Cholinergic Signaling Attenuates Pro-Inflammatory Interleukin-8 Response in Colonic Epithelial Cells.

Infants affected by Hirschsprung disease (HSCR), a neurodevelopmental congenital disorder, lack ganglia of the intrinsic enteric nervous system (aganglionosis) in a variable length of the colon, and are prone to developing severe Hirschsprung-associated enterocolitis (HAEC). HSCR patients typically show abnormal dense innervation of extrinsic cholinergic nerve fibers throughout the aganglionic rectosigmoid. Cholinergic signaling has been reported to reduce inflammatory response. Consequently, a sparse extrinsic cholinergic innervation in the mucosa of the rectosigmoid correlates with increased inflammatory immune cell frequencies and higher incidence of HAEC in HSCR patients. However, whether cholinergic signals influence the pro-inflammatory immune response of intestinal epithelial cells (IEC) is unknown. Here, we analyzed colonic IEC isolated from 43 HSCR patients with either a low or high mucosal cholinergic innervation density (fiber-low versus fiber-high) as well as from control tissue. Compared to fiber-high samples, IEC purified from fiber-low rectosigmoid expressed significantly higher levels of IL-8 but not TNF-α, IL-10, TGF-β1, Muc-2 or tight junction proteins. IEC from fiber-low rectosigmoid showed higher IL-8 protein concentrations in cell lysates as well as prominent IL-8 immunoreactivity compared to IEC from fiber-high tissue. Using the human colonic IEC cell line SW480 we demonstrated that cholinergic signals suppress lipopolysaccharide-induced IL-8 secretion via the alpha 7 nicotinic acetylcholine receptor (a7nAChR). In conclusion, we showed for the first time that the presence of a dense mucosal cholinergic innervation is associated with decreased secretion of IEC-derived pro-inflammatory IL-8 in the rectosigmoid of HSCR patients likely dependent on a7nAChR activation. Owing to the association between IL-8 and enterocolitis-prone, fiber-low HSCR patients, targeted therapies against IL-8 might be a promising immunotherapy candidate for HAEC treatment

The Anaphase-Promoting Complex/Cyclosome Is Required for Anaphase Progression in Multinucleated Ashbya gossypii Cells

Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself

Water permeability is a measure of severity in acute appendicitis

Acute appendicitis is the most common indication for pediatric abdominal emergency surgery. Determination of the severity of appendicitis on clinical grounds is challenging. Complicated appendicitis presenting with perforation, abscess or diffuse peritonitis is not uncommon. The question remains why and when acute appendicitis progresses to perforation. The aim of this study was to assess the impact of water permeability on the severity of appendicitis. We show that AQP1 expression and water permeability in appendicitis correlate with the stage of inflammation and systemic infection parameters, leading eventually to perforation of the appendix. AQP1 is also expressed within the ganglia of the enteric nervous system and ganglia count increases with inflammation. Severity of appendicitis can be correlated with water permeability measured by AQP1 protein expression and increase of ganglia count in a progressive manner. This introduces the question if regulation of water permeability can present novel curative or ameliorating therapeutic options

A minimum number of autoimmune T cells to induce autoimmunity?

While autoimmune T cells are present in most individuals, only a minority of the population suffers from an autoimmune disease. To better appreciate the limits of T cell tolerance, we carried out experiments to determine how many autoimmune T cells are required to initiate an experimental autoimmune disease. Variable numbers of autoimmune OT-I T cells were transferred into RIP-OVA mice, which were injected with antigen-loaded DCs in a single footpad; this restricted T cell priming to a few OT-I T cells that are present in the draining popliteal lymph node. Using selective plane illumination microscopy (SPIM) we counted the number of OT-I T cells present in the popliteal lymph node at the time of priming. Analysis of our data suggests that a single autoimmune T cell cannot induce an experimental autoimmune disease, but a "quorum" of 2-5 autoimmune T cells clearly has this capacity

Lack of Mucosal Cholinergic Innervation Is Associated With Increased Risk of Enterocolitis in Hirschsprung's Disease

BACKGROUND & AIMS Hirschsprung's disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric neuronal cells (ganglia) in the distal gut. The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown. METHODS We enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal acetylcholinesterase-positive innervation into low-fiber and high-fiber patient groups. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using Luminex analysis and fluorescence-activated cell sorting analysis of colonic tissue and immune cells. Using confocal immunofluorescence microscopy, macrophages were identified in close proximity to nerve fibers and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic tissue using 16S-rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation. RESULTS The presence of mucosal nerve fiber innervation correlated with reduced T-helper 17 cytokines and cell frequencies. In high-fiber tissue, macrophages co-localized with nerve fibers and expressed significantly less interleukin 23 than macrophages from low-fiber tissue. HSCR patients lacking mucosal nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis. CONCLUSIONS The mucosal fiber phenotype might serve as a prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new therapeutic options

Gut microbiota modulate T cell trafficking into human colorectal cancer

OBJECTIVE Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers. DESIGN Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing. RESULTS CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival. CONCLUSIONS Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues