NLRP6-associated host microbiota composition impacts in the intestinal barrier to systemic dissemination of Brucella abortus.

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.

Identifying functionally relevant candidate genes for inflexible ethanol intake in mice and humans using a guilt-by-association approach.

Gene prioritization approaches are useful tools to explore and select candidate genes in transcriptome studies. Knowing the importance of processes such as neuronal activity, intracellular signal transduction, and synapse plasticity to the development and maintenance of compulsive ethanol drinking, the aim of the present study was to explore and identify functional candidate genes associated with these processes in an animal model of inflexible pattern of ethanol intake. To do this, we applied a guilt-by-association approach, using the GUILDify and ToppGene software, in our previously published microarray data from the prefrontal cortex (PFC) and striatum of inflexible drinker mice. We then tested some of the prioritized genes that showed a tissue-specific pattern in postmortem brain tissue (PFC and nucleus accumbens (NAc)) from humans with alcohol use disorder (AUD). In the mouse brain, we prioritized 44 genes in PFC and 26 in striatum, which showed opposite regulation patterns in PFC and striatum. The most prioritized of them (i.e., Plcb1 and Prkcb in PFC, and Dnm2 and Lrrk2 in striatum) were associated with synaptic neuroplasticity, a neuroadaptation associated with excessive ethanol drinking. The identification of transcription factors among the prioritized genes suggests a crucial role for Irf4 in the pattern of regulation observed between PFC and striatum. Lastly, the differential transcription of IRF4 and LRRK2 in PFC and nucleus accumbens in postmortem brains from AUD compared to control highlights their involvement in compulsive ethanol drinking in humans and mice.

Interaction between high-fat diet and ethanol intake leads to changes on the fecal microbiome.

It is known that high-fat diet and alcohol intake can modulate the gut microbiota and consequently affect physiological processes such as fat storage and conditional behavior. However, the effects of the interaction between high-fat diet, its withdrawal and ethanol intake in gut microbiota remain unclear. To address this question, we used an animal model in which C57BL/6 mice were fed on standard (AIN93G) or high-sugar and -butter (HSB) diet for 8 weeks. Then, a protocol of free choice between water and a 10% alcohol solution was introduced, and the HSB diet was replaced with AIN93G in two experimental groups. This model allowed us to distinguish the individual effects of HSB diet and ethanol, and the effects of its interaction on the microbiome. The interaction of those factors was the main driver in the structure changes of the fecal microbial community. HSB diet and ethanol consumption directly affected the abundance of Firmicutes and Actinobacteria phylum, and Clostridiaceae and Coriobacteriaceae family. On the other hand, we also showed that abundance of Bacteroidales_S24-7 family and the Firmicutes/Bacteroidetes ratio were affected only by HSB diet consumption and that ethanol consumption was uniquely responsible for the bacterial translocation to the liver, indicating a breaking of the gut barrier. Finally, we also pointed out that the withdrawal of the HSB diet affects the preference for alcohol and shows a structural resilience in the fecal microbiome. These results highlight the importance of the gut microbiome modulation and its possible role on the phenotype developed by animals.

High-fat diet withdrawal modifies alcohol preference and transcription of dopaminergic and GABAergic receptors.

The bidirectional and positive relation between the ingestion of fat and alcohol has become the subject of extensive discussion. However, this relation is more studied in animal models of binge eating with intermittent access of high-fat diet or in a model of short period of this diet consumption. Here, we developed a model to elucidate how chronic high-fat diet and its withdrawal influence alcohol intake (two-bottle choice) and anxiety behavior (marble burying test). In the first experimental stage, animals were fed on standard (AIN93G) or high sugar and butter (HSB) diet for 8 weeks. Then, a protocol of free-choice between water and a 10% alcohol solution was introduced, and the HSB diet was replaced with AIN93G in two experimental groups. The result obtained with this model point out that the relation among high-fat diet consumption and alcohol intake appears to depend on the presence or absence of the diet when alcohol intake is evaluated, and that an imbalance in the mesocorticolimbic dopaminergic pathway, observed by the transcriptional regulation of the dopamine receptors (Drd1/Drd2) and GABAB receptors subunit (Gabbr1/Gabbr2), can be driving the alcohol intake.