RESUMO
Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain-containing protein (PumA) of the multi-drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF-κB, a property transferable to non-PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll-like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin-associated protein 1 (UBAP1), a component of the endosomal-sorting complex required for transport I (ESCRT-I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Evasão da Resposta Imune , Glicoproteínas de Membrana/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Pseudomonas aeruginosa/patogenicidade , Receptores de Interleucina-1/antagonistas & inibidores , Receptores Toll-Like/antagonistas & inibidores , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Humanos , Pseudomonas aeruginosa/imunologiaRESUMO
BACKGROUND: Gut dysbiosis due to the adverse effects of antibiotics affects outcomes of lung infection. Previous murine models relied on significant depletion of both gut and lung microbiota, rendering the analysis of immune gut-lung cross-talk difficult. Here, we study the effects of antibiotic-induced gut dysbiosis without lung dysbiosis on lung immunity and the consequences on acute P. aeruginosa lung infection. METHODS: C57BL6 mice received 7 days oral vancomycin-colistin, followed by normal regimen or fecal microbial transplant or Fms-related tyrosine kinase 3 ligand (Flt3-Ligand) over 2 days, and then intra-nasal P. aeruginosa strain PAO1. Gut and lung microbiota were studied by next-generation sequencing, and lung infection outcomes were studied at 24 h. Effects of vancomycin-colistin on underlying immunity and bone marrow progenitors were studied in uninfected mice by flow cytometry in the lung, spleen, and bone marrow. RESULTS: Vancomycin-colistin administration induces widespread cellular immunosuppression in both the lung and spleen, decreases circulating hematopoietic cytokine Flt3-Ligand, and depresses dendritic cell bone marrow progenitors leading to worsening of P. aeruginosa lung infection outcomes (bacterial loads, lung injury, and survival). Reversal of these effects by fecal microbial transplant shows that these alterations are related to gut dysbiosis. Recombinant Flt3-Ligand reverses the effects of antibiotics on subsequent lung infection. CONCLUSIONS: These results show that gut dysbiosis strongly impairs monocyte/dendritic progenitors and lung immunity, worsening outcomes of P. aeruginosa lung infection. Treatment with a fecal microbial transplant or immune stimulation by Flt3-Ligand both restore lung cellular responses to and outcomes of P. aeruginosa following antibiotic-induced gut dysbiosis.
Assuntos
Antibacterianos/efeitos adversos , Disbiose/complicações , Terapia de Imunossupressão/efeitos adversos , Pneumonia/etiologia , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Disbiose/etiologia , Disbiose/fisiopatologia , Terapia de Imunossupressão/métodos , Pulmão/microbiologia , Pulmão/fisiopatologia , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Pneumonia/fisiopatologia , Pseudomonas aeruginosa/efeitos dos fármacos , Vancomicina/efeitos adversos , Vancomicina/farmacologiaRESUMO
While antibiotic use is a risk factor of carbapenemase-producing Enterobacteriaceae (CPE) acquisition, the importance of timing of antibiotic administration relative to CPE exposure remains unclear. In a murine model of gut colonization by New Delhi metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae, a single injection of clindamycin within at most 1 week before or after CPE exposure induced colonization persisting up to 100 days. The timing of antibiotic administration relative to CPE exposure may be relevant to infection control and antimicrobial stewardship approaches.
Assuntos
Antibacterianos/administração & dosagem , Proteínas de Bactérias/metabolismo , Infecções por Enterobacteriaceae/tratamento farmacológico , Enterobacteriaceae/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , beta-Lactamases/metabolismo , Animais , Modelos Animais de Doenças , Enterobacteriaceae/metabolismo , Controle de Infecções/métodos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/metabolismo , Camundongos , Testes de Sensibilidade Microbiana/métodosRESUMO
While NLRC4-dependent sensing of intracellular Gram-negative pathogens such as Salmonella enterica serovar typhimurium is a beneficial host response, NLRC4-dependent sensing of the Pseudomonas aeruginosa type 3 secretion system (T3SS) has been shown to be involved in pathogenicity. In mice, different pathogen-associated microbial patterns are sensed by the combination of the NLRC4-inflammasome with different neuronal apoptosis inhibitory proteins (NAIPs). NAIP2 is involved in sensing PscI, an inner-rod protein of the P. aeruginosa T3SS. Surprisingly, only a single human NAIP (hNAIP) has been found. Moreover, there is no description of hNAIP-NLRC4 inflammasome recognition of T3SS inner-rod proteins in humans. Here, we show that the P. aeruginosa T3SS inner-rod protein PscI and needle protein PscF are both sensed by the hNAIP-NLRC4 inflammasome in human macrophages and PBMCs from healthy donors, allowing caspase-1 and IL-1ß maturation and resulting in a robust inflammatory response. TLR4 and TLR2 are involved in redundantly sensing these two T3SS components.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Inflamassomos/metabolismo , Macrófagos/imunologia , Proteína Inibidora de Apoptose Neuronal/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Sistemas de Secreção Tipo III/metabolismo , Animais , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-1beta/metabolismo , Macrófagos/microbiologia , Camundongos , Moléculas com Motivos Associados a Patógenos/imunologia , Células THP-1 , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Sistemas de Secreção Tipo III/imunologiaRESUMO
The synthesis of eight perylenediimide-based glycoclusters was readily performed from hexa- and tetra-propargylated cores through azide-alkyne "click" conjugation. Variations in the carbohydrate epitope (Glc, Gal, Man, Fuc) and the linker arm provided molecular diversity. Interactions with LecA and LecB, two proteins involved in the adhesion of Pseudomonas aeruginosa to host tissues, were evaluated by microcalorimetry (ITC). In both cases high affinities were obtained with Kd values in the nanomolar range. Further evaluation of their anti-adhesive properties using cultured epithelial cells demonstrated their potent anti-adhesive activities against Pseudomonas aeruginosa with only 30-40% residual adhesion observed. The fluorescence properties of the PDI core were then investigated by confocal microscopy on cell-bacteria cultures. However, the red fluorescence signal of the PDI-based glycocluster was too weak to provide significant data. The present study provides another type of anti-adhesive glycocluster against bacterial infection with a large aromatic PDI core.
Assuntos
Adesinas Bacterianas/efeitos dos fármacos , Glicoconjugados/farmacologia , Imidas/farmacologia , Lectinas/antagonistas & inibidores , Perileno/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Calorimetria , Adesão Celular/efeitos dos fármacos , Glicoconjugados/síntese química , Glicoconjugados/química , Imidas/síntese química , Imidas/química , Ligantes , Estrutura Molecular , Perileno/síntese química , Perileno/química , Perileno/farmacologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/citologiaRESUMO
The export of bacterial toxins across the bacterial envelope requires the assembly of complex, membrane-embedded protein architectures. Pseudomonas aeruginosa employs type III secretion (T3S) injectisome to translocate exotoxins directly into the cytoplasm of a target eukaryotic cell. This multi-protein channel crosses two bacterial membranes and extends further as a needle through which the proteins travel. We show in this work that PscI, proposed to form the T3S system (T3SS) inner rod, possesses intrinsic properties to polymerize into flexible and regularly twisted fibrils and activates IL-1ß production in mouse bone marrow macrophages in vitro. We also found that point mutations within C-terminal amphipathic helix of PscI alter needle assembly in vitro and T3SS function in cell infection assays, suggesting that this region is essential for an efficient needle assembly. The overexpression of PscF partially compensates for the absence of the inner rod in PscI-deficient mutant by forming a secretion-proficient injectisome. All together, we propose that the polymerized PscI in P. aeruginosa optimizes the injectisome function by anchoring the needle within the envelope-embedded complex of the T3S secretome and - contrary to its counterpart in Salmonella - is not involved in substrate switching.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Polimerização , Transporte Proteico , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/genéticaRESUMO
BACKGROUND: Pseudomonas aeruginosa (Pa) is a Gram-negative bacteria frequently involved in healthcare-associated pneumonia with poor clinical outcome. To face the announced post-antibiotic era due to increasing resistance and lack of new antibiotics, new treatment strategies have to be developed. Immunomodulation of the host response involved in outcome could be an alternative therapeutic target in Pa-induced lung infection. Kynurenines are metabolites resulting from tryptophan catabolism and are known for their immunomodulatory properties. Pa catabolizes tryptophan through the kynurenine pathway. Interestingly, many host cells also possess the kynurenine pathway, whose metabolites are known to control immune system homeostasis. Thus, bacterial metabolites may interfere with the host's immune response. However, the kynurenine pathway in Pa, including functional enzymes, types and amounts of secreted metabolites remains poorly known. Using liquid chromatography coupled to mass spectrometry and different strains of Pa, we determined types and levels of metabolites produced by Pa ex vivo in growth medium, and the relevance of this production in vivo in a murine model of acute lung injury. RESULTS: Ex vivo, Pa secretes clinically relevant kynurenine levels (µM to mM). Pa also secretes kynurenic acid and 3-OH-kynurenine, suggesting that the bacteria possess both a functional kynurenine aminotransferase and kynurenine monooxygenase. The bacterial kynurenine pathway is the major pathway leading to anthranilate production both ex vivo and in vivo. In the absence of the anthranilate pathway, the kynurenine pathway leads to kynurenic acid production. CONCLUSION: Pa produces and secretes several metabolites of the kynurenine pathway. Here, we demonstrate the existence of new metabolic pathways leading to synthesis of bioactive molecules, kynurenic acid and 3-OH-kynurenine in Pa. The kynurenine pathway in Pa is critical to produce anthranilate, a crucial precursor of some Pa virulence factors. Metabolites (anthranilate, kynurenine, kynurenic acid) are produced at sustained levels both ex vivo and in vivo leading to a possible immunomodulatory interplay between bacteria and host. These data may imply that pulmonary infection with bacteria highly expressing the kynurenine pathway enzymes could influence the equilibrium of the host's tryptophan metabolic pathway, known to be involved in the immune response to infection. Further studies are needed to explore the effects of these metabolic changes on the pathophysiology of Pa infection.
Assuntos
Pseudomonas aeruginosa/metabolismo , Triptofano/metabolismo , Lesão Pulmonar Aguda/microbiologia , Animais , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Imunidade Inata , Ácido Cinurênico/metabolismo , Cinurenina/metabolismo , Redes e Vias Metabólicas , Camundongos , Murinae , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Transaminases/metabolismo , ortoaminobenzoatos/metabolismoRESUMO
Efficient wound healing is required to maintain the integrity of the intestinal epithelial barrier because of its constant exposure to a large variety of environmental stresses. This process implies a partial cell depolarization and the acquisition of a motile phenotype that involves rearrangements of the actin cytoskeleton. Here we address how polarized enterocytes harboring actin-rich apical microvilli undergo extensive cell remodeling to drive injury repair. Using live imaging technologies, we demonstrate that enterocytes in vitro and in vivo rapidly depolarize their microvilli at the wound edge. Through its F-actin-severing activity, the microvillar actin-binding protein villin drives both apical microvilli disassembly in vitro and in vivo and promotes lamellipodial extension. Photoactivation experiments indicate that microvillar actin is mobilized at the lamellipodium, allowing optimal migration. Finally, efficient repair of colonic mechanical injuries requires villin severing of F-actin, emphasizing the importance of villin function in intestinal homeostasis. Thus, villin severs F-actin to ensure microvillus depolarization and enterocyte remodeling upon injury. This work highlights the importance of specialized apical pole disassembly for the repolarization of epithelial cells initiating migration.
Assuntos
Actinas/química , Enterócitos/citologia , Proteínas dos Microfilamentos/fisiologia , Actinas/metabolismo , Animais , Apoptose , Diferenciação Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Endoscopia , Enterócitos/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/metabolismo , Fenótipo , Suínos , CicatrizaçãoRESUMO
The murine norovirus (MNV) is a recently discovered mouse pathogen, representing the most common contaminant in laboratory mouse colonies. Nevertheless, the effects of MNV infection on biomedical research are still unclear. We tested the hypothesis that MNV infection could alter immune response in mice with acute lung infection. Here we report that co-infection with MNV increases survival of mice with Pseudomonas aeruginosa acute lung injury and decreases in vivo production of pro-inflammatory cytokines. Our results suggest that MNV infection can deeply modify the parameters studied in conventional models of infection and lead to false conclusions in experimental models.
Assuntos
Infecções por Caliciviridae/imunologia , Citocinas/metabolismo , Imunomodulação , Norovirus/fisiologia , Pneumonia/imunologia , Infecções por Pseudomonas/imunologia , Doenças dos Roedores/imunologia , Doença Aguda , Animais , Infecções por Caliciviridae/virologia , Coinfecção/imunologia , Coinfecção/microbiologia , Coinfecção/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Doenças dos Roedores/microbiologia , Doenças dos Roedores/virologiaRESUMO
RATIONALE: Pseudomonas aeruginosa, a major problem pathogen responsible for severe infections in critically ill patients, triggers, through a functional type-3 secretion system (T3SS), the activation of an intracellular cytosolic sensor of innate immunity, NLRC4. Although the NLRC4-inflammasome-dependent response contributes to increased clearance of intracellular pathogens, it seems that NLRC4 inflammasome activation decreases the clearance of P. aeruginosa, a mainly extracellular pathogen. OBJECTIVES: We sought to determine the underlying mechanisms of this effect of the activation of NLRC4 by P. aeruginosa. METHODS: We established acute lung injury in wild-type and Nlrc4(-/-) mice using sublethal intranasal inocula of P. aeruginosa strain CHA expressing or not a functional T3SS. We studied 96-hour survival, lung injury, bacterial clearance from the lungs, cytokine secretion in bronchoalveolar lavage, lung antimicrobial peptide expression by quantitative polymerase chain reaction, and flow cytometry analysis of lung cells. MEASUREMENTS AND MAIN RESULTS: Nlrc4(-/-) mice showed enhanced bacterial clearance and decreased lung injury contributing to increased survival against extracellular P. aeruginosa strain expressing a functional T3SS. The mechanism involved decreased NLRC4-inflammasome-driven IL-18 secretion attenuating lung injury caused by excessive neutrophil recruitment. Additionally, in the lungs of Nlrc4(-/-) mice secretion of IL-17 by innate immune cells was increased and responsible for increased expression of lung epithelial antimicrobial peptides. Furthermore, IL-18 secretion was found to repress IL-17 and IL-17-driven lung antimicrobial peptide expression. CONCLUSIONS: We report a new role of the T3SS apparatus itself, independently of exotoxin translocation. Through NLRC4 inflammasome activation, the T3SS promotes IL-18 secretion, which dampens a beneficial IL-17-mediated antimicrobial host response.
Assuntos
Lesão Pulmonar Aguda/microbiologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Inflamassomos/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/metabolismo , Animais , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/química , Caspase 1/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Imunidade Inata , Interleucina-17/metabolismo , Interleucina-18/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
RATIONALE: Pattern recognition receptors are attractive targets for vaccine adjuvants, and polymorphisms of the innate receptor NOD1 have been associated with allergic asthma. OBJECTIVES: To elucidate whether NOD1 agonist may favor allergic asthma in humans through activation of dendritic cells, and to evaluate the mechanisms involved using an in vivo model. METHODS: NOD1-primed dendritic cells from allergic and nonallergic donors were characterized in vitro on their phenotype, cytokine secretion, and Th2 polarizing ability. The in vivo relevance was examined in experimental allergic asthma, and the mechanisms were assessed using transfer of NOD1-conditioned dendritic cells from wild-type or CCL17-deficient mice. MEASUREMENTS AND MAIN RESULTS: NOD1 priming of human dendritic cells promoted a Th2 polarization profile that involved the production of CCL17 and CCL22 in nonallergic subjects but only CCL17 in allergic patients, without requiring allergen costimulation. Moreover, NOD1-primed dendritic cells from allergic donors exhibited enhanced maturation that led to abnormal CCL22 and IL-10 secretion compared with nonallergic donors. In mice, systemic NOD1 ligation exacerbated allergen-induced experimental asthma by amplifying CCL17-mediated Th2 responses in the lung. NOD1-mediated sensitization of purified murine dendritic cells enhanced production of CCL17 and CCL22, but not of thymic stromal lymphopoietin and IL-33, in vitro. Consistently, adoptive transfer of NOD1-conditioned dendritic cells exacerbated the Th2 pulmonary response in a CCL17-dependent manner in vivo. CONCLUSIONS: Data from this study unveil a deleterious role of NOD1 in allergic asthma through direct induction of CCL17 by dendritic cells, arguing for a need to address vaccine formulation safety issues related to allergy.
Assuntos
Asma/imunologia , Quimiocina CCL17/imunologia , Quimiocina CCL22/imunologia , Células Dendríticas/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Alérgenos/imunologia , Animais , Asma/genética , Asma/prevenção & controle , Modelos Animais de Doenças , Feminino , Humanos , Técnicas In Vitro , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/genética , Fenótipo , Polimorfismo Genético , Células Th2/imunologia , Regulação para Cima/imunologiaRESUMO
The colonic epithelium self-renews every 3 to 5 d, but our understanding of the underlying processes preserving wound healing from carcinogenesis remains incomplete. Here, we demonstrate that Nod-like receptor pyrin domain-containing protein 6 (NLRP6) suppresses inflammation and carcinogenesis by regulating tissue repair. NLRP6 was primarily produced by myofibroblasts within the stem-cell niche in the colon. Although NLRP6 expression was lowered in diseased colon, NLRP6-deficient mice were highly susceptible to experimental colitis. Upon injury, NLRP6 deficiency deregulated regeneration of the colonic mucosa and processes of epithelial proliferation and migration. Consistently, absence of NLRP6 accelerated colitis-associated tumor growth in mice. A gene-ontology analysis on a whole-genome expression profiling revealed a link between NLRP6 and self-renewal of the epithelium. Collectively, the integrity of the epithelial barrier is preserved by NLRP6 that may be manipulated to develop drugs capable of preventing adenoma formation in inflammatory bowel diseases.
