RESUMO
Dendritic cells (DCs) participate in the pathogenesis of several diseases. We investigated DCs and the connection between mucosa and joints in a murine model of Yersinia enterocolitica O:3-induced reactive arthritis (ReA) in TNFRp55-/- mice. DCs of mesenteric lymph nodes (MLN) and joint regional lymph nodes (RLN) were analyzed in TNFRp55-/- and wild-type mice. On day 14 after Y. enterocolitica infection (arthritis onset), we found that under TNFRp55 deficiency, migratory (MHChighCD11c+) DCs increased significantly in RLN. Within these RLN, resident (MHCintCD11c+) DCs increased on days 14 and 21. Similar changes in both migratory and resident DCs were also detected on day 14 in MLN of TNFRp55-/- mice. In vitro, LPS-stimulated migratory TNFRp55-/- DCs of MLN increased IL-12/23p40 compared with wild-type mice. In addition, TNFRp55-/- bone marrow-derived DCs in a TNFRp55-/- MLN microenvironment exhibited higher expression of CCR7 after Y. enterocolitica infection. The major intestinal DC subsets (CD103+CD11b-, CD103-CD11b+, and CD103+CD11b+) were found in the RLN of Y. enterocolitica-infected TNFRp55-/- mice. Fingolimod (FTY720) treatment of Y. enterocolitica-infected mice reduced the CD11b- subset of migratory DCs in RLN of TNFRp55-/- mice and significantly suppressed the severity of ReA in these mice. This result was associated with decreased articular IL-12/23p40 and IFN-γ levels. In vitro FTY720 treatment downregulated CCR7 on Y. enterocolitica-infected bone marrow-derived DCs and purified MLN DCs, which may explain the mechanism underlying the impairment of DCs in RLN induced by FTY720. Taken together, data indicate the migration of intestinal DCs to RLN and the contribution of these cells in the immunopathogenesis of ReA, which may provide evidence for controlling this disease.
Assuntos
Artrite Reativa/imunologia , Células Dendríticas/imunologia , Linfonodos/imunologia , Mesentério/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Yersiniose/imunologia , Yersinia enterocolitica/imunologia , Animais , Artrite Reativa/metabolismo , Células Dendríticas/metabolismo , Linfonodos/metabolismo , Masculino , Mesentério/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proibitinas , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Transdução de Sinais/imunologia , Receptores Chamariz do Fator de Necrose Tumoral/imunologia , Yersiniose/metabolismoRESUMO
In order to monitor the occurrence of zoonotic agents in pig herds as well as to improve herd health management, the development of new cost-effective diagnostic methods for pigs is necessary. In this study, a protein microarray-based assay for the simultaneous detection of immunoglobulin G (IgG) antibodies against different zoonotic agents and pathogens causing production diseases in pigs was developed. Therefore, antigens of ten different important swine pathogens (Toxoplasma gondii, Yersinia enterocolitica, Salmonella spp., Trichinella spp., Mycobacterium avium, Hepatitis E virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, the porcine reproductive and respiratory syndrome virus, Influenza A virus) were spotted and covalently immobilized as 'antigen-spots' on microarray chips in order to test pig serum for the occurrence of antibodies. Pig serum was sampled at three German abattoirs and ELISA tests for the different pathogens were conducted with the purpose of creating a panel of reference samples for microarray analysis. To evaluate the accuracy of the antigens on the microarray, receiver operating characteristic (ROC) curve analysis using the ELISA test results as reference was performed for the different antigens. High area under curve values were achieved for the antigens of two zoonotic agents: Toxoplasma gondii (0.91), Yersinia enterocolitica (0.97) and for three production diseases: Actinobacillus pleuropneumoniae (0.77), Mycoplasma hyopneumoniae (0.94) and the porcine reproductive and respiratory syndrome virus (0.87). With the help of the newly developed microarray assay, collecting data on the occurrence of antibodies against zoonotic agents and production diseases in pig herds could be minimized to one measurement, resulting in an efficient screening test.
Assuntos
Imunoglobulina G/sangue , Programas de Rastreamento/veterinária , Análise Serial de Proteínas/veterinária , Doenças dos Suínos/diagnóstico , Zoonoses/diagnóstico , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antiprotozoários/sangue , Anticorpos Antivirais/sangue , Programas de Rastreamento/métodos , Miniaturização , Análise Serial de Proteínas/métodos , Testes Sorológicos/métodos , Testes Sorológicos/veterinária , Sus scrofa/imunologia , Suínos , Doenças dos Suínos/imunologia , Toxoplasma/imunologia , Trichinella/imunologia , Yersinia enterocolitica/imunologia , Zoonoses/imunologiaRESUMO
The early response to bacterial infection requires cytokine responses by immune cells. In this issue of Cell Host & Microbe, Seo et al. (2018) demonstrate that TNF-TNFR superfamily molecules LIGHT and HVEM stimulate early IFN-γ production by type 3 innate lymphoid cells, which are critical for defense against Yersinia enterocolitica.
Assuntos
Células Alógenas/imunologia , Imunidade Inata , Linfócitos/imunologia , Linfotoxina-alfa/imunologia , Membro 14 de Receptores do Fator de Necrose Tumoral/imunologia , Animais , Humanos , Interferon gama/imunologia , Enteropatias/imunologia , Enteropatias/microbiologia , Yersiniose/imunologia , Yersinia enterocolitica/imunologiaRESUMO
Dendritic cells (DCs) play critical functions in the initiation of immune responses. Understanding their role in reactive arthritis (ReA) will help delineate the pathogenesis of this arthropathy. In early studies, we detected IL-12/23p40 deregulation in Yersinia entercolitica (Ye)-induced ReA in TNFRp55-deficient (TNFRp55-/-) mice. In this study, we assessed the contribution of DCs in this overproduction. First, greater levels of IL-12/23p40, IFN-γand IL-17A were confirmed in supernatants of lipopolysaccharide (LPS)-stimulated TNFRp55-/-splenocytes obtained on arthritis onset (day 14 after Ye infection). Later, DCs were identified as a precise source of IL-12/23p40 since increased frequency of splenic IL-12/23p40+DCs was detected in TNFRp55-/- mice. After robust in vivo amplification of DCs by injection of Fms-like tyrosine kinase 3-Ligand (Flt3L)-transfected BL16 melanoma, DCs were purified. These cells recapitulated the higher production of IL-12/23p40 under TNFRp55deficiency. In agreement with these results, TNFRp55-/- DCs promoted Th1 and Th17 programs by co-culture with WT CD4+lymphocytes. A mechanistic study demonstrated that JNK and p38 MAPK pathways are involved in IL-12/23p40 overproduction in purified TNFRp55-/- DCs as well as in the JAWS II cell line. This deregulation was once again attributed to TNFRp55 deficiency since CAY10500, a specific inhibitor of this pathway, compromised TNF-mediated IL-12/23p40 control in LPS-stimulated WT DCs. Simultaneously, this inhibition reduced IL-10 production, suggesting its role mediating IL-12/23p40 regulation by TNFRp55 pathway. These results provide experimental data on the existence of a TNFRp55-mediated anti-inflammatory circuit in DCs. Moreover, these cells may be considered as a novel target in the treatment of ReA.
Assuntos
Artrite Reativa/imunologia , Células Dendríticas/imunologia , Subunidade p40 da Interleucina-12/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Células Th1/citologia , Células Th17/citologia , Receptores Chamariz do Fator de Necrose Tumoral/genética , Yersiniose/complicações , Yersinia enterocolitica/imunologia , Animais , Artrite Reativa/patologia , Linhagem Celular , Polaridade Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proibitinas , Baço/imunologia , Yersiniose/imunologiaRESUMO
Yersinia enterocolitica is an enteropathogenic bacterium that causes gastrointestinal disorders, as well as extraintestinal manifestations. To subvert the host's immune response, Y. enterocolitica uses a type III secretion system consisting of an injectisome and effector proteins, called Yersinia outer proteins (Yops), that modulate activation, signaling, and survival of immune cells. In this article, we show that galectin-1 (Gal-1), an immunoregulatory lectin widely expressed in mucosal tissues, contributes to Y. enterocolitica pathogenicity by undermining protective antibacterial responses. We found higher expression of Gal-1 in the spleen and Peyer's patches of mice infected orogastrically with Y. enterocolitica serotype O:8 compared with noninfected hosts. This effect was prevented when mice were infected with Y. enterocolitica lacking YopP or YopH, two critical effectors involved in bacterial immune evasion. Consistent with a regulatory role for this lectin during Y. enterocolitica pathogenesis, mice lacking Gal-1 showed increased weight and survival, lower bacterial load, and attenuated intestinal pathology compared with wild-type mice. These protective effects involved modulation of NF-κB activation, TNF production, and NO synthesis in mucosal tissue and macrophages, as well as systemic dysregulation of IL-17 and IFN-γ responses. In vivo neutralization of these proinflammatory cytokines impaired bacterial clearance and eliminated host protection conferred by Gal-1 deficiency. Finally, supplementation of recombinant Gal-1 in mice lacking Gal-1 or treatment of wild-type mice with a neutralizing anti-Gal-1 mAb confirmed the immune inhibitory role of this endogenous lectin during Y. enterocolitica infection. Thus, targeting Gal-1-glycan interactions may contribute to reinforce antibacterial responses by reprogramming innate and adaptive immune mechanisms.
Assuntos
Galectina 1/metabolismo , Interações Hospedeiro-Patógeno , Yersiniose/imunologia , Yersinia enterocolitica/imunologia , Animais , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Galectina 1/antagonistas & inibidores , Galectina 1/genética , Galectina 1/imunologia , Interferon gama/sangue , Interferon gama/imunologia , Interleucina-17/sangue , Interleucina-17/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Intestinos/patologia , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/microbiologia , Nódulos Linfáticos Agregados/patologia , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/genética , Baço/imunologia , Baço/microbiologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Induction of adaptive immunity leads to the establishment of immunological memory; however, how innate immunity regulates memory T cell function remains obscure. Here we show a previously undefined mechanism in which innate and adaptive immunity are linked by TIR domain-containing adapter-inducing beta interferon (TRIF) during establishment and reactivation of memory T cells against Gram-negative enteropathogens. Absence of TRIF in macrophages (MÏs) but not dendritic cells led to a predominant generation of CD4(+) central memory T cells that express IL-17 during enteric bacterial infection in mice. TRIF-dependent type I interferon (IFN) signaling in T cells was essential to Th1 lineage differentiation and reactivation of memory T cells. TRIF activated memory T cells to facilitate local neutrophil influx and enhance bacterial elimination. These results highlight the importance of TRIF as a mediator of the innate and adaptive immune interactions in achieving the protective properties of memory immunity against Gram-negative bacteria and suggest TRIF as a potential therapeutic target.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Memória Imunológica , Yersiniose/imunologia , Yersinia enterocolitica/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células Dendríticas/imunologia , Humanos , Interferon gama/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Yersiniose/genética , Yersiniose/microbiologia , Yersinia enterocolitica/genéticaRESUMO
Lactobacillus plantarum C4, previously isolated from kefir and characterized as a potential probiotic strain, was tested for its protective and immunomodulatory capacity in a murine model of yersiniosis. The inoculation of BALB/c mice with a low pathogenicity serotype O9 strain of Yersinia enterocolitica results in a prolonged intestinal infection with colonization of Peyer's patches. Pretreatment with C4 was without effect on fecal excretion of yersiniae, but shortened the colonization of Peyer's patches. This protective effect was associated with pro-inflammatory status in the intestinal mucosa (TNF-α production in infected mice was increased by C4) and an increase in total IgA secretion. At a systemic level, C4 did not promote a pro-inflammatory response, although production of the immunoregulatory cytokine IFN-γ was enhanced. These findings suggest that L. plantarum C4 can increase resistance to intestinal infections through its immunomodulatory activity.
