RESUMO
Mucosal barrier integrity and pathogen clearance is a complex process influenced by both Th17 and Treg cells. Previously, we had described the DNA methylation profile of Th17 cells and identified Zinc finger protein (Zfp)362 to be uniquely demethylated. Here, we generated Zfp362-/- mice to unravel the role of Zfp362 for Th17 cell biology. Zfp362-/- mice appeared clinically normal, showed no phenotypic alterations in the T-cell compartment, and upon colonization with segmented filamentous bacteria, no effect of Zfp362 deficiency on Th17 cell differentiation was observed. By contrast, Zfp362 deletion resulted in increased frequencies of colonic Foxp3+ Treg cells and IL-10+ and RORγt+ Treg cell subsets in mesenteric lymph nodes. Adoptive transfer of naïve CD4+ T cells from Zfp362-/- mice into Rag2-/- mice resulted in a significantly lower weight loss when compared with controls receiving cells from Zfp362+/+ littermates. However, this attenuated weight loss did not correlate with alterations of Th17 cells but instead was associated with an increase of effector Treg cells in mesenteric lymph nodes. Together, these results suggest that Zfp362 plays an important role in promoting colonic inflammation; however, this function is derived from constraining the effector function of Treg cells rather than directly promoting Th17 cell differentiation.
Assuntos
Linfócitos T Reguladores , Células Th17 , Camundongos , Animais , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Diferenciação Celular , Inflamação/metabolismo , Redução de Peso , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismoRESUMO
Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. All three itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.
Assuntos
Vírus da Influenza A/imunologia , Macrófagos/imunologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Succinatos/farmacologia , Células A549 , Animais , Carboxiliases/deficiência , Carboxiliases/imunologia , Citocinas/genética , Citocinas/imunologia , Humanos , Macrófagos/virologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Células THP-1RESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1010219.].
RESUMO
The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage.
Assuntos
Citrobacter rodentium/crescimento & desenvolvimento , Infecções por Enterobacteriaceae/metabolismo , Ácidos Graxos/metabolismo , Microbioma Gastrointestinal , Animais , CamundongosRESUMO
Cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunidade nas Mucosas , Vacinas contra Influenza/imunologia , Muromegalovirus/imunologia , Administração Intranasal , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocinas/biossíntese , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Produtos do Gene env/administração & dosagem , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Vetores Genéticos , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Células NIH 3T3 , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Infections with enterohemorrhagic Escherichia coli (EHEC) cause disease ranging from mild diarrhea to hemolytic-uremic syndrome (HUS) and are the most common cause of renal failure in children in high-income countries. The severity of the disease derives from the release of Shiga toxins (Stx). The use of antibiotics to treat EHEC infections is generally avoided, as it can result in increased stx expression. Here, we systematically tested different classes of antibiotics and found that their influence on stx expression and release varies significantly. We assessed a selection of these antibiotics in vivo using the Citrobacter rodentium Ïstx2dact mouse model and show that stx2d-inducing antibiotics resulted in weight loss and kidney damage despite clearance of the infection. However, several non-Stx-inducing antibiotics cleared bacterial infection without causing Stx-mediated pathology. Our results suggest that these antibiotics might be useful in the treatment of EHEC-infected human patients and decrease the risk of HUS development.
Assuntos
Injúria Renal Aguda/prevenção & controle , Antibacterianos/uso terapêutico , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Toxina Shiga II/metabolismo , Injúria Renal Aguda/microbiologia , Animais , Citrobacter rodentium/genética , Citrobacter rodentium/metabolismo , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Síndrome Hemolítico-Urêmica/tratamento farmacológico , Síndrome Hemolítico-Urêmica/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Toxina Shiga II/genética , Toxina Shiga II/toxicidadeRESUMO
Effector CD4+ T cell subsets, whose differentiation is facilitated by distinct cytokine cues, amplify the corresponding type of inflammatory response. Regulatory T (Treg) cells integrate environmental cues to suppress particular types of inflammation. In this regard, STAT3, a transcription factor essential for T helper 17 (Th17) cell differentiation, is necessary for Treg cell-mediated control of Th17 cell responses. Here, we showed that anti-inflammatory interleukin-10 (IL-10), and not proinflammatory IL-6 and IL-23 cytokine signaling, endowed Treg cells with the ability to suppress pathogenic Th17 cell responses. Ablation of the IL-10 receptor in Treg cells resulted in selective dysregulation of Th17 cell responses and colitis similar to that observed in mice harboring STAT3-deficient Treg cells. Thus, Treg cells limit Th17 cell inflammation by serving as principal amplifiers of negative regulatory circuits operating in immune effector cells.
Assuntos
Colite/imunologia , Interleucina-10/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Linhagem da Célula , Colite/patologia , Interleucina-10/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Receptores de Interleucina-10/deficiência , Receptores de Interleucina-10/imunologia , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/citologiaRESUMO
Pseudomonas aeruginosa is an environmental microorganism and a causative agent of diverse acute and chronic, biofilm-associated infections. Advancing research-based knowledge on its adaptation to conditions within the human host is bound to reveal novel strategies and targets for therapeutic intervention. Here, we investigated the traits that P. aeruginosa PA14 as well as a virulence attenuated ΔlasR mutant need to survive in selected murine infection models. Experimentally, the genetic programs that the bacteria use to adapt to biofilm-associated versus acute infections were dissected by passaging transposon mutant libraries through mouse lungs (acute) or mouse tumours (biofilm-infection). Adaptive metabolic changes of P. aeruginosa were generally required during both infection processes. Counter-selection against flagella expression was observed during acute lung infections. Obviously, avoidance of flagella-mediated activation of host immunity is advantageous for the wildtype bacteria. For the ΔlasR mutant, loss of flagella did not confer a selective advantage. Apparently, other pathogenesis mechanisms are active in this virulence attenuated strain. In contrast, the infective process of P. aeruginosa in the chronic biofilm model apparently required expression of flagellin. Together, our findings imply that the host immune reactions against the infectious agent are very decisive for acuteness and duration of the infectious disease. They direct disease outcome.
Assuntos
Flagelos/fisiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Animais , Biofilmes , Doença Crônica , Flagelos/genética , Camundongos , Camundongos Endogâmicos BALB C , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/microbiologia , VirulênciaRESUMO
AIDS-related lymphomas (ARLs) are expected to increase in the future since combined antiretroviral therapy (cART) enhances the life expectancy of HIV-1-infected (HIV+) patients but does not affect the occurrence of ARLs to the same extent as that of other tumors. Lymphangiogenesis is essential in supporting growth and metastatic spreading of ARLs. HIV-1 does not infect the neoplastic B cells, but HIV-1 proteins have been hypothesized to play a key role in sustaining a prolymphangiogenic microenvironment in lymphoid organs. The HIV-1 matrix protein p17 is detected in blood and accumulates in the germinal centers of lymph nodes of HIV+ patients under successful cART. The viral protein displays potent lymphangiogenic activity in vitro and in vivo This is, at least in part, mediated by the secretion of the lymphangiogenic factor endothelin-1, suggesting that activation of a secretory pathway sustains the lymphangiogenic activity of p17. Here, we show that the p17 lymphangiogenic activity occurs on human lymph node-derived lymphatic endothelial cells (LN-LECs) under stress conditions only and relies entirely on activation of an autophagy-based pathway. In fact, induction of autophagy by p17 promotes lymphangiogenesis, whereas pharmacological and genetic inhibition of autophagy inhibits p17-triggered lymphangiogenesis. Similarly, the vasculogenic activity of p17 was totally inhibited in autophagy-incompetent mice. Our findings reveal a previously unrecognized role of autophagy in lymphangiogenesis and open the way to identify novel treatment strategies aimed at inhibiting aberrant tumor-driven lymphangiogenesis in HIV+ patients.IMPORTANCE AIDS-related lymphomas (ARLs) are the most common malignancies in HIV-1-infected (HIV+) patients after the introduction of combined antiretroviral therapy (cART). Lymphangiogenesis is of critical importance in sustaining growth and metastasis of ARLs. Indeed, enhanced lymphangiogenesis occurs in the lymph nodes of HIV+ patients under successful cART. The HIV-1 matrix protein p17 is detected in blood and accumulates in the lymph node germinal centers even in the absence of virus replication. Several findings suggest a key role for p17 as a microenvironmental factor capable of promoting lymphangiogenesis. Here, we show that p17 promotes lymphangiogenesis of human lymph node-derived lymphatic endothelial cells (LN-LECs). The lymphangiogenic activity of p17 is sustained by an autophagy-based pathway that enables LN-LECs to release prolymphangiogenic factors into the extracellular microenvironment. Our findings indicate that specific targeting of autophagy may provide an important new tool for treating ARLs.
Assuntos
Autofagia , Células Endoteliais/efeitos dos fármacos , Antígenos HIV/metabolismo , Linfangiogênese , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Background: To successfully limit pathogen dissemination, an immunological link between the entry tissue of the pathogen and the underlying secondary lymphoid organs (SLOs) needs to be established to prime adaptive immune responses. Here, the prerequisite of CCR7 to mount host immune responses within SLOs during gastrointestinal Yersinia pseudotuberculosis infection to limit pathogen spread was investigated. Methods: Survival, bacterial dissemination, and intestinal and systemic pathology of wild-type and CCR7-/- mice were assessed and correlated to the presence of immune cell subsets and cytokine responses throughout the course of infection. Results: The CCR7-/- mice show a significantly higher morbidity and are more prone to pathogen dissemination and intestinal and systemic inflammation during the oral route of infection. Significant impact of CCR7 deficiency over the course of infection on several immunological parameters were observed (ie, elevated neutrophil-dominated innate immune response in Peyer's patches, limited dendritic cell migration to mesenteric lymph nodes [mLNs] causing reduced T cell-mediated adaptive immune responses (in particular Th17-like responses) in mLNs). Conclusions: Our work indicates that CCR7 is required to mount a robust immune response against enteropathogenic Y. pseudotuberculosis by promoting Th17-like responses in mLNs.
Assuntos
Predisposição Genética para Doença , Receptores CCR7/imunologia , Células Th17/imunologia , Infecções por Yersinia pseudotuberculosis/imunologia , Animais , Movimento Celular , Células Dendríticas/imunologia , Interações Hospedeiro-Patógeno/genética , Intestinos/imunologia , Intestinos/microbiologia , Linfonodos/imunologia , Linfonodos/microbiologia , Camundongos , Células Mieloides/imunologia , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/microbiologia , Receptores CCR7/genética , Yersinia pseudotuberculosis , Infecções por Yersinia pseudotuberculosis/genéticaRESUMO
Coagulation is a mechanism for wound healing after injury. Several recent studies delineate an additional role of the intrinsic pathway of coagulation, also known as the contact system, in the early innate immune response against bacterial infections. In this study, we investigated the role of factor XIII (FXIII), which is activated upon coagulation induction, during Streptococcus pyogenes-mediated skin and soft tissue infections. FXIII has previously been shown to be responsible for the immobilization of bacteria within a fibrin network which may prevent systemic bacterial dissemination. In order to investigate if the FXIII-mediated entrapment of S. pyogenes also influences the disease outcome we used a murine S. pyogenes M1 skin and soft tissue infection model. Here, we demonstrate that a lack of FXIII leads to prolonged clotting times, increased signs of inflammation, and elevated bacterial dissemination. Moreover, FXIII-deficient mice show an impaired survival when compared with wildtype animals. Additionally, local reconstitution of FXIII-deficient mice with a human FXIII-concentrate (Fibrogammin®P) could reduce the systemic complications, suggesting a protective role for FXIII during early S. pyogenes skin infection. FXIII therefore might be a possible therapeutically application to support the early innate immune response during skin infections caused by S. pyogenes.
Assuntos
Fator XIII/metabolismo , Interações Hospedeiro-Patógeno , Imunidade Inata , Dermatopatias Bacterianas/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Antígenos de Bactérias , Proteínas da Membrana Bacteriana Externa , Proteínas de Transporte , Modelos Animais de Doenças , Fator XIII/genética , Feminino , Camundongos Endogâmicos CBA , Camundongos Knockout , Dermatopatias Bacterianas/microbiologia , Dermatopatias Bacterianas/patologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/patogenicidade , Análise de SobrevidaRESUMO
Patients suffering from cystic fibrosis (CF) are commonly affected by chronic Pseudomonas aeruginosa biofilm infections. This is the main cause for the high disease severity. In this study, we demonstrate that P. aeruginosa is able to efficiently colonize murine solid tumors after intravenous injection and to form biofilms in this tissue. Biofilm formation was evident by electron microscopy. Such structures could not be observed with transposon mutants, which were defective in biofilm formation. Comparative transcriptional profiling of P. aeruginosa indicated physiological similarity of the bacteria in the murine tumor model and the CF lung. The efficacy of currently available antibiotics for treatment of P. aeruginosa-infected CF lungs, such as ciprofloxacin, colistin, and tobramycin, could be tested in the tumor model. We found that clinically recommended doses of these antibiotics were unable to eliminate wild-type P. aeruginosa PA14 while being effective against biofilm-defective mutants. However, colistin-tobramycin combination therapy significantly reduced the number of P. aeruginosa PA14 cells in tumors at lower concentrations. Hence, we present a versatile experimental system that is providing a platform to test approved and newly developed antibiofilm compounds.
Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Colistina/farmacologia , Fibrose Cística/tratamento farmacológico , Fibrose Cística/microbiologia , Modelos Animais de Doenças , Feminino , Pneumopatias/tratamento farmacológico , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana/métodos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Tobramicina/farmacologiaRESUMO
Recent work has shown that coagulation and innate immunity are tightly interwoven host responses that help eradicate an invading pathogen. Some bacterial species, including Staphylococcus aureus, secrete pro-coagulant factors that, in turn, can modulate these immune reactions. Such mechanisms may not only protect the micro-organism from a lethal attack, but also promote bacterial proliferation and the establishment of infection. Our data showed that coagulase-positive S. aureus bacteria promoted clotting of plasma which was not seen when a coagulase-deficient mutant strain was used. Furthermore, in vitro studies showed that this ability constituted a mechanism that supported the aggregation, survival and persistence of the micro-organism within the fibrin network. These findings were also confirmed when agglutination and persistence of coagulase-positive S. aureus bacteria at the local focus of infection were studied in a subcutaneous murine infection model. In contrast, the coagulase-deficient S. aureus strain which was not able to induce clotting failed to aggregate and to persist in vivo. In conclusion, our data suggested that coagulase-positive S. aureus have evolved mechanisms that prevent their elimination within a fibrin clot.
Assuntos
Coagulação Sanguínea , Fibrina/metabolismo , Evasão da Resposta Imune , Infecções Estafilocócicas/sangue , Staphylococcus aureus/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coagulase/genética , Coagulase/metabolismo , Fibrina/genética , Humanos , Camundongos , Camundongos Endogâmicos CBA , Viabilidade Microbiana , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but the functional consequences of this toxin for host-pathogen interactions during the infection are unknown. In the present study we show that CNFY has a strong influence on virulence. We demonstrate that the CNFY toxin is thermo-regulated and highly expressed in all colonized lymphatic tissues and organs of orally infected mice. Most strikingly, we found that a cnfY knock-out variant of a naturally toxin-expressing Y. pseudotuberculosis isolate is strongly impaired in its ability to disseminate into the mesenteric lymph nodes, liver and spleen, and has fully lost its lethality. The CNFY toxin contributes significantly to the induction of acute inflammatory responses and to the formation of necrotic areas in infected tissues. The analysis of the host immune response demonstrated that presence of CNFY leads to a strong reduction of professional phagocytes and natural killer cells in particular in the spleen, whereas loss of the toxin allows efficient tissue infiltration of these immune cells and rapid killing of the pathogen. Addition of purified CNFY triggers formation of actin-rich membrane ruffles and filopodia, which correlates with the activation of the Rho GTPases, RhoA, Rac1 and Cdc42. The analysis of type III effector delivery into epithelial and immune cells in vitro and during the course of the infection further demonstrated that CNFY enhances the Yop translocation process and supports a role for the toxin in the suppression of the antibacterial host response. In summary, we highlight the importance of CNFY for pathogenicity by showing that this toxin modulates inflammatory responses, protects the bacteria from attacks of innate immune effectors and enhances the severity of a Yersinia infection.
Assuntos
Toxinas Bacterianas/metabolismo , Neuropeptídeos/metabolismo , Infecções por Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Toxinas Bacterianas/genética , Ativação Enzimática/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neuropeptídeos/genética , Transporte Proteico , Yersinia pseudotuberculosis/genética , Infecções por Yersinia pseudotuberculosis/genética , Infecções por Yersinia pseudotuberculosis/patologia , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTPRESUMO
Recognition of nucleic acids by TLR9 requires its trafficking from the endoplasmic reticulum to endolysosomal compartments and its subsequent proteolytic processing. Both processes depend on interactions of TLR9 with the polytopic endoplasmic reticulum-resident protein UNC93B1. To examine the intracellular behavior of TLR9 in primary APCs, we generated transgenic mice expressing a TLR9-GFP fusion. The TLR9-GFP transgene is functional and is proteolytically processed in resting bone marrow-derived macrophages (BMDMs), dendritic cells, and B cells. Inhibition of cleavage impairs TLR9-dependent responses in all primary APCs analyzed. The kinetics of TLR9-GFP processing in BMDMs and B cells differs: in B cells, proteolysis occurs at a faster rate, consistent with an almost exclusive localization to endolysosomes at the resting state. In contrast to the joint requirement for cathepsins L and S for TLR9 cleavage in macrophages, TLR9-GFP cleavage depends on cathepsin L activity in B cells. As expected, in BMDMs and B cells from UNC93B1 (3d) mutant mice, cleavage of TLR9-GFP is essentially blocked, and the expression level of UNC93B1 appears tightly correlated with TLR9-GFP cleavage. We conclude that proteolysis is a universal requirement for TLR9 activation in the primary cell types tested, however the cathepsin requirement, rate of cleavage, and intracellular behavior of TLR9 varies. The observed differences in trafficking indicate the possibility of distinct modes of endosomal content sampling to facilitate initiation of TLR-driven responses in APCs.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Animais , Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lisossomos/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Transgênicos , Estabilidade Proteica , Transporte Proteico , Proteólise , Transdução de Sinais , TransgenesRESUMO
BACKGROUND: Allergic asthma is a chronic inflammatory airway disease caused by exposure to airborne allergens. In order to develop novel therapies for allergic asthma, models that are relevant to human disease are needed. METHODS: Female BALB/c mice were presensitised subcutaneously with alum-adsorbed recombinant cat allergen Fel d 1, followed by intranasal challenges with cat dander extract spiked with recombinant Fel d 1 for 7 weeks. For reference, mice were presensitised and challenged with ovalbumin following the same protocol. Airway hyperresponsiveness, serum antibodies, airway inflammation and cell infiltration, and cytokines in lung tissue and bronchoalveolar lavage were measured. RESULTS: Mice presensitised with recombinant Fel d 1 and challenged with cat dander extract or presensitised and challenged with ovalbumin showed airway hyperresponsiveness in response to metacholine. Mice of the cat allergen model showed influx of neutrophils, eosinophils and lymphocytes in bronchoalveolar lavage, combined with increased levels of IL-17a and increased IL-4 mRNA expression in lung tissue. In contrast, mice sensitised and challenged with ovalbumin showed a predominant influx of eosinophils in bronchoalveolar lavage and had an increased expression of IL-5 in lung tissue. Both protocols induced features of lung tissue remodelling and allergen-specific antibody responses. CONCLUSIONS: The presented mouse model for cat allergen-induced asthma exhibits hallmarks of chronic allergic asthma, like airway hyperresponsiveness, a mixed neutrophilic/eosinophilic infiltration in bronchoalveolar lavage, expression of IL-17a and signs of remodelling in lung tissue. The model will provide a relevant platform for the development of novel treatment strategies.
Assuntos
Asma/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Linfócitos/imunologia , Neutrófilos/imunologia , Remodelação das Vias Aéreas , Animais , Anticorpos/sangue , Hiper-Reatividade Brônquica , Gatos , Células Cultivadas , Citocinas/metabolismo , Alérgenos Animais/imunologia , Feminino , Glicoproteínas/imunologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologiaRESUMO
Gut-draining mesenteric and celiac lymph nodes (mLNs and celLNs) critically contribute to peripheral tolerance toward food and microbial antigens by supporting the de novo induction of regulatory T cells (Tregs). These tolerogenic properties of mLNs and celLNs are stably imprinted within stromal cells (SCs) by microbial signals and vitamin A (VA), respectively. Here, we report that a single, transient gastrointestinal infection in the neonatal, but not adult, period durably abrogates the efficient Treg-inducing capacity of celLNs by altering the subset composition and gene expression profile of celLNSCs. These cells carry information about the early-life pathogen encounter until adulthood and durably instruct migratory dendritic cells entering the celLN with reduced tolerogenic properties. Mechanistically, transiently reduced VA levels cause long-lasting celLN functional impairment, which can be rescued by early-life treatment with VA. Together, our data highlight the therapeutic potential of VA to prevent sequelae post gastrointestinal infections in infants.
Assuntos
Linfonodos , Linfócitos T Reguladores , Vitamina A , Animais , Linfonodos/imunologia , Linfonodos/patologia , Linfonodos/efeitos dos fármacos , Vitamina A/farmacologia , Vitamina A/uso terapêutico , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Camundongos , Animais Recém-Nascidos , Tolerância Imunológica/efeitos dos fármacos , Células Dendríticas/imunologia , Camundongos Endogâmicos C57BL , FemininoRESUMO
IMPORTANCE: There is a strong need to find novel treatment options against urinary tract infections associated with antimicrobial resistance. This study evaluates two atypical tetracyclines, namely chelocardin (CHD) and amidochelocardin (CDCHD), with respect to their pharmacokinetics and pharmacodynamics. We show CHD and CDCHD are cleared at high concentrations in mouse urine. Especially, CDCHD is highly effective in an ascending urinary tract infection model, suggesting further preclinical evaluation.
Assuntos
Antibacterianos , Infecções Urinárias , Animais , Camundongos , Testes de Sensibilidade Microbiana , Antibacterianos/uso terapêutico , Antibacterianos/farmacocinética , Tetraciclinas/farmacologia , Tetraciclinas/uso terapêutico , Infecções Urinárias/tratamento farmacológicoRESUMO
The Klebsiella oxytoca species complex is part of the human microbiome, especially during infancy and childhood. K. oxytoca species complex strains can produce enterotoxins, namely, tilimycin and tilivalline, while also contributing to colonization resistance (CR). The relationship between these seemingly contradictory roles is not well understood. Here, by coupling ex vivo assays with CRISPR-mutagenesis and various mouse models, we show that K. oxytoca provides CR against Salmonella Typhimurium. In vitro, the antimicrobial activity against various Salmonella strains depended on tilimycin production and was induced by various simple carbohydrates. In vivo, CR against Salmonella depended on toxin production in germ-free mice, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo. Finally, dulcitol utilization was essential for toxin-independent CR in gnotobiotic mice. Together, this demonstrates that nutrient availability is key to both toxin-dependent and substrate-driven competition between K. oxytoca and Salmonella.
Assuntos
Klebsiella oxytoca , Infecções por Salmonella , Salmonella typhimurium , Klebsiella oxytoca/genética , Klebsiella oxytoca/metabolismo , Animais , Camundongos , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/efeitos dos fármacos , Humanos , Modelos Animais de Doenças , Enterotoxinas/metabolismo , Enterotoxinas/genética , Feminino , Camundongos Endogâmicos C57BL , Infecções por Klebsiella/microbiologia , Microbiota , Microbioma Gastrointestinal , Antibiose , BenzodiazepinonasRESUMO
Transfer of the gut microbiota from wild to laboratory mice alters the host's immune status and enhances resistance to infectious and metabolic diseases, but understanding of which microbes and how they promote host fitness is only emerging. Our analysis of metagenomic sequencing data reveals that Helicobacter spp. are enriched in wild compared with specific-pathogen-free (SPF) and conventionally housed mice, with multiple species commonly co-colonizing their hosts. We create laboratory mice harboring three non-SPF Helicobacter spp. to evaluate their effect on mucosal immunity and colonization resistance to the enteropathogen Citrobacter rodentium. Our experiments reveal that Helicobacter spp. interfere with C. rodentium colonization and attenuate C. rodentium-induced gut inflammation in wild-type (WT) mice, even preventing lethal infection in Rag2-/- SPF mice. Further analyses suggest that Helicobacter spp. interfere with tissue attachment of C. rodentium, putatively by reducing the availability of mucus-derived sugars. These results unveil pivotal protective functions of wild mouse microbiota constituents against intestinal infection.