The inflammasome is hypothesized to be a key mediator of the response to physiological and psychological stressors, and its dysregulation may be implicated in major depressive disorder. Inflammasome activation causes the maturation of caspase-1 and activation of interleukin (IL)-1β and IL-18, two proinflammatory cytokines involved in neuroimmunomodulation, neuroinflammation and neurodegeneration. In this study, C57BL/6 mice with genetic deficiency or pharmacological inhibition of caspase-1 were screened for anxiety- and depressive-like behaviors, and locomotion at baseline and after chronic stress. We found that genetic deficiency of caspase-1 decreased depressive- and anxiety-like behaviors, and conversely increased locomotor activity and skills. Caspase-1 deficiency also prevented the exacerbation of depressive-like behaviors following chronic stress. Furthermore, pharmacological caspase-1 antagonism with minocycline ameliorated stress-induced depressive-like behavior in wild-type mice. Interestingly, chronic stress or pharmacological inhibition of caspase-1 per se altered the fecal microbiome in a very similar manner. When stressed mice were treated with minocycline, the observed gut microbiota changes included increase in relative abundance of Akkermansia spp. and Blautia spp., which are compatible with beneficial effects of attenuated inflammation and rebalance of gut microbiota, respectively, and the increment in Lachnospiracea abundance was consistent with microbiota changes of caspase-1 deficiency. Our results suggest that the protective effect of caspase-1 inhibition involves the modulation of the relationship between stress and gut microbiota composition, and establishes the basis for a gut microbiota–inflammasome–brain axis, whereby the gut microbiota via inflammasome signaling modulate pathways that will alter brain function, and affect depressive- and anxiety-like behaviors. Our data also suggest that further elucidation of the gut microbiota–inflammasome–brain axis may offer novel therapeutic targets for psychiatric disorders.

Sequencing-based studies of the human faecal microbiota are increasingly common. Appropriate storage of sample material is essential to avoid the introduction of post-collection bias in microbial community composition. Rapid freezing to −80 °C is commonly considered to be best-practice. However, this is not feasible in many studies, particularly those involving sample collection in participants’ homes. We determined the extent to which a range of stabilisation and storage strategies maintained the composition of faecal microbial community structure relative to freezing to −80 °C. Refrigeration at 4 °C, storage at ambient temperature and the use of several common preservative buffers (RNAlater, OMNIgene.GUT, Tris-EDTA) were assessed relative to freezing. Following 72 hours of storage, faecal microbial composition was assessed by 16 S rRNA amplicon sequencing. Refrigeration was associated with no significant alteration in faecal microbiota diversity or composition. However, samples stored using other conditions showed substantial divergence compared to −80 °C control samples. Aside from refrigeration, the use of OMNIgene.GUT resulted in the least alteration, while the greatest change was seen in samples stored in Tris-EDTA buffer. The commercially available OMNIgene.GUT kit may provide an important alternative where refrigeration and cold chain transportation is not available.

Asthma pathophysiology and treatment responsiveness are predicted by inflammatory phenotype. However, the relationship between airway microbiology and asthma phenotype is poorly understood. We aimed to characterize the airway microbiota in patients with symptomatic stable asthma and relate composition to airway inflammatory phenotype and other phenotypic characteristics. The microbial composition of induced sputum specimens collected from adult patients screened for a multicenter randomized controlled trial was determined by using 16S rRNA gene sequencing. Inflammatory phenotypes were defined by sputum neutrophil and eosinophil cell proportions. Microbiota were defined by using α- and β-diversity measures, and interphenotype differences were identified by using similarity of percentages, network analysis, and taxon fold change. Phenotypic predictors of airway microbiology were identified by using multivariate linear regression. Microbiota composition was determined in 167 participants and classified as eosinophilic (n = 84), neutrophilic (n = 14), paucigranulocytic (n = 60), or mixed neutrophilic-eosinophilic (n = 9) asthma phenotypes. Airway microbiology was significantly less diverse (P = .022) and more dissimilar (P = .005) in neutrophilic compared with eosinophilic participants. Sputum neutrophil proportions, but not eosinophil proportions, correlated significantly with these diversity measures (α-diversity: Spearman r = -0.374, P < .001; β-diversity: r = 0.238, P = .002). Interphenotype differences were characterized by a greater frequency of pathogenic taxa at high relative abundance and reduced Streptococcus, Gemella, and Porphyromonas taxa relative abundance in patients with neutrophilic asthma. Multivariate regression confirmed that sputum neutrophil proportion was the strongest predictor of microbiota composition. Neutrophilic asthma is associated with airway microbiology that is significantly different from that seen in patients with other inflammatory phenotypes, particularly eosinophilic asthma. Differences in microbiota composition might influence the response to antimicrobial and steroid therapies and the risk of lung infection.

Serotonin (5-hydroxytryptamine or 5-HT) is a multifunctional bioamine with important signaling roles in a range of physiological pathways. Almost all of the 5-HT in our bodies is synthesized in specialized enteroendocrine cells within the gastrointestinal (GI) mucosa called enterochromaffin (EC) cells. These cells provide all of our circulating 5-HT. We have long appreciated the important contributions of 5-HT within the gut, including its role in modulating GI motility. However, evidence of the physiological and clinical significance of gut-derived 5-HT outside of the gut has recently emerged, implicating 5-HT in regulation of glucose homeostasis, lipid metabolism, bone density, and diseases associated with metabolic syndrome, such as obesity and type 2 diabetes. Although a new picture has developed in the last decade regarding the various metabolic roles of peripheral serotonin, so too has our understanding of the physiology of EC cells. Given that they are scattered throughout the lining of the GI tract within the epithelial cell layer, these cells are typically difficult to study. Advances in isolation procedures now allow the study of pure EC-cell cultures and single cells, enabling studies of EC-cell physiology to occur. EC cells are sensory cells that are capable of integrating cues from ingested nutrients, the enteric nervous system, and the gut microbiome. Thus, levels of peripheral 5-HT can be modulated by a multitude of factors, resulting in both local and systemic effects for the regulation of a raft of physiological pathways related to metabolism and obesity.

The rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites. A workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types. The application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts.