Shobhit Dhawan

Mast cells trigger epithelial barrier dysfunction, bacterial translocation and postoperative ileus in a mouse model

Background  Abdominal surgery involving bowel manipulation commonly results in inflammation of the bowel wall, which leads to impaired intestinal motility and postoperative ileus (POI). Mast cells have shown to play a key role in the pathogenesis of POI in mouse models and human studies. We studied whether mast cells contribute to the pathogenesis of POI by eliciting intestinal barrier dysfunction.

Genetic deletion of dectin-1 does not affect the course of murine experimental colitis

BackgroundIt is believed that inflammatory bowel diseases (IBD) result from an imbalance in the intestinal immune response towards the luminal microbiome. Dectin-1 is a widely expressed pattern recognition receptor that recognizes fungi and upon recognition it mediates cytokine responses and skewing of the adaptive immune system. Hence, dectin-1 may be involved in the pathogenesis of IBD.MethodsWe assessed the responses of dectin-1 deficient macrophages to the intestinal microbiota and determined the course of acute DSS and chronic Helicobacter hepaticus induced colitis in dectin-1 deficient mice.ResultsWe show that the mouse intestinal microbiota contains fungi and the cytokine responses towards this microbiota were significantly reduced in dectin-1 deficient macrophages. However, in two different colitis models no significant differences in the course of inflammation were found in dectin-1 deficient mice compared to wild type mice.ConclusionsTogether our data suggest that, although at the immune cell level there is a difference in response towards the intestinal flora in dectin-1 deficient macrophages, during intestinal inflammation this response seems to be redundant since dectin-1 deficiency in mice does not affect intestinal inflammation in experimental colitis.

Acetylcholine-producing T cells in the intestine regulate antimicrobial peptide expression and microbial diversity

The cholinergic anti-inflammatory pathway reduces systemic tumor necrosis factor (TNF) via acetylcholine-producing memory T cells in the spleen. These choline acetyltransferase (ChAT)-expressing T cells are also found in the intestine, where their function is unclear. We aimed to characterize these cells in mouse and human intestine and delineate their function. We made use of the ChAT-enhanced green fluorescent protein (eGFP) reporter mice. CD4Cre mice were crossed to ChATfl/fl mice to achieve specific deletion of ChAT in CD4+ T cells. We observed that the majority of ChAT-expressing T cells in the human and mouse intestine have characteristics of Th17 cells and coexpress IL17A, IL22, and RORC The generation of ChAT-expressing T cells was skewed by dendritic cells after activation of their adrenergic receptor β2 To evaluate ChAT T cell function, we generated CD4-specific ChAT-deficient mice. CD4ChAT-/- mice showed a reduced level of epithelial antimicrobial peptides lysozyme, defensin A, and ang4, which was associated with an enhanced bacterial diversity and richness in the small intestinal lumen in CD4ChAT-/- mice. We conclude that ChAT-expressing T cells in the gut are stimulated by adrenergic receptor activation on dendritic cells. ChAT-expressing T cells may function to mediate the host AMP secretion, microbial growth and expansion.

Cholinergic receptor activation on epithelia protects against cytokine-induced barrier dysfunction

Various types of cholinergic receptors are expressed on intestinal epithelia. Their function is not completely understood. We hypothesize that cholinergic receptor activation on epithelium may serve a protective function in cytokine‐induced barrier dysfunction.

Adrenergic innervation regulates intestinal microbiota diversity via generation of cholinergic Th17 lymphocytes

Background: Although, it is usually difficult to differentiate multiple system atrophy (MSA) from Parkinson’s disease (PD), we have previously reported that post-void residuals and sphincter electromyography were useful for differentiation 1), . However, we did not know which is more appropriate in differentiating MSA from PD. Aims: We aimed to perform receiver operating characteristic analysis to determine the ability of sphincter electromyography (mean duration) and post-void residuals (free flow study and pressure-flow study) for distinguishing multiple system atrophy from Parkinson’s disease. Methods: We retrospectively reviewed 241 case records where both urodynamic study and sphincter electromyography were performed in patients with multiple system atrophy (n = 147) and Parkinsons disease (n = 94). We performed receiver operating characteristic analysis of the data sets. Results: The area under the curve (AUC) used to differentiate multiple system atrophy from Parkinson’s disease was 0.79 in post-void residuals during pressure flow study, 0.73 in post-void residuals during freeflow study and 0.69 in mean duration of sphincter electromyography, respectively; these values were statistically significant. Conclusion: The present results suggested that post-void residuals were more appropriate than mean duration of sphincter electromyography in differentiating MSA from PD. In particular, the AUC in postvoid residuals under pressure flow study was larger than that of under free-flow study. 1) Yamamoto T, Sakakibara R, Uchiyama T, Yamaguchi C, Ohno S, Nomura F, Yanagisawa M, Hattori T, Kuwabara S. Time-dependent changes and gender differences in urinary dysfunction in patients with multiple system atrophy. Neurourol Urodyn. 2014 Jun;33(5):516-23. 2) Yamamoto T, Sakakibara R, Uchiyama T, Yamaguchi C, Nomura F, Ito T, YanagisawaM, YanoM, Awa Y, Yamanishi T, Hattori T, Kuwabara S. Receiver operating characteristic analysis of sphincter electromyography for parkinsonian syndrome. Neurourol Urodyn. 2012 Sep;31(7):1128-34.

