Optical Imaging of the Small Intestine Immune Compartments Across Scales

The limitations of 2D microscopy constrain our ability to observe and understand tissue-wide networks that are, by nature, 3-dimensional. Optical projection tomography enables the acquisition of large volumes (ranging from micrometres to centimetres) in various tissues, with label-free capacities for the observation of auto-fluorescent signals as well fluorescent-labelled targets of interest in multiple channels. We present a multi-modal workflow for the characterization of both structural and quantitative parameters of the mouse small intestine. As proof of principle, we evidence its applicability for imaging the mouse intestinal immune compartment and surrounding mucosal structures. We quantify the volumetric size and spatial distribution of Isolated Lymphoid Follicles (ILFs) and quantify density of villi throughout centimetre long segments of intestine. Furthermore, we exhibit the age- and microbiota-dependence for ILF development, and leverage a technique that we call reverse-OPT for identifying and homing in on regions of interest. Several quantification capabilities are displayed, including villous density in the autofluorescent channel and the size and spatial distribution of the signal of interest at millimetre-scale volumes. The concatenation of 3D image acquisition with the reverse-OPT sample preparation and a 2D high-resolution imaging modality adds value to interpretations made in 3D. This cross-modality referencing technique is found to provide accurate localisation of ROIs and to add value to interpretations made in 3D. Importantly, OPT may be used to identify sparsely-distributed regions of interest in large volumes whilst retaining compatibility with high-resolution microscopy modalities, including confocal microscopy. We believe this pipeline to be approachable for a wide-range of specialties, and to provide a new method for characterisation of the mouse intestinal immune compartment.

Identification of SSRI-evoked antidepressant sensory signals by decoding vagus nerve activity

The vagus nerve relays mood-altering signals originating in the gut lumen to the brain. In mice, an intact vagus is required to mediate the behavioural effects of both intraluminally applied selective serotonin reuptake inhibitors and a strain of Lactobacillus with antidepressant-like activity. Similarly, the prodepressant effect of lipopolysaccharide is vagus nerve dependent. Single vagal fibres are broadly tuned to respond by excitation to both anti- and prodepressant agents, but it remains unclear how neural responses encode behaviour-specific information. Here we demonstrate using ex vivo experiments that for single vagal fibres within the mesenteric neurovascular bundle supplying the mouse small intestine, a unique neural firing pattern code is common to both chemical and bacterial vagus-dependent antidepressant luminal stimuli. This code is qualitatively and statistically discernible from that evoked by lipopolysaccharide, a non-vagus-dependent antidepressant or control non-antidepressant Lactobacillus strain and are not affected by sex status. We found that all vagus dependent antidepressants evoked a decrease in mean spike interval, increase in spike burst duration, decrease in gap duration between bursts and increase in intra-burst spike intervals. Our results offer a novel neuronal electrical perspective as one explanation for mechanisms of action of gut-derived vagal dependent antidepressants. We expect that our ex vivo individual vagal fibre recording model will improve the design and operation of new, extant electroceutical vagal stimulation devices currently used to treat major depression. Furthermore, use of this vagal antidepressant code should provide a valuable screening tool for novel potential oral antidepressant candidates in preclinical animal models.

Spatial gene expression maps of the intestinal lymphoid follicle and associated epithelium identify zonated expression programs

The intestine is lined with isolated lymphoid follicles (ILFs) that facilitate sampling of luminal antigens to elicit immune responses. Technical challenges related to the scarcity and small sizes of ILFs and their follicle-associated epithelium (FAE) impeded the characterization of their spatial gene expression programs. Here, we combined RNA sequencing of laser capture microdissected tissues with single-molecule transcript imaging to obtain a spatial gene expression map of the ILF and its associated FAE in the mouse small intestine. We identified zonated expression programs in both follicles and FAEs, with a decrease in enterocyte antimicrobial and absorption programs and a partial induction of expression programs normally observed at the villus tip. We further identified Lepr+ subepithelial telocytes at the FAE top, which are distinct from villus tip Lgr5+ telocytes. Our analysis exposes the epithelial and mesenchymal cell states associated with ILFs.

Bioinformatic Analysis of lncRNA Mediated Competitive Endogenous RNA Network in Intestinal Ischemia/reperfusion Injury

BackgroundRecently, an increasing number of studies have reported the roles of competitive endogenous RNA (ceRNA) networks in ischemia/reperfusion (I/R) injury, which include the liver, kidney, heart, brain, and intestine. However, the functions and mechanisms of long non-coding RNAs (lncRNAs), which serve as ceRNA networks in intestinal I/R injury, are still unclear. MethodsIn this study, bioinformatics methods were used to filter and construct the lncRNA-miRNA-mRNA networks in intestinal I/R injury. RNA expression data were retrieved from NCBI GEO datasets, the expression profiles between mouse small intestine with superior mesenteric artery occlusion and Sham operation was analyzed, and 189 microRNA differential expressed genes(miDEGs) were discovered successfully from miRNA GEO dataset (GSE83701). Next, targeted lncRNAs and mRNA in the database were matched based on miDEGs. Then, lncRNA-miRNA-mRNA networks were constructed with Cytoscape. The hub lncRNA-miRNA-mRNA networks were selected via Cytoscape plug-in CytoHubba and intersected mRNAs of datasets GSE96733 and GSE83701. Finally, the vital nodes of the ceRNA networks were validated by qPCR. ResultsThe 1700020114Rik/mmu-miR-7a-5p/Klf4 axis was postulated to play a potential role in intestinal I/R injury.ConclusionThe results shed novel insight into the molecular mechanism of ceRNA networks in intestinal I/R injury and highlighted the potential of 170002700020114Rik/mmu-miR-7a-5p/Klf4 ceRNA network in the prevention and treatment of intestinal I/R injury.

NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus

Clostridium perfringens type F strains causing non-foodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence to, and growth using, Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI -carrying vs. non- nanI -carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, vs. their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells vs HT29 cells. Those effects involved mucus production by MTX-E12 cells since mucus reduction using N-acetyl-cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

Robust Acquisition of High Resolution Spatial Transcriptomes from Preserved Tissues with Immunofluorescence Based Laser Capture Microdissection

The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue. However, obtaining comprehensive transcriptomic data based on established phenotypes while retaining this spatial information has been challenging. Here we present a general and robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to enable acquisition of finely resolved spatial transcriptomes with as few as tens of cells from snap-frozen or RNAlater-treated tissues, overcoming the long-standing problem of significant RNA degradation during this lengthy process. The efficacy of this approach is exemplified by the characterization of differences at the transcript isoform level between cells at the tip versus the main capillary body of the mouse small intestine lacteal. With the extensive repertoire of phenotype-specific antibodies that are presently available, our method provides a powerful means by which spatially resolved cellular states can be delineated in situ with preserved tissues. Moreover, such high quality spatial transcriptomes defined by immuno-markers can be used to compare with clusters obtained from single-cell RNAseq studies of dissociated cells as well as applied to bead-based spatial transcriptomics approaches that require such information a priori for cell identification.

Organoids and Colorectal Cancer

Organoids were first established as a three-dimensional cell culture system from mouse small intestine. Subsequent development has made organoids a key system to study many human physiological and pathological processes that affect a variety of tissues and organs. In particular, organoids are becoming very useful tools to dissect colorectal cancer (CRC) by allowing the circumvention of classical problems and limitations, such as the impossibility of long-term culture of normal intestinal epithelial cells and the lack of good animal models for CRC. In this review, we describe the features and current knowledge of intestinal organoids and how they are largely contributing to our better understanding of intestinal cell biology and CRC genetics. Moreover, recent data show that organoids are appropriate systems for antitumoral drug testing and for the personalized treatment of CRC patients.