods Mol Biol ; 2314: 649-702, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235675

RESUMO

Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully understood and require further investigations. Therefore, increasing the knowledge of this process will help to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances in imaging techniques and images-based analysis allow the rapid quantification of biological objects in complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to track the bacillus within host cell settings.


Assuntos
Células Dendríticas/microbiologia , Células Epiteliais/microbiologia , Ensaios de Triagem em Larga Escala/métodos , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fagócitos/microbiologia , Tuberculose/microbiologia , Animais , Células Dendríticas/metabolismo , Testes Diagnósticos de Rotina , Células Epiteliais/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Mycobacterium tuberculosis/patogenicidade , Estresse Oxidativo , Fagócitos/metabolismo , Espécies Reativas de Oxigênio , Tuberculose/metabolismo
8.
Nat Immunol ; 22(7): 839-850, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34168371

RESUMO

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.


Assuntos
Hanseníase Virchowiana/imunologia , Hanseníase Tuberculoide/imunologia , Mycobacterium leprae/imunologia , Pele/imunologia , Adolescente , Adulto , Idoso , Feminino , Fibroblastos/imunologia , Fibroblastos/microbiologia , Fibroblastos/patologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Queratinócitos/imunologia , Queratinócitos/microbiologia , Queratinócitos/patologia , Hanseníase Virchowiana/genética , Hanseníase Virchowiana/microbiologia , Hanseníase Virchowiana/patologia , Hanseníase Tuberculoide/genética , Hanseníase Tuberculoide/microbiologia , Hanseníase Tuberculoide/patologia , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/patogenicidade , RNA-Seq , Análise de Célula Única , Pele/microbiologia , Pele/patologia , Linfócitos T/imunologia , Linfócitos T/microbiologia , Linfócitos T/patologia , Transcriptoma
9.
Nat Commun ; 12(1): 3816, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155215

RESUMO

To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.


Assuntos
Antituberculosos/farmacologia , Citosol/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Antituberculosos/farmacocinética , Diarilquinolinas/farmacocinética , Diarilquinolinas/farmacologia , Sinergismo Farmacológico , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Microscopia Eletrônica , Mutação , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Pirazinamida/farmacocinética , Sistemas de Secreção Tipo VII/genética
10.
Res Vet Sci ; 138: 1-10, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34087563

RESUMO

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.


Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Feminino , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Virulência/genética , Fatores de Virulência/metabolismo
11.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068595

RESUMO

Systemic inflammation, from gut translocation of organismal molecules, might worsen uremic complications in acute kidney injury (AKI). The monitoring of gut permeability integrity and/or organismal molecules in AKI might be clinically beneficial. Due to the less prominence of Candida albicans in human intestine compared with mouse gut, C. albicans were orally administered in bilateral nephrectomy (BiN) mice. Gut dysbiosis, using microbiome analysis, and gut permeability defect (gut leakage), which was determined by fluorescein isothiocyanate-dextran and intestinal tight-junction immunofluorescent staining, in mice with BiN-Candida was more severe than BiN without Candida. Additionally, profound gut leakage in BiN-Candida also resulted in gut translocation of lipopolysaccharide (LPS) and (1→3)-ß-D-glucan (BG), the organismal components from gut contents, that induced more severe systemic inflammation than BiN without Candida. The co-presentation of LPS and BG in mouse serum enhanced inflammatory responses. As such, LPS with Whole Glucan Particle (WGP, a representative BG) induced more severe macrophage responses than LPS alone as determined by supernatant cytokines and gene expression of downstream signals (NFκB, Malt-1 and Syk). Meanwhile, WGP alone did not induced the responses. In parallel, WGP (with or without LPS), but not LPS alone, accelerated macrophage ATP production (extracellular flux analysis) through the upregulation of genes in mitochondria and glycolysis pathway (using RNA sequencing analysis), without the induction of cell activities. These data indicated a WGP pre-conditioning effect on cell energy augmentation. In conclusion, Candida in BiN mice accelerated gut translocation of BG that augmented cell energy status and enhanced pro-inflammatory macrophage responses. Hence, gut fungi and BG were associated with the enhanced systemic inflammation in acute uremia.