Assuntos
Proliferação de Células , Neoplasias Colorretais/genética , Células Epiteliais/metabolismo , Receptores de Superfície Celular/genética , Animais , Movimento Celular/genética , Colite/genética , Colite/metabolismo , Colite/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Mucosa Intestinal/lesões , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cicatrização/genéticaRESUMO
OBJECTIVE: Inflammatory bowel diseases (IBD) have been intrinsically linked to a deregulated cytokine network, but novel therapeutic principles are urgently needed. Here we identify the interleukin (IL)-33 and its receptor ST2 as key negative regulators of wound healing and permeability in the colon of mice. DESIGN: Expression of IL-33 and ST2 was determined by qRT-PCR, ELISA, immunohistochemistry and western-blot analysis. Wild-type and St2(-/-) mice were used in wound healing experiments and in two experimental models of IBD triggered by 2,4,6-trinitrobenzene sulphonic acid or dextran sodium sulphate (DSS). Neutralisation of ST2 was performed by using a specific blocking antibody. RESULTS: Nuclear localisation and enhanced expression of IL-33 in myofibroblasts and enterocytes was linked to disease involvement independently of inflammation, while the expression of ST2 was primarily restricted to the colonic epithelia. In two experimental models of IBD, genetic ablation of ST2 significantly improved signs of colitis, while a sustained epithelial expression of the cyto-protective factor connexin-43 was observed in DSS-treated St2-deficient mice. Unexpectedly, absence of ST2 in non-hematopoietic cells was sufficient to protect against colitis. Consistently, specific inhibition of endogenous ST2-mediated signalling by treatment with neutralising antibody improved DSS-induced colitis. In addition, IL-33 treatment impaired epithelial barrier permeability in vitro and in vivo, whereas absence of ST2 enhanced wound healing response upon acute mechanical injury in the colon. CONCLUSIONS: Our study unveiled a novel non-hematopoietic function of IL-33 in epithelial barrier function and wound healing. Therefore, blocking the IL-33/ST2 axis may represent an efficient therapy in IBD.
Assuntos
Colite Ulcerativa/etiologia , Interleucinas/fisiologia , Receptores de Interleucina/fisiologia , Animais , Western Blotting , Células CACO-2 , Colite Ulcerativa/fisiopatologia , Colite Ulcerativa/terapia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Reação em Cadeia da Polimerase em Tempo Real , Cicatrização/fisiologiaRESUMO
Crohn's disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for intestinal homeostasis in response to both dietary- and microbiota-derived signals. Its role in host defense remains unknown, however. We show that PPARgamma functions as an antimicrobial factor by maintaining constitutive epithelial expression of a subset of beta-defensin in the colon, which includes mDefB10 in mice and DEFB1 in humans. Colonic mucosa of Ppargamma mutant animals shows defective killing of several major components of the intestinal microbiota, including Candida albicans, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli. Neutralization of the colicidal activity using an anti-mDefB10 blocking antibody was effective in a PPARgamma-dependent manner. A functional promoter variant that is required for DEFB1 expression confers strong protection against Crohn's colitis and ileocolitis (odds ratio, 0.559; P = 0.018). Consistently, colonic involvement in CD is specifically linked to reduced expression of DEFB1 independent of inflammation. These findings support the development of PPARgamma-targeting therapeutic and/or nutritional approaches to prevent colonic inflammation by restoring antimicrobial immunity in CD.
Assuntos
Bactérias/imunologia , Colo/imunologia , Colo/microbiologia , Fungos/imunologia , Imunidade Inata/imunologia , PPAR gama/metabolismo , Animais , Linhagem Celular , Colo/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Regulação da Expressão Gênica , Frequência do Gene/genética , Genótipo , Humanos , Íleo/imunologia , Íleo/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Imunológicos , PPAR gama/deficiência , Regiões Promotoras Genéticas/genética , Ligação Proteica , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
Carbapenemase-producing Enterobacterales (CPE) are spreading rapidly in hospital settings. Asymptomatic CPE gut colonisation may be associated with dysbiosis and gut-lung axis alterations, which could impact lung infection outcomes. In this study, in male C57BL/6JRj mice colonised by CPE, we characterise the resulting gut dysbiosis, and analyse the lung immune responses and outcomes of subsequent Pseudomonas aeruginosa lung infection. Asymptomatic gut colonisation by CPE leads to a specific gut dysbiosis and increases the severity of P. aeruginosa lung infection through lower numbers of alveolar macrophages and conventional dendritic cells. CPE-associated dysbiosis is characterised by a near disappearance of the Muribaculaceae family and lower levels of short-chain fatty acids. Faecal microbiota transplantation restores immune responses and outcomes of lung infection outcomes, demonstrating the involvement of CPE colonisation-induced gut dysbiosis in altering the immune gut-lung axis, possibly mediated by microbial metabolites such as short-chain fatty acids.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Camundongos , Masculino , Klebsiella pneumoniae , Disbiose , Camundongos Endogâmicos C57BL , Pulmão , Ácidos Graxos VoláteisRESUMO
BACKGROUND AND AIMS: NOD2 has emerged as a critical player in the induction of both Th1 and Th2 responses for potentiation and polarisation of antigen-dependent immunity. Loss-of-function mutations in the NOD2-encoding gene and deregulation of its downstream signalling pathway have been linked to Crohn's disease. Although it is well documented that NOD2 is capable of sensing bacterial muramyl dipeptide, it remains counter-intuitive to link development of overt intestinal inflammation to a loss of bacterial-induced inflammatory response. We hypothesised that a T helper bias could also contribute to an autoimmune-like colitis different from inflammation that is fully fledged by Th1 type cells. METHODS: An oedematous bowel wall with a mixed Th1/Th2 response was induced in mice by intrarectal instillation of the haptenating agent oxazolone. Survival and clinical scoring were evaluated. At several time points after instillation, colonic damage was assessed by macroscopic and microscopic observations. To evaluate the involvement of NOD2 in immunochemical phenomena, quantitative polymerase chain reaction [PCR] and flow cytometry analysis were performed. Bone marrow chimera experimentation allowed us to evaluate the role of haematopoietic/non-hematopoietic NOD2-expressing cells. RESULTS: Herein, we identified a key regulatory circuit whereby NOD2-mediated sensing of a muramyl dipeptide [MDP] by radio-resistant cells improves colitis with a mixed Th1/Th2 response that is induced by oxazolone. Genetic ablation of either Nod2 or Ripk2 precipitated oxazolone colitis that is predominantly linked to a lack of interferon-gamma. Bone marrow chimera experiments revealed that inactivation of Nod2 signalling in non-haematopoietic cells is causing a biased M1-M2 polarisation of macrophages and a decreased frequency of splenic regulatory T cells that correlates with an impaired activation of CD4â +â T cells within mesenteric lymph nodes. Mechanistically, mice were protected from oxazolone-induced colitis upon administration of MDP in an interleukin-1- and interleukin-23-dependent manner. CONCLUSIONS: These findings indicate that Nod2 signalling may prevent pathological conversion of T helper cells for maintenance of tissue homeostasis.
Assuntos
Colite , Oxazolona , Camundongos , Animais , Oxazolona/efeitos adversos , Acetilmuramil-Alanil-Isoglutamina/efeitos adversos , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Colite/metabolismo , Inflamação , Transdução de Sinais , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismoRESUMO
Biofilms are highly tolerant to antimicrobials and host immune defense, enabling pathogens to thrive in hostile environments. The diversity of microbial biofilm infections requires alternative and complex treatment strategies. In a previous work we demonstrated that the human Atrial Natriuretic Peptide (hANP) displays a strong anti-biofilm activity toward Pseudomonas aeruginosa and that the binding of hANP by the AmiC protein supports this effect. This AmiC sensor has been identified as an analog of the human natriuretic peptide receptor subtype C (h-NPRC). In the present study, we evaluated the anti-biofilm activity of the h-NPRC agonist, osteocrin (OSTN), a hormone that displays a strong affinity for the AmiC sensor at least in vitro. Using molecular docking, we identified a pocket in the AmiC sensor that OSTN reproducibly docks into, suggesting that OSTN might possess an anti-biofilm activity as well as hANP. This hypothesis was validated since we observed that OSTN dispersed established biofilm of P. aeruginosa PA14 strain at the same concentrations as hANP. However, the OSTN dispersal effect is less marked than that observed for the hANP (-61% versus -73%). We demonstrated that the co-exposure of P. aeruginosa preformed biofilm to hANP and OSTN induced a biofilm dispersion with a similar effect to that observed with hANP alone suggesting a similar mechanism of action of these two peptides. This was confirmed by the observation that OSTN anti-biofilm activity requires the activation of the complex composed by the sensor AmiC and the regulator AmiR of the ami pathway. Using a panel of both P. aeruginosa laboratory reference strains and clinical isolates, we observed that the OSTN capacity to disperse established biofilms is highly variable from one strain to another. Taken together, these results show that similarly to the hANP hormone, OSTN has a strong potential to be used as a tool to disperse P. aeruginosa biofilms.
RESUMO
Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.
Assuntos
Infecções por Caliciviridae , Colite , Gastroenterite , Microbioma Gastrointestinal , Proteína Adaptadora de Sinalização NOD2 , Animais , Camundongos , Infecções por Caliciviridae/imunologia , Colite/induzido quimicamente , Colite/virologia , Gastroenterite/imunologia , Gastroenterite/virologia , Proteína Adaptadora de Sinalização NOD2/metabolismoRESUMO
Bacteria of the genus Achromobacter are environmental germs, with an unknown reservoir. It can become opportunistic pathogens in immunocompromised patients, causing bacteremia, meningitis, pneumonia, or peritonitis. In recent years, Achromobacter xylosoxidans has emerged with increasing incidence in patients with cystic fibrosis (CF). Recent studies showed that A. xylosoxidans is involved in the degradation of the respiratory function of patients with CF. The respiratory ecosystem of patients with CF is colonized by bacterial species that constantly fight for space and access to nutrients. The type VI secretion system (T6SS) empowers this constant bacterial antagonism, and it is used as a virulence factor in several pathogenic bacteria. This study aimed to investigate the prevalence of the T6SS genes in A. xylosoxidans isolated in patients with CF. We also evaluated clinical and molecular characteristics of T6SS-positive A. xylosoxidans strains. We showed that A. xylosoxidans possesses a T6SS gene cluster and that some environmental and clinical isolates assemble a functional T6SS nanomachine. A. xylosoxidans T6SS is used to target competing bacteria, including other CF-specific pathogens. Finally, we demonstrated the importance of the T6SS in the internalization of A. xylosoxidans in lung epithelial cells and that the T6SS protein Hcp is detected in the sputum of patients with CF. Altogether, these results suggest for the first time a role of T6SS in CF-lung colonization by A. xylosoxidans and opens promising perspective to target this virulence determinant as innovative theranostic options for CF management.
Assuntos
Achromobacter denitrificans , Fibrose Cística , Infecções por Bactérias Gram-Negativas , Sistemas de Secreção Tipo VI , Achromobacter denitrificans/genética , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Ecossistema , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Pulmão , Sistemas de Secreção Tipo VI/genética , Fatores de Virulência/genéticaRESUMO
Pseudomonas aeruginosa biofilms cause chronic, antibiotic tolerant infections in wounds and lungs. Numerous recent studies demonstrate that bacteria can detect human communication compounds through specific sensor/receptor tools that modulate bacterial physiology. Consequently, interfering with these mechanisms offers an exciting opportunity to directly affect the infection process. It is shown that the human hormone Atrial Natriuretic Peptide (hANP) both prevents the formation of P. aeruginosa biofilms and strongly disperses established P. aeruginosa biofilms. This hANP action is dose-dependent with a strong effect at low nanomolar concentrations and takes effect in 30-120 min. Furthermore, although hANP has no antimicrobial effect, it acts as an antibiotic adjuvant. hANP enhances the antibiofilm action of antibiotics with diverse modes of action, allowing almost full biofilm eradication. The hANP effect requires the presence of the P. aeruginosa sensor AmiC and the AmiR antiterminator regulator, indicating a specific mode of action. These data establish the activation of the ami pathway as a potential mechanism for P. aeruginosa biofilm dispersion. hANP appears to be devoid of toxicity, does not enhance bacterial pathogenicity, and acts synergistically with antibiotics. These data show that hANP is a promising powerful antibiofilm weapon against established P. aeruginosa biofilms in chronic infections.