Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.

Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3(Cre/+) , devoid of mast cells but with intact Kit signaling.

Oxazolone-induced colitis has been frequently used in literature as a model of IBD, but insights into the underlying immune response and pathological features are surprisingly still very limited. Vagus nerve stimulation (VNS) has proven to be effective in innate and Th1/Th17 predominant inflammatory models, including pre-clinical models of colitis, however to what extent VNS is also effective in ameliorating Th2-driven colitis remains to be studied. In the present study, we therefore further characterized the immune response in oxazolone-induced colitis and investigated the potential therapeutic effect of VNS.

Vagus nerve (VN) stimulation is currently evaluated as a novel approach to treat immune-mediated disorders. The optimal stimulation parameters, however, largely depend on the VN composition potentially impacting on its clinical translation. Hence, we evaluated whether morphological differences exist between the cervical and abdominal VNs across different species.

The physiological functions of mast cells remain largely an enigma. In the context of barrier damage, mast cells are integrated in type 2 immunity and, together with immunoglobulin E (IgE), promote allergic diseases. Allergic symptoms may, however, facilitate expulsion of allergens, toxins and parasites and trigger future antigen avoidance1-3. Here, we show that antigen-specific avoidance behaviour in inbred mice4,5 is critically dependent on mast cells; hence, we identify the immunological sensor cell linking antigen recognition to avoidance behaviour. Avoidance prevented antigen-driven adaptive, innate and mucosal immune activation and inflammation in the stomach and small intestine. Avoidance was IgE dependent, promoted by Th2 cytokines in the immunization phase and by IgE in the execution phase. Mucosal mast cells lining the stomach and small intestine rapidly sensed antigen ingestion. We interrogated potential signalling routes between mast cells and the brain using mutant mice, pharmacological inhibition, neural activity recordings and vagotomy. Inhibition of leukotriene synthesis impaired avoidance, but overall no single pathway interruption completely abrogated avoidance, indicating complex regulation. Collectively, the stage for antigen avoidance is set when adaptive immunity equips mast cells with IgE as a telltale of past immune responses. On subsequent antigen ingestion, mast cells signal termination of antigen intake. Prevention of immunopathology-causing, continuous and futile responses against per se innocuous antigens or of repeated ingestion of toxins through mast-cell-mediated antigen-avoidance behaviour may be an important arm of immunity.

Background: We previously showed increased susceptibility to dextran sulfate sodium (DSS)-induced colitis in vagotomized mice. Here, we evaluated whether vagus nerve stimulation (VNS) is able to reduce the severity of DSS colitis and aimed to unravel the mechanism involved. Methods: Colitis was induced in wild type mice by 2.5% DSS administration in drinking water for 5 days. VNS (5 Hz, 1 ms, 1 mA) was applied 1 day prior to and after 4 days of DSS administration to evaluate changes in epithelial integrity and inflammatory response, respectively. Epithelial integrity was assessed using TUNEL and Ki67 staining. Monocytes, immature and mature macrophages were sorted from colonic samples and gene expression levels of pro-inflammatory cytokines were studied. Results: VNS applied prior to DSS administration (i.e., prophylactic VNS) reduced disease activity index (VNS 0.8 ± 0.6 vs. sham 2.8 ± 0.7, p < 0.001, n = 5) and tended to improve histology score. Prophylactic VNS significantly increased epithelial cell proliferation and diminished apoptosis compared to sham stimulation. VNS applied at day 4 during DSS administration (i.e., therapeutic VNS) decreased the influx of monocytes, monocyte-derived macrophages and neutrophils, and significantly reduced pro-inflammatory cytokine expression (i.e., Tnfα and Cxcl1) in immature macrophages compared to sham stimulation. Conclusions: A single period of VNS applied prior to DSS exposure reduced DSS-induced colitis by an improvement in epithelial integrity. On the other hand, VNS applied during the inflammatory phase of DSS colitis reduced cytokine expression in immature macrophages. Our data further underscores the potential of VNS as novel therapeutic approach for inflammatory bowel diseases.

The vagus nerve has emerged as an important modulator of the intestinal immune system. Its anti-inflammatory properties have been previously shown in innate and Th1/Th17 predominant inflammatory models. To what extent the vagus nerve is of importance in Th2 inflammatory responses like food allergy is still unclear. In this study, we therefore aimed to investigate the effect of vagotomy (VGX) and vagus nerve stimulation (VNS), on the development and severity of experimental food allergy.

Macrophages are highly heterogeneous tissue-resident immune cells that perform a variety of tissue-supportive functions. The current paradigm dictates that intestinal macrophages are continuously replaced by incoming monocytes that acquire a pro-inflammatory or tissue-protective signature. Here, we identify a self-maintaining population of macrophages that arise from both embryonic precursors and adult bone marrow-derived monocytes and persists throughout adulthood. Gene expression and imaging studies of self-maintaining macrophages revealed distinct transcriptional profiles that reflect their unique localization (i.e., closely positioned to blood vessels, submucosal and myenteric plexus, Paneth cells, and Peyer's patches). Depletion of self-maintaining macrophages resulted in morphological abnormalities in the submucosal vasculature and loss of enteric neurons, leading to vascular leakage, impaired secretion, and reduced intestinal motility. These results provide critical insights in intestinal macrophage heterogeneity and demonstrate the strategic role of self-maintaining macrophages in gut homeostasis and intestinal physiology.