Assuntos
Injúria Renal Aguda/metabolismo , Candida albicans/metabolismo , Inflamação/sangue , Proteoglicanas/sangue , Injúria Renal Aguda/genética , Injúria Renal Aguda/microbiologia , Animais , Candida/metabolismo , Candida albicans/patogenicidade , Disbiose/sangue , Metabolismo Energético , Humanos , Inflamação/microbiologia , Inflamação/patologia , Inflamação/cirurgia , Lipopolissacarídeos/sangue , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Microbiota/genética , Nefrectomia/efeitos adversos
12.
Commun Biol ; 4(1): 627, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035436

RESUMO

Patients with Crohn's disease exhibit abnormal colonization of the intestine by adherent invasive E. coli (AIEC). They adhere to epithelial cells, colonize them and survive inside macrophages. It appeared recently that AIEC LF82 adaptation to phagolysosomal stress involves a long lag phase in which many LF82 cells become antibiotic tolerant. Later during infection, they proliferate in vacuoles and form colonies harboring dozens of LF82 bacteria. In the present work, we investigated the mechanism sustaining this phase of growth. We found that intracellular LF82 produced an extrabacterial matrix that acts as a biofilm and controls the formation of LF82 intracellular bacterial communities (IBCs) for several days post infection. We revealed the crucial role played by the pathogenicity island encoding the yersiniabactin iron capture system to form IBCs and for optimal LF82 survival. These results illustrate that AIECs use original strategies to establish their replicative niche within macrophages.


Assuntos
Doença de Crohn/microbiologia , Células Epiteliais/microbiologia , Fagossomos/metabolismo , Animais , Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Doença de Crohn/metabolismo , Células Epiteliais/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/patogenicidade , Infecções por Escherichia coli/metabolismo , Microbioma Gastrointestinal , Humanos , Mucosa Intestinal/microbiologia , Intestinos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Fagossomos/fisiologia , Células RAW 264.7
13.
Microb Pathog ; 158: 104969, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34044047

RESUMO

Burkholderia pseudomallei is the etiological agent of melioidosis, which is an emerging infectious disease endemic to many tropical regions. Autophagy is an intrinsic cellular process that degrades cytoplasmic components and plays an important role in protecting the host against pathogens. Like many intracellular pathogens, B. pseudomallei can evade the autophagy-dependent cellular clearance. However, the underlying mechanism remains unclear. In this study, we applied a combination of multiple assays to monitor autophagy processes and found that B. pseudomallei induced an incomplete autophagic flux and eliminate autophagy clearance in macrophages by blocking autophagosome-lysosome fusion. Based on a high-throughput microarray screening, we found that LIPA (lysosomal acid LIPAse A) was downregulated during B. pseudomallei infection. MiR-146a was then identified to be specifically upregulated upon infection with B. pseudomallei and further regulated LIPA expression by interacting with 3'UTR of LIPA. Furthermore, overexpression of miR-146a contributed to the defect of autophagic flux caused by B. pseudomallei and was beneficial for the survival of B. pseudomallei in macrophages. Therefore, our findings suggest that miR-146a inhibits autophagy via posttranscriptional suppression of LIPA expression to maintain B. pseudomallei survival in macrophages.


Assuntos
Burkholderia pseudomallei , Macrófagos/microbiologia , Melioidose , MicroRNAs , Esterol Esterase , Animais , Autofagia , Burkholderia pseudomallei/genética , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , Células RAW 264.7
14.
Commun Biol ; 4(1): 548, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972668

RESUMO

Mitochondrial function and innate immunity are intimately linked; however, the mechanisms how mitochondrion-shaping proteins regulate innate host defense remains largely unknown. Herein we show that mitofusin-2 (MFN2), a mitochondrial fusion protein, promotes innate host defense through the maintenance of aerobic glycolysis and xenophagy via hypoxia-inducible factor (HIF)-1α during intracellular bacterial infection. Myeloid-specific MFN2 deficiency in mice impaired the antimicrobial and inflammatory responses against mycobacterial and listerial infection. Mechanistically, MFN2 was required for the enhancement of inflammatory signaling through optimal induction of aerobic glycolysis via HIF-1α, which is activated by mitochondrial respiratory chain complex I and reactive oxygen species, in macrophages. MFN2 did not impact mitophagy during infection; however, it promoted xenophagy activation through HIF-1α. In addition, MFN2 interacted with the late endosomal protein Rab7, to facilitate xenophagy during mycobacterial infection. Our findings reveal the mechanistic regulations by which MFN2 tailors the innate host defense through coordinated control of immunometabolism and xenophagy via HIF-1α during bacterial infection.


Assuntos
Infecções Bacterianas/imunologia , GTP Fosfo-Hidrolases/fisiologia , Glicólise , Imunidade Inata/imunologia , Macroautofagia , Macrófagos/imunologia , Mitocôndrias/imunologia , Animais , Bactérias/crescimento & desenvolvimento , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
15.
Commun Biol ; 4(1): 520, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947954

RESUMO

During infectious diseases, small subpopulations of bacterial pathogens enter a non-replicating (NR) state tolerant to antibiotics. After phagocytosis, intracellular Salmonella enterica serovar Typhimurium (STM) forms persisters able to subvert immune defenses of the host. Physiological state and sensing properties of persisters are difficult to analyze, thus poorly understood. Here we deploy fluorescent protein reporters to detect intracellular NR persister cells, and to monitor their stress response on single cell level. We determined metabolic properties of NR STM during infection and demonstrate that NR STM persisters sense their environment and respond to stressors. Since persisters showed a lower stress response compared to replicating (R) STM, which was not consequence of lower metabolic capacity, the persistent state of STM serves as protective niche. Up to 95% of NR STM were metabolically active at beginning of infection, very similar to metabolic capacity of R STM. Sensing and reacting to stress with constant metabolic activity supports STM to create a more permissive environment for recurrent infections. Stress sensing and response of persister may be targeted by new antimicrobial approaches.


Assuntos
Antibacterianos/farmacologia , Macrófagos/imunologia , Infecções por Salmonella/imunologia , Salmonella enterica/crescimento & desenvolvimento , Estresse Fisiológico , Animais , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/microbiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética
16.
Front Immunol ; 12: 573266, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34046027

RESUMO

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.


Assuntos
Proteínas de Bactérias/metabolismo , Epigênese Genética , Perfilação da Expressão Gênica , Macrófagos/metabolismo , Streptococcus pneumoniae/metabolismo , Estreptolisinas/metabolismo , Proteínas de Bactérias/genética , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Histonas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Metilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteômica/métodos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/fisiologia , Estreptolisinas/genética
17.
FASEB J ; 35(6): e21543, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34046950