Assuntos
Produtos Fermentados do Leite/microbiologia , Lactobacillus plantarum/isolamento & purificação , Lactobacillus plantarum/fisiologia , Probióticos , Yersiniose/prevenção & controle , Yersinia enterocolitica , Animais , Citocinas/imunologia , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Imunoglobulina A Secretora/imunologia , Imunomodulação , Interferon gama/sangue , Interferon gama/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Lactobacillus plantarum/crescimento & desenvolvimento , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/microbiologia , Fator de Necrose Tumoral alfa/imunologia , Yersiniose/imunologia , Yersinia enterocolitica/crescimento & desenvolvimento , Yersinia enterocolitica/imunologiaRESUMO
DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Mielopoese/imunologia , Yersiniose/imunologia , Yersinia enterocolitica/imunologia , Animais , Células Dendríticas/patologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Interferon gama/imunologia , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Yersiniose/patologiaRESUMO
The aim of this study was to find out if reactive arthritis was involved in the aetiology of chronic closed lock of the temporomandibular joint (TMJ) by looking for bacterial antigens in the synovial membrane of the TMJ, and by studying the antibody serology and carriage of human leucocyte antigen (HLA) B27 in patients with chronic closed lock. Patients with reciprocal clicking and healthy subjects acted as controls. We studied a total of 43 consecutive patients, 15 with chronic closed lock, 13 with reciprocal clicking, and 15 healthy controls with no internal derangements of the TMJ. Venous blood samples were collected from all subjects for measurement of concentrations of HLA tissue antigen and serology against Chlamydia trachomatis, Yersinia enterocolitica, Salmonella spp., Campylobacter jejuni, and Mycoplasma pneumoniae. Samples of synovial tissue from patients with closed lock and reciprocal clicking were obtained during discectomy and divided into two pieces, the first of which was tested by strand displacement amplification for the presence of C trachomatis, and the second of which was analysed for the presence of species-specific bacterial DNA using 16s rRNA pan-polymerase chain reaction (PCR). There were no significant differences between the groups in the incidence of antibodies against M pneumoniae, Salmonella spp. or Y enterocolitica. No patient had antibodies towards C trachomatis or C jejuni. We found no bacterial DNA in the synovial fluid from any patient. The HLA B27 antigen was present in 2/15 subjects in both the closed lock and control groups, and none in the reciprocal clicking group. In conclusion, reactive arthritis does not seem to be the mechanism of internal derangement of the TMJ.
Assuntos
Artrite Reativa/microbiologia , Luxações Articulares/microbiologia , Transtornos da Articulação Temporomandibular/microbiologia , Adulto , Idoso , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/análise , Artrite Reativa/genética , Campylobacter jejuni/genética , Campylobacter jejuni/imunologia , Estudos de Casos e Controles , Chlamydia trachomatis/genética , Chlamydia trachomatis/imunologia , DNA Bacteriano/análise , Feminino , Antígeno HLA-B27/análise , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Luxações Articulares/imunologia , Masculino , Pessoa de Meia-Idade , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/imunologia , Medição da Dor/métodos , Amplitude de Movimento Articular/fisiologia , Salmonella/genética , Salmonella/imunologia , Membrana Sinovial/imunologia , Transtornos da Articulação Temporomandibular/imunologia , Yersinia enterocolitica/genética , Yersinia enterocolitica/imunologia , Adulto JovemRESUMO
The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2 x 10(6) TCID50 was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.
Assuntos
Proteínas E2 de Adenovirus/imunologia , Adesinas Bacterianas/imunologia , Peste Suína Clássica/imunologia , Vacinas Virais/imunologia , Adenovírus Humanos/imunologia , Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/genética , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Vírus da Febre Suína Clássica/imunologia , Glicoproteínas/biossíntese , Glicoproteínas/imunologia , Células HEK293 , Humanos , Distribuição Aleatória , Suínos , Vacinação , Potência de Vacina , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Replicação Viral/genética , Yersinia enterocolitica/imunologia , Yersinia pseudotuberculosis/imunologiaRESUMO
How intestinal epithelial cells (IECs) recognize pathogens and activate inflammasomes at intestinal surfaces is poorly understood. We hypothesized that IECs use integrin receptors to recognize pathogens and initiate inflammation within the intestinal tract. We find that IECs infected with Yersinia enterocolitica, an enteric pathogen, use ß1 integrins as pathogen recognition receptors detecting the bacterial adhesin invasin (Inv). The Inv-integrin interaction provides the first signal for NLRP3 inflammasome activation with the type three secretion system translocon providing the second signal for inflammasome activation, resulting in release of IL-18. During infection, Yersinia employs two virulence factors, YopE and YopH, to counteract Inv-mediated integrin-dependent inflammasome activation. Furthermore, NLRP3 inflammasome activation in epithelial cells requires components of the focal adhesion complex signaling pathway, focal adhesion kinase, and rac1. The binding of Inv to ß1 integrins rapidly induces IL-18 mRNA expression, suggesting integrins provide a first signal for NLRP3 inflammasome activation. These data suggest integrins function as pathogen recognition receptors on IECs to rapidly induce inflammasome-derived IL-18-mediated responses.
Assuntos
Células Epiteliais/imunologia , Inflamassomos/imunologia , Inflamassomos/metabolismo , Integrina alfa5beta1/fisiologia , Mucosa Intestinal/imunologia , Transdução de Sinais/imunologia , Yersinia enterocolitica/imunologia , Adesinas Bacterianas/metabolismo , Adesinas Bacterianas/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/farmacologia , Células CACO-2 , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ligação Proteica/imunologia , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/genética , Fatores de Virulência/fisiologia , Yersinia enterocolitica/genéticaRESUMO
Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3/metabolismo , Doença de Crohn/genética , Polimorfismo de Nucleotídeo Único/genética , Proteólise , Motivos de Aminoácidos , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/química , Caspase 3/deficiência , Caspase 3/genética , Linhagem Celular , Células Cultivadas , Doença de Crohn/patologia , Citocinas/imunologia , Ativação Enzimática , Feminino , Privação de Alimentos , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Estresse Fisiológico , Yersinia enterocolitica/imunologiaRESUMO
Neonatal animals are generally very susceptible to infection with bacterial pathogens. However, we recently reported that neonatal mice are highly resistant to orogastric infection with Yersinia enterocolitica. Here, we show that proinflammatory responses greatly exceeding those in adults arise very rapidly in the mesenteric lymph nodes (MLN) of neonates. High-level induction of proinflammatory gene expression occurred in the neonatal MLN as early as 18 h postinfection. Marked innate phagocyte recruitment was subsequently detected at 24 h postinfection. Enzyme-linked immunosorbent spot assay (ELISPOT) analyses indicated that enhanced inflammation in neonatal MLN is contributed to, in part, by an increased frequency of proinflammatory cytokine-secreting cells. Moreover, both CD11b(+) and CD11b(-) cell populations appeared to play a role in proinflammatory gene expression. The level of inflammation in neonatal MLN was also dependent on key bacterial components. Y. enterocolitica lacking the virulence plasmid failed to induce innate phagocyte recruitment. In contrast, tumor necrosis factor alpha (TNF-α) protein expression and neutrophil recruitment were strikingly higher in neonatal MLN after infection with a yopP-deficient strain than with wild-type Y. enterocolitica, whereas only modest increases occurred in adults. This hyperinflammatory response was associated with greater colonization of the spleen and higher mortality in neonates, while there was no difference in mortality among adults. This model highlights the dynamic levels of inflammation in the intestinal lymphoid tissues and reveals the protective (wild-type strain) versus harmful (yopP-deficient strain) consequences of inflammation in neonates. Moreover, these results reveal that the neonatal intestinal lymphoid tissues have great potential to rapidly mobilize innate components in response to infection with bacterial enteropathogens.
Assuntos
Linfonodos/imunologia , Linfonodos/microbiologia , Linfadenite Mesentérica/imunologia , Linfadenite Mesentérica/microbiologia , Yersiniose/imunologia , Yersiniose/patologia , Yersinia enterocolitica/imunologia , Animais , Animais Recém-Nascidos , Perfilação da Expressão Gênica , Inflamação , Linfonodos/patologia , Macrófagos/imunologia , Linfadenite Mesentérica/patologia , Camundongos , Baço/microbiologia , Análise de Sobrevida , Yersiniose/microbiologiaRESUMO
BACKGROUND: The pathogenic Yersinia species exhibit a primarily extracellular lifestyle through manipulation of host signaling pathways that regulate pro-inflammatory gene expression and cytokine release. To identify host genes that are targeted by Yersinia during the infection process, we performed an RNA interference (RNAi) screen based on recovery of host NF-κB-mediated gene activation in response to TNF-α stimulation upon Y. enterocolitica infection. RESULTS: We screened shRNAs against 782 genes in the human kinome and 26 heat shock genes, and identified 19 genes that exhibited ≥ 40% relative increase in NF-κB reporter gene activity. The identified genes function in multiple cellular processes including MAP and ERK signaling pathways, ion channel activity, and regulation of cell growth. Pre-treatment with small molecule inhibitors specific for the screen hits c-KIT and CKII recovered NF-κB gene activation and/or pro-inflammatory TNF-α cytokine release in multiple cell types, in response to either Y. enterocolitica or Y. pestis infection. CONCLUSIONS: We demonstrate that pathogenic Yersinia exploits c-KIT signaling in a T3SS-dependent manner to downregulate expression of transcription factors EGR1 and RelA/p65, and pro-inflammatory cytokines. This study is the first major functional genomics RNAi screen to elucidate virulence mechanisms of a pathogen that is primarily dependent on extracellular-directed immunomodulation of host signaling pathways for suppression of host immunity.
Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Yersinia enterocolitica/imunologia , Yersinia enterocolitica/patogenicidade , Linhagem Celular , Citocinas/biossíntese , Regulação para Baixo , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Humanos , Fator de Transcrição RelA/biossínteseRESUMO
Brucella abortus and Yersinia enterocolitica serotype O:9 serologically cross-react in the immune response with the host; therefore, our aim was to compare the immune responses to these two pathogens. We selected typical B. abortus and Y. enterocolitica O:9 strains to study the cytokine immune response and the histopathological changes in livers and spleens of BALB/c mice. The data showed the cytokine responses to the two strains of pathogens were different, where the average levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-α) were higher with B. abortus infections than with Y. enterocolitica O:9 infections, especially for IFN-γ, while the IL-10 level was lower and the levels of IL-1ß, IL-4, IL-5, and IL-6 were similar. The histopathological effects in the livers and spleens of the BALB/c mice with B. abortus and Y. enterocolitica O:9 infections were similar; however, the pathological changes in the liver were greater with B. abortus infections, while damage in the spleen was greater with Y. enterocolitica O:9 infections. These observations show that different cytokine responses and histopathological changes occur with B. abortus and Y. enterocolitica O:9 infections.
Assuntos
Brucella abortus/imunologia , Brucelose/imunologia , Citocinas/metabolismo , Yersiniose/imunologia , Yersinia enterocolitica/imunologia , Animais , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Interleucina-6/metabolismo , Fígado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Yersinia enterocolitica is a human pathogen that is ubiquitous in livestock, especially pigs. The bacteria are able to colonize the intestinal tract of a variety of mammalian hosts, but the severity of induced gut-associated diseases (yersiniosis) differs significantly between hosts. To gain more information about the individual virulence determinants that contribute to colonization and induction of immune responses in different hosts, we analyzed and compared the interactions of different human- and animal-derived isolates of serotypes O:3, O:5,27, O:8, and O:9 with murine, porcine, and human intestinal cells and macrophages. The examined strains exhibited significant serotype-specific cell binding and entry characteristics, but adhesion and uptake into different host cells were not host specific and were independent of the source of the isolate. In contrast, survival and replication within macrophages and the induced proinflammatory response differed between murine, porcine, and human macrophages, suggesting a host-specific immune response. In fact, similar levels of the proinflammatory cytokine macrophage inflammatory protein 2 (MIP-2) were secreted by murine bone marrow-derived macrophages with all tested isolates, but the equivalent interleukin-8 (IL-8) response of porcine bone marrow-derived macrophages was strongly serotype specific and considerably lower in O:3 than in O:8 strains. In addition, all tested Y. enterocolitica strains caused a considerably higher level of secretion of the anti-inflammatory cytokine IL-10 by porcine than by murine macrophages. This could contribute to limiting the severity of the infection (in particular of serotype O:3 strains) in pigs, which are the primary reservoir of Y. enterocolitica strains pathogenic to humans.
Assuntos
Interações Hospedeiro-Patógeno , Yersinia enterocolitica/classificação , Yersinia enterocolitica/imunologia , Animais , Aderência Bacteriana , Células Cultivadas , Citocinas/metabolismo , Endocitose , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Sorotipagem , Suínos , Yersinia enterocolitica/isolamento & purificaçãoRESUMO
Extracellular Gram-negative pathogenic bacteria target essential cytoplasmic processes of eukaryotic cells by using effector protein delivery systems such as the type III secretion system (T3SS). These secretion systems directly inject effector proteins into the host cell cytoplasm. Among the T3SS-dependent Yop proteins of pathogenic Yersinia, the function of the effector protein YopM remains enigmatic. In a recent study, we demonstrated that recombinant YopM from Yersinia enterocolitica enters host cells autonomously without the presence of bacteria and thus identified YopM as a novel bacterial cell-penetrating protein. Following entry YopM down-regulates expression of pro-inflammatory cytokines such as tumor necrosis factor α. These properties earmark YopM for further development as a novel anti-inflammatory therapeutic. To elucidate the uptake and intracellular targeting mechanisms of this bacterial cell-penetrating protein, we analyzed possible routes of internalization employing ultra-cryo electron microscopy. Our results reveal that under physiological conditions, YopM enters cells predominantly by exploiting endocytic pathways. Interestingly, YopM was detected free in the cytosol and inside the nucleus. We could not observe any colocalization of YopM with secretory membranes, which excludes retrograde transport as the mechanism for cytosolic release. However, our findings indicate that direct membrane penetration and/or an endosomal escape of YopM contribute to the cytosolic and nuclear localization of the protein. Surprisingly, even when endocytosis is blocked, YopM was found to be associated with endosomes. This suggests an intracellular endosome-associated transport of YopM.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Transporte Biológico Ativo , Compartimento Celular , Peptídeos Penetradores de Células/imunologia , Peptídeos Penetradores de Células/metabolismo , Endocitose , Endossomos/metabolismo , Endossomos/ultraestrutura , Células HeLa , Humanos , Imunossupressores/imunologia , Imunossupressores/metabolismo , Microscopia Imunoeletrônica , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Yersinia enterocolitica/imunologia , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidadeRESUMO
We investigated the lethality of pathogenic Yersinia enterocolitica bioserotypes 1B/O:8 and 2/O:9 in susceptible BALB/C and resistant C57BL/6 mice; the cytokine alterations and histopathological changes were observed comparing the two strains in BALB/C mice. The data showed the 50% lethal dose (LD50) for the pathogenic Y. enterocolitica bioserotype 1B/O:8 was 10³ cfu in both BALB/C and C57BL/6 mice; while the LD50 for the 2/O:9 was 108 cfu in BALB/C mice and 109 cfu in C57BL/6 mice, a large difference. After infection with the two strains in BALB/C mice, GM-CSF (granulocyte-macrophage colony stimulating factor), IFN-γ (interferon-γ), IL-1ß (interleukin-1ß), IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and TNF-α (tumor necrosis factor-α) appeared as a cytokine storm in a short period, reached peak values, and then quickly decreased. This appeared important for the immune response and cytokine immunopathogenesis in pathogenic Y. enterocolitica infections. In the initial infection stage, GM-CSF, IL-6, and TNF-α of 2/O:9 were higher than 1B/O:8; and subsequently the status was reversed. However, levels of IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-10, IL-12 following infection with 1B/O:8 were always higher than with 2/O:9. The histopathological changes in the liver and spleen in BALB/C mice infected with the two strains were similar at different times and doses. These observations show the different immunological effects and changes for pathogenic Y. enterocolitica 1B/O:8 and 2/O:9 infections using the mouse model.
Assuntos
Citocinas/biossíntese , Yersiniose/imunologia , Yersinia enterocolitica/imunologia , Yersinia enterocolitica/patogenicidade , Animais , Feminino , Fígado/imunologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Sorotipagem , Especificidade da Espécie , Baço/imunologia , Baço/patologia , Virulência/imunologia , Yersiniose/microbiologia , Yersiniose/patologia , Yersinia enterocolitica/classificaçãoRESUMO
Yersinia enterocolitica is an important human pathogen. Yersiniosis, caused by Y. enterocolitica, has become more prevalent globally in recent years. Prevention of yersiniosis still remains a challenge, and an efficacious and safe vaccine that confers protection against this enteric pathogen needs to be developed. In this study, a novel vaccine based on the bacterial ghost, in combination with mutation of the Y. enterocolitica msbB gene, was developed and the immunopotency of this vaccine was evaluated in mice. Significant levels of IgG1/IgG2a antibodies and IL-4/IFN-γ cytokines were detected after mice were administered this vaccine intragastrically, indicating that a Th1/Th2-mediated mixed immune response was stimulated. Importantly, mutation of the msbB gene efficiently reduced secretion of the proinflammatory cytokines IL-1ß, IL-6 and TNF-α, suggesting a reduction in inflammatory reaction caused by lipopolysaccharide. In addition, when challenged with a dose that was 100-fold the minimal lethal dose of the virulent wild strain of Y. enterocolitica, this mutated ghost vaccine was capable of eliciting the same effective protection (80%) in comparison with the non-mutated ghost strain, and the survival time was extended by at least two days. Together, our results demonstrated that this novel ghost bacterial strain could be used as a safe and effective vaccine against Y. enterocolitica.
Assuntos
Proteínas de Bactérias/genética , Vacinas Bacterianas/imunologia , Mediadores da Inflamação/imunologia , Inflamação/imunologia , Mutação/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Formação de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Feminino , Imunoglobulina G/imunologia , Interferon gama , Interleucina-4/imunologia , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação/imunologia , Células Th1/imunologia , Células Th2/imunologia , Fator de Necrose Tumoral alfa/imunologia , Yersiniose/imunologia , Yersiniose/prevenção & controleRESUMO
Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.