Sa1842 Cholinergic Agonists Modulate Cytokine Induced Myosin Light Chain Phosphorylation and Barrier Dysfunction in Intestinal Epithelium

G A A b st ra ct s studies led us to hypothesize that suppression of the TGF β /Smad signaling pathway by TLR stimulation may contribute to a complex triggering mechanism leading to autoimmune inflammation. Methods: Dendritic cells were differentiated from the bone marrow of C57/ BL6 mice in the presence of GM-CSF and IL-4 (BMDCs). The cells were then treated with LPS (10-100 ng/ml) from commensal or pathogenic E. coli, or with different TLR agonists for various time points followed by TGF β stimulation (10 ng/ml) for 30 min. Cell lysates were prepared andWestern Blotting was performed to detect the proteins of interest. Results: Stimulation of dendritic cells with TLR1/2, TLR3, TLR4 and TLR5 agonists followed by TGFβ treatment resulted in significant inhibition TGFβ/Smad signaling pathway based on pSer465/467 Smad2 (pSmad2) induction as compared to cells treated with TGF β alone. TLR stimulation did not affect the expression of membrane-bound TGFβR subunits. The effects of higher concentration of LPS (100ng/mL) were not TLR4-dependent, thus indicating both TLR2 and TLR4 involvement. Although the effects of all concentrations of LPS were independent of MyD88, they were contingent on the expression of TIR-domain-containing adaptor-inducing interferon-b (TRIF) adapter, as determined with BMDC from respective knockout mice. Conclusions: Our studies indicate clearly that TLR-induced DC maturation antagonizes the TGFβ pathway through a TRIF-dependent mechanism. Further studies are aimed to reveal the exact mechanism involved and the impact of acute or chronic infections as potential trigger in individuals genetically predisposed to autoimmune disorders.

320 Neuro-Immune Regulation of TH17 Lymphocytes in Mouse Intestine

Background: Upon allergen encounter, food allergy typically leads to mast cell degranulation, an influx of Th2 immune cells together with increases in antigen specific antibodies. We showed that vagal nerve stimulation (VNS) increased Tregs and reduced mast cell activation in a mouse model of food allergy (DDW 2012). Here we aimed to test the hypothesis that, in contrast to VNS, vagotomy (VGX) would worsen food allergy. Methods: We sensitised balb/c mice to ovalbumin (OVA) adsorbed to aluminium-hydroxide (day 0&14) followed by intra-gastric gavages with OVA, from day 28 onwards. Sub-diaphragmatic VGX was performed at least one week prior to OVA gavage, together with pyloroplasty, where pyloroplasty alone served as control. Diarrhoea was scored for 1hr by analysing pellets, where 0 was normal faecal pellets, and 3 represents over diarrhoea. We determined serum mast cell proteases (mouse mast cell protease 1, MMCP-1), antibody titres (OVA specific IgG1 and IgG2a) and identified immune T-cell subsets in the mesenteric lymph nodes, lamina propria (LP) and Peyers patches. Cytokines released upon OVA challenge from mesenteric lymph nodes were measured by cytometric bead array. Further, qPCR was used to determine cytokine mRNA. OVA-specific Treg proliferation was assessed by adoptive transfer of Naive DO11.10 T-cells. Results: VGX increased serum MMCP-1 levels from 1120 ± 136 vs 2841 ± 964 ng/mL (n=7, p,0.05) 1hr after the fourth OVA gavage, coinciding with increased diarrhoea scores (from 1.1 to 2.8 arbitrary score, P , 0.05) but no change in serum IgG1 or IgG2a. VGX did not change the population of Tregs (CD25+/foxp3+) in the MLN (14.2 ± 1.9 controls vs VGX 15.0 ± 2.0% of CD4+) or LP (45.4 ± 2.9 to 42.0 ± 8.0% of CD4+). Similarly, no change in Th1 (IFN γ+), Th17 (IL-17+) or Th2 (IL-4+/IL-5+) cells was found after VGX in the mesenteric lymph nodes as compared to control. OVA specific Tregs (CD25+/foxp3+% of CD4+/DO11.10+) in the mesenteric lymph nodes (4.6 ± 0.4 to 6.2 ± 0.7%), LP (81.1 ± 7.0 vs. 80.5 ± 4.2%) or Peyers patches (24.5 ± 8.1 vs. 26.7 ± 6.4%) remained unchanged after VGX compared to controls (n=8-12). Jejunal mRNA levels (over RPL32 expression) for IL-13, IL-5, TNFα and IFNγ were not affected by VGX. Conclusion: As opposed to VNS, VGX increases the severity of food allergy, as shown by increased mast cell activation and diarrhoea scores compared to sham operated mice. CD4+ T cell skewing and cytokine production was however not affected by VGX. Our data indicate that reduced vagal tone renders mice more susceptible to develop food allergy further underscoring the role of the vagal innervation in modulating the mucosal immune system.