RESUMO

Clinically, bone destruction caused by Mycobacterium tuberculosis was serious especially in patients with vitamin D (VD) deficiency. However, the role of VD in M. tuberculosis-induced bone destruction remains clear. In this context, we investigate the role of VD and vitamin D receptor (VDR) in the M. tuberculosis-induced bone destruction. First, we infected RAW264.7 and bone marrow-derived macrophages (BMMs) with Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) in vitro. Then, we activated VDR through VD administration. TRAP and FAK staining, bone resorption assays, immunofluorescence staining, qPCR, and western blot were carried out. In vivo, the M. tuberculosis-induced osteolytic model on the murine skull was established and the µCT and histological analyses were performed. We found that VDR and TRAP were upregulated in bone tuberculosis tissue and proved that M. tuberculosis infection promoted osteoclastogenesis in RAW264.7 and BMMs. VD could inhibit osteoclasts differentiation, fusion, and bone resorption dose-dependently. However, when VDR was knocked down, the inhibitory effect of VD on osteoclasts disappeared. In mechanism, activation of VDR inhibits the phosphorylation of IκB α, thereby inhibiting NFκB signaling pathway and alleviating osteoclastogenesis. Furthermore, in the skull osteolysis model, VD administration reduced osteolysis, but not in VDR-/- mice. Our study, for the first time, demonstrates that activation of VDR by VD administration inhibits M. tuberculosis-induced bone destruction. Our results reveal that VD and VDR are potential therapeutic targets for M. tuberculosis-induced bone destruction, and are of great clinical significance for the development of new therapeutic strategies.


Assuntos
Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , NF-kappa B/antagonistas & inibidores , Osteólise/prevenção & controle , Receptores de Calcitriol/metabolismo , Tuberculose/complicações , Vitamina D/administração & dosagem , Animais , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Osteólise/etiologia , Osteólise/metabolismo , Osteólise/patologia , Receptores de Calcitriol/genética , Tuberculose/microbiologia , Vitaminas/administração & dosagem
18.
Front Immunol ; 12: 624360, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33841405

RESUMO

The gut-liver axis has been increasingly recognized as a major autoimmunity modulator. However, the implications of intestinal barrier in the pathogenesis of autoimmune hepatitis (AIH) remain elusive. Here, we investigated the functional role of gut barrier and intestinal microbiota for hepatic innate immune response in AIH patients and murine models. In this study, we found that AIH patients displayed increased intestinal permeability and pronounced RIP3 activation of liver macrophages. In mice models, intestinal barrier dysfunction increased intestinal bacterial translocation, thus amplifying the hepatic RIP3-mediated innate immune response. Furthermore, GSK872 dampened RIP3 activation and ameliorated the activation and accumulation of liver macrophages in vitro and in vivo experiments. Strikingly, broad-spectrum antibiotic ablation significantly alleviated RIP3 activation and liver injury, highlighting the causal role of intestinal microbiota for disease progression. Our results provided a potentially novel mechanism of immune tolerance breakage in the liver via the gut-liver axis. In addition, we also explored the therapeutic and research potentials of regulating the intestinal microbiota for the therapy of AIH.


Assuntos
Microbioma Gastrointestinal , Hepatite Autoimune/enzimologia , Intestinos/microbiologia , Fígado/enzimologia , Ativação de Macrófagos , Macrófagos/enzimologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Idoso , Animais , Antibacterianos/farmacologia , Translocação Bacteriana , Células CACO-2 , Estudos de Casos e Controles , Modelos Animais de Doenças , Disbiose , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Hepatite Autoimune/imunologia , Hepatite Autoimune/microbiologia , Hepatite Autoimune/prevenção & controle , Humanos , Imunidade Inata , Macrófagos do Fígado/enzimologia , Macrófagos do Fígado/imunologia , Macrófagos do Fígado/microbiologia , Fígado/imunologia , Fígado/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Permeabilidade , Células RAW 264.7 , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais
19.
Front Immunol ; 12: 625586, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33841407

