Search

Brucella abortus is a Gram-negative bacterium responsible for a worldwide zoonotic infection—Brucellosis, which has been associated with high morbidity rate in humans and severe economic losses in infected livestock. The natural route of infection is through oral and nasal mucosa but the invasion process through host gut mucosa is yet to be understood. Studies have examined the role of NLRP6 (NOD-like receptor family pyrin domain-containing-6 protein) in gut homeostasis and defense against pathogens. Here, we investigated the impact of gut microbiota and NLRP6 in a murine model of Ba oral infection. Nlrp6-/- and wild-type (WT) mice were infected by oral gavage with Ba and tissues samples were collected at different time points. Our results suggest that Ba oral infection leads to significant alterations in gut microbiota. Moreover, Nlrp6-/- mice were more resistant to infection, with decreased CFU in the liver and reduction in gut permeability when compared to the control group. Fecal microbiota transplantation from WT and Nlrp6-/- into germ-free mice reflected the gut permeability phenotype from the donors. Additionally, depletion of gut microbiota by broad-spectrum-antibiotic treatment prevented Ba replication in WT while favoring bacterial growth in Nlrp6-/-. Finally, we observed higher eosinophils in the gut and leukocytes in the blood of infected Nlrp6-/- compared to WT-infected mice, which might be associated to the Nlrp6-/- resistance phenotype. Altogether, these results indicated that gut microbiota composition is the major factor involved in the initial stages of pathogen host replication and partially also by the resistance phenotype observed in Nlrp6 -/- mice regulating host inflammation against Ba infection., Author summary Brucella abortus (Ba) is an intracellular bacterium that causes zoonotic and clinical problems worldwide. Although the common route of infection is through oral and nasal, the mechanisms toward the gastrointestinal mucosa response is still unexplored. It is well known that microbiota promotes and maintains host intestinal homeostasis during bacterial infections. However, mechanisms by which the gut microbiota affects the Ba infection have not yet been demonstrated. Here, we provide significant insights into the relationship between gut microbiota and B. abortus oral infection and demonstrate the gut microbiota contribution to the gut permeability and dissemination of Ba. Furthermore, we investigated the participation of the gut microbiota from Nlrp6 deficient mice, on the gut permeability and Ba infection. Substantial experiments performed, mostly in vivo, showed that gut microbiota alterations promote gut barrier disruption, as indicated by increased gut permeability after Ba oral infection. Thus, our work highlights the role of gut mucosal environment through gut microbiota and Nlrp6 molecule involved in host innate immune responses to Ba infection.

The ST2 receptor plays an important role in the gut such as permeability regulation, epithelium regeneration, and promoting intestinal immune modulation. Here, we studied the role of ST2 receptor in a murine model of oral infection with Brucella abortus, its influence on gut homeostasis and control of bacterial replication. Balb/c (wild-type, WT) and ST2 deficient mice (ST2-/-) were infected by oral gavage and the results were obtained at 3 and 14 days post infection (dpi). Our results suggest that ST2-/- are more resistant to B. abortus infection, as a lower bacterial colony-forming unit (CFU) was detected in the livers and spleens of knockout mice, when compared to WT. Additionally, we observed an increase in intestinal permeability in WT-infected mice, compared to ST2-/- animals. Breakage of the intestinal epithelial barrier and bacterial dissemination might be associated with the presence of the ST2 receptor, since, in the knockout mice no change in intestinal permeability was observed after infection. Together with enhanced resistance to infection, ST2-/- produced greater levels of IFN-&gamma, and TNF-&alpha, in the small intestine, compared to WT mice. Nevertheless, in the systemic model of infection ST2 plays no role in controlling Brucella replication in vivo. Our results suggest that the ST2 receptor is involved in the invasion process of B. abortus by the mucosa in the oral infection model.

Microbiota-derived acetate coordinates innate immune responses during intestinal Clostridium difficile infection through its cognate receptor FFAR2. Acetate accelerates early neutrophil recruitment and increases ILC3 expression of the IL-1 receptor, boosting ILC3 production of IL-22 in response to neutrophil-derived IL-1β., Antibiotic-induced dysbiosis is a key predisposing factor for Clostridium difficile infections (CDIs), which cause intestinal disease ranging from mild diarrhea to pseudomembranous colitis. Here, we examined the impact of a microbiota-derived metabolite, short-chain fatty acid acetate, on an acute mouse model of CDI. We found that administration of acetate is remarkably beneficial in ameliorating disease. Mechanistically, we show that acetate enhances innate immune responses by acting on both neutrophils and ILC3s through its cognate receptor free fatty acid receptor 2 (FFAR2). In neutrophils, acetate-FFAR2 signaling accelerates their recruitment to the inflammatory sites, facilitates inflammasome activation, and promotes the release of IL-1β; in ILC3s, acetate-FFAR2 augments expression of the IL-1 receptor, which boosts IL-22 secretion in response to IL-1β. We conclude that microbiota-derived acetate promotes host innate responses to C. difficile through coordinate action on neutrophils and ILC3s., Graphical Abstract

Innate immune response against Brucella abortus involves activation of Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Among the NLRs involved in the recognition of B. abortus are NLRP3 and AIM2. Here, we demonstrate that B. abortus triggers non-canonical inflammasome activation dependent on caspase-11 and gasdermin-D (GSDMD). Additionally, we identify that Brucella-LPS is the ligand for caspase-11 activation. Interestingly, we determine that B. abortus is able to trigger pyroptosis leading to pore formation and cell death, and this process is dependent on caspase-11 and GSDMD but independently of caspase-1 protease activity and NLRP3. Mice lacking either caspase-11 or GSDMD were significantly more susceptible to infection with B. abortus than caspase-1 knockout or wild-type animals. Additionally, guanylate-binding proteins (GBPs) present in mouse chromosome 3 participate in the recognition of LPS by caspase-11 contributing to non-canonical inflammasome activation as observed by the response of Gbpchr3-/- BMDMs to bacterial stimulation. We further determined by siRNA knockdown that among the GBPs contained in mouse chromosome 3, GBP5 is the most important for Brucella LPS to be recognized by caspase-11 triggering IL-1β secretion and LDH release. Additionally, we observed a reduction in neutrophil, dendritic cell and macrophage influx in spleens of Casp11-/- and Gsdmd-/- compared to wild-type mice, indicating that caspase-11 and GSDMD are implicated in the recruitment and activation of immune cells during Brucella infection. Finally, depletion of neutrophils renders wild-type mice more susceptible to Brucella infection. Taken together, these data suggest that caspase-11/GSDMD-dependent pyroptosis triggered by B. abortus is important to infection restriction in vivo and contributes to immune cell recruitment and activation., Author summary Brucella abortus is the causative agent of brucellosis, a zoonotic disease that affects both humans and cattle. In humans, it is characterized by undulant fever and chronic symptoms as arthritis, endocarditis, and meningitis, while in cattle it causes abortion and infertility. Due to its difficult diagnosis and treatment, it leads to severe economic losses and human suffering. Recently, a novel non-canonical inflammasome pathway was described that involves sensing of bacterial LPS by an intracellular receptor termed caspase-11 and leads to pyroptosis and non-canonical NLRP3 inflammasome activation. Here, we show that B. abortus or its purified LPS is able to activate the non-canonical inflammasome. In this process, activated caspase-11 leads to GSDMD-dependent pyroptosis. Moreover, this pathway was dependent of IFN-induced GBP proteins, mainly GBP5. To analyze the role of caspase-1, caspase-11 and GSDMD in controlling B. abortus infection, we infected knockout (KO) mice for these molecules and we observed that caspase-11 and GSDMD KO animals were more susceptible to infection compared to wild-type animals. Casp11-/- and Gsdmd-/- animals also recruited less immune cells in mouse spleens compared to wild-type animals in response to B. abortus. Thus, caspase-11 and GSDMD are major components of the innate immune system to restrict B. abortus in vivo. This pathway of bacterial sensing by the host immune system is important to future development of drugs and vaccines that may contribute to the control of brucellosis worldwide.

Absent in melanoma 2 (AIM2) is a sensor of cytosolic dsDNA and is responsible for the activation of inflammatory and host immune responses to DNA viruses and intracellular bacteria. AIM2 is a member of the hematopoietic interferon-inducible nuclear proteins with a 200 amino-acid repeat (HIN200) family, containing a pyrin domain (PYD) at the N-terminus. Several studies have demonstrated that AIM2 is responsible for host defense against intracellular bacteria such as Francisella tularensis, Listeria monocytogenes and Mycobacerium tuberculosis. However, the role of AIM2 in host defenses against Brucella is poorly understood. In this study, we have shown that AIM2 senses Brucella DNA in dendritic cells to induce pyroptosis and regulates type I IFN. Confocal microscopy of infected cells revealed co-localization between Brucella DNA and endogenous AIM2. Dendritic cells from AIM2 KO mice infected with B. abortus showed impaired secretion of IL-1β as well as compromised caspase-1 cleavage. AIM2 KO mice displayed increased susceptibility to B. abortus infection in comparison to wild-type mice, and this susceptibility was associated with defective IL-1β production together with reduced IFN-γ responses. In summary, the increased bacterial burden observed in vivo in AIM2 KO animals confirmed that AIM2 is essential for an effective innate immune response against Brucella infection.