RESUMO

Background: Helicobacter pylori infection is the main cause of chronic gastritis in children. Little is known about the effect of Helicobacter pylori on microbiota and immunity. This study was aimed at characterizing stomach microbiota and immune-regulatory properties of children with Helicobacter pylori colonization. Methods: We studied 122 children who had undergone gastric endoscopy due to gastrointestinal symptoms, 57 were diagnosed with Helicobacter pylori infection. Endoscopic mucosal biopsy samples were obtained for DNA and RNA extraction. Microbiomes were analyzed by 16S rRNA profiling, with the differentially expressed genes analyzed using RNA sequencing. The RNA-sequencing results of selected genes were validated by qRT-PCR. Results: Bacterial diversity of Helicobacter pylori-positive gastric specimens were lower than those of negative, and both groups were clearly separated according to beta diversity. Helicobacter pylori-positive group significantly reduced proportions of six phyla and eight genera; only Helicobacter taxa were more abundant in Helicobacter pylori-negative group. Gastric tissues RNA sequencing showed increased expression of multiple immune response genes in Helicobacter pylori -infection. Helicobacter pylori -infected children with restructured gastric microbiota had higher levels of FOXP3, IL-10, TGF-ß1 and IL-17A expressions, which were consistent with increased CD4+T cell and macrophagocyte, compared with non-infected children. Conclusions: Presence of Helicobacter pylori significantly influences gastric microbiota and results in lower abundance of multiple taxonomic levels in children. Meanwhile, it affects gastric immune environment and promotes the occurrence of gastritis. Clinical Trial Registration: [http://www.chictr.org.cn], identifier [ChiCTR1800015190].


Assuntos
Duodeno/microbiologia , Mucosa Gástrica/microbiologia , Gastrite/microbiologia , Microbioma Gastrointestinal , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Imunidade nas Mucosas , Mucosa Intestinal/microbiologia , Adolescente , Fatores Etários , Biópsia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Estudos de Casos e Controles , Criança , Duodeno/imunologia , Disbiose , Endoscopia Gastrointestinal , Feminino , Fatores de Transcrição Forkhead/análise , Mucosa Gástrica/imunologia , Gastrite/diagnóstico , Gastrite/imunologia , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Interações Hospedeiro-Patógeno , Humanos , Interleucina-10/análise , Interleucina-17/análise , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Ribotipagem , Fator de Crescimento Transformador beta1/análise
20.
Am J Respir Cell Mol Biol ; 65(2): 157-166, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33848452

RESUMO

Cystic fibrosis (CF) is characterized by chronic airway infection, inflammation, and tissue damage that lead to progressive respiratory failure. NLRP3 and NLRC4 are cytoplasmic pattern recognition receptors that activate the inflammasome, initiating a caspase-1-mediated response. We hypothesized that gain-of-function inflammasome responses are associated with worse outcomes in children with CF. We genotyped nonsynonymous variants in NLRP3 and the NLRC4 pathway from individuals in the EPIC (Early Pseudomonas Infection Control) Observational Study cohort and tested for association with CF outcomes. We generated knockouts of NLRP3 and NLRC4 in human macrophage-like cells and rescued knockouts with wild-type or variant forms of NLRP3 and NLRC4. We identified a SNP in NLRP3, p.(Q705K), that was associated with a higher rate of P. aeruginosa colonization (N = 609; P = 0.01; hazard ratio, 2.3 [Cox model]) and worsened lung function over time as measured by forced expiratory volume in 1 second (N = 445; P = 0.001 [generalized estimating equation]). We identified a SNP in NLRC4, p.(A929S), that was associated with a lower rate of P. aeruginosa colonization as part of a composite of rare variants (N = 405; P = 0.045; hazard ratio, 0.68 [Cox model]) and that was individually associated with protection from lung function decline (P < 0.001 [generalized estimating equation]). Rescue of the NLRP3 knockout with the p.(Q705K) variant produced significantly more IL-1ß in response to NLRP3 stimulation than rescue with the wild type (P = 0.020 [Student's t test]). We identified a subset of children with CF at higher risk of early lung disease progression. Knowledge of these genetic modifiers could guide therapies targeting inflammasome pathways.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Ligação ao Cálcio/genética , Fibrose Cística , Inflamassomos/genética , Macrófagos/microbiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Polimorfismo de Nucleotídeo Único , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa , Criança , Pré-Escolar , Fibrose Cística/genética , Fibrose Cística/microbiologia , Feminino , Humanos , Inflamassomos/metabolismo , Masculino , Células THP-1 , Células U937
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