Brucella abortus é uma bactéria gram-negativa, intracelular facultativa e causadora da Brucelose que é adquirida através da ingestão de carne, leite e seus derivados contaminados. Além disso, é uma zoonose bacteriana das mais difundidas no mundo, levando ao aborto em animais domésticos e de criação. Estudos recentes sugerem que o NLRP6, membro da família de receptores do tipo NOD, tem importante papel na regulação da resposta imune, renovação das células epiteliais e na composição da microbiota intestinal. Nesse contexto, o objetivo deste trabalho foi estudar os mecanismos envolvidos no processo de invasão e estabelecimento da infecção Brucella abortus por via oral, explorando o papel da molécula NLRP6 no processo de homeostase intestinal frente à infecção. Para isso, animais selvagens (WT) e deficientes para NLRP6 (Nlrp6-/-) foram inoculados via intragástrica com B. abortus cepa virulenta S2308 e amostras de diferentes tecidos foram coletadas em diferentes tempos. Como resultados, foi observado que animais Nlrp6-/- foram mais resistentes à infecção oral, apresentando redução da carga bacteriana no fígado, em relação aos WT. Entretanto, esse fenótipo não foi observado quando a infecção se deu por via intraperitoneal, sugerindo que o NLRP6 seja importante na fase de invasão de B. abortus através da mucosa intestinal. Além disso, a infecção oral induziu um aumento da permeabilidade intestinal nos animais WT infectados, mas não nos Nlrp6-/-. Foi observado, também, alterações significativas na composição da microbiota intestinal durante a infecção em ambos os grupos de animais, sugerindo-se um quadro de disbiose, e, portanto, participação da microbiota intestinal no contexto do processo infeccioso por B. abortus. Ademais, o transplante fecal da microbiota proveniente dos WT e Nlrp6-/- infectados para animais germ-free resultou no fenótipo de permeabilidade similar ao dos grupos doadores. No entanto, a depleção da microbiota por antibióticos de amplo-espectro nos animais WT preveniu a quebra de barreira epitelial intestinal e reduziu a carga bacteriana sistêmica. Por outro lado, a depleção em Nlrp6-/- favoreceu a invasão da B. abortus, mesmo sem alteração da permeabilidade intestinal. Em resumo, esses resultados indicam que a infecção oral por B. abortus altera a homeostase intestinal em favorecimento a sua invasão e estabelecimento de infecção sistêmica, e um dos mecanismos envolvidos nesse processo é dependente da molécula NLRP6. O NLRP6 mostrou-se importante no controle da sobrevida da bactéria B. abortus, na manutenção da barreira epitelial e na regulação negativa da resposta imune frente a infecção por via oral pela Brucella abortus, na qual a microbiota intestinal parece exercer um papel determinante Brucella abortus is a gram-negative, facultative intracellular bacterium that causes Brucellosis acquired through ingestion of meat, milk and its contaminated derivatives. In addition, it one of the most widespread bacterial zoonosis, leading to abortion in domestic and farmed animals. Recent studies suggest that the NLRP6, a member of NOD-like receptor family, plays an important role in the regulation of immune response, epithelial cell turnover and in the composition of the gut microbiota. In this context, the objective of this work was to study the mechanisms involved in the invasion and establishment of oral Brucella abortus infection, exploring the role of NLRP6 in the intestinal homeostasis during infection. Wild-type (WT) and NLRP6 (Nlrp6-/-) deficient animals were inoculated intragastrically with B. abortus virulent strain S2308 and samples from different tissues were collected at different times. As results, it was observed that Nlrp6-/- mice were more resistant to oral infection, presenting reduction of bacterial load in the liver when compared to WT animals. However, this phenotype was not observed when the infection occurred intraperitoneally, suggesting that NLRP6 is important in the phase of B. abortus invasion through the intestinal mucosa. In addition, oral infection induced an increase in intestinal permeability in infected WT mice, but not in Nlrp6-/-. Significant changes in the composition of the intestinal microbiota during infection in both groups of animals were also observed, suggesting dysbiosis. Therefore, our findings suggest the participation of the gut microbiota in the context of B. abortus infection. Additionally, fecal transplantation of the microbiota from the infected WT and Nlrp6-/- to germ-free mice resulted in similar donor permeability phenotype. However, depletion of the microbiota by broad-spectrum antibiotics in WT animals prevent leaky gut and reduced systemic bacterial load. On the other hand, depletion of the microbiota in Nlrp6-/- favored the invasion of B. abortus, even without intestinal permeability change. In summary, these results indicate that oral infection by Brucella abortus alters intestinal homeostasis in favor of its invasion and establishment of systemic infection in a mechanism dependent of NLRP6. NLRP6 is important in the control of bacteria survival, in the maintenance of the intestinal epithelial barrier and in the negative regulation of the immune response against oral infection caused by Brucella abortus, in which the gut microbiota seems to play a critical role