The nervous system plays a profound regulatory role in maintaining appropriate immune responses by signaling to immune cells. These immune cells, including B- and T-cells, can further act as intermediary messengers, with subsets of B- and T-cells expressing choline acetyltransferase (ChAT), the enzyme required for acetylcholine (ACh) synthesis. Neural control of ACh release from ChAT+ T-cells can have powerful immune implications, regulating lymphocyte trafficking, inflammation, and prevent death due to experimental septic shock. Although ACh release from T-cells has been proposed to occur following norepinephrine (NE) released from sympathetic nerve terminals in the spleen, it is unknown how this communication occurs. While it was proposed that tyrosine hydroxylase (TH+) axons form synapse-like structures with ChAT+ T-cells, there is scant evidence to support or refute this phenomenon. With this in mind, we sought to determine the relative abundance of ChAT+ B- and T-cells in close proximity to TH+ axons, and determine what factors contribute to their localization in the spleen. Using confocal microscopy of tissue sections and three-dimensional imaging of intact spleen, we confirmed that ChAT+ B-cells exceed the number of ChAT+ T-cells, and overall few ChAT+ B- or T-cells are located close to TH+ fibers compared to total numbers. The organized location of ChAT+ lymphocytes within the spleen suggested that these cells were recruited by chemokine gradients. We identified ChAT+ B- and T-cells express the chemokine receptor CXCR5; indicating that these cells can respond to CXCL13 produced by stromal cells expressing the β2 adrenergic receptor in the spleen. Our findings suggest that sympathetic innervation contributes to organization of ChAT+ immune cells in the white pulp of the spleen by regulating CXCL13. Supporting this contention, chemical sympathectomy significantly reduced expression of this chemokine. Together, we demonstrated that there does not appear to be a basis for synaptic neuro-immune communication, and that sympathetic innervation can modulate immune function through altering stromal cell chemokine production.

The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium . These ChAT + T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C . rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1β, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ, or epithelial expression of antimicrobial peptides, compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C . rodentium infection, inducible nitric oxide synthase ( Nos2 ) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro . These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C . rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses.

Background Neurons are an integral component of the immune system that functions to coordinate responses to bacterial pathogens. Sensory nociceptive neurons that can detect bacterial pathogens are found throughout the body with dense innervation of the intestinal tract. Methods In this study, we assessed the role of these nerves in the coordination of host defenses to Citrobacter rodentium. Selective ablation of nociceptive neurons significantly increased bacterial burden 10 days postinfection and delayed pathogen clearance. Results Because the sensory neuropeptide CGRP (calcitonin gene-related peptide) regulates host responses during infection of the skin, lung, and small intestine, we assessed the role of CGRP receptor signaling during C rodentium infection. Although CGRP receptor blockade reduced certain proinflammatory gene expression, bacterial burden and Il-22 expression was unaffected. Conclusions Our data highlight that sensory nociceptive neurons exert a significant host protective role during C rodentium infection, independent of CGRP receptor signaling.

Osteosarcoma remains the most common primary bone tumour in dogs with half of affected dogs unable to survive 1 year beyond diagnosis. New therapeutic options are needed to improve outcomes for this disease. Recent investigations into potential therapeutic targets have focused on cell surface molecules with little clear therapeutic benefit. Transcription factors and protein interactions represent underdeveloped areas of therapeutic drug development. We have utilized allosteric inhibitors of the core binding factor transcriptional complex, comprised of core binding factor beta (CBFβ) and RUNX2, in four canine osteosarcoma cell lines Active inhibitor compounds demonstrate anti‐tumour activities with concentrations demonstrated to be achievable in vivo while an inactive, structural analogue has no activity. We show that CBFβ inhibitors are capable of inducing apoptosis, inhibiting clonogenic cell growth, altering cell cycle progression and impeding migration and invasion in a cell line‐dependent manner. These effects coincide with a reduced interaction between RUNX2 and CBFβ and alterations in expression of RUNX2 target genes. We also show that addition of CBFβ inhibitors to the commonly used cytotoxic chemotherapeutic drugs doxorubicin and carboplatin leads to additive and/or synergistic anti‐proliferative effects in canine osteosarcoma cell lines. Taken together, we have identified the interaction between components of the core binding factor transcriptional complex, RUNX2 and CBFβ, as a potential novel therapeutic target in canine osteosarcoma and provide justification for further investigations into the anti‐tumour activities we describe here.

Core binding factor beta (CBFβ) functions as a binding partner to the RUNX family of DNA binding transcription factors (RUNX1‐3) and acts as a transcriptional co‐activator by allosterically increasing their DNA binding. According to The Cancer Genome Atlas Program (TCGA) and Genotype‐Tissue Expression (GTEx) datasets, high CBFβ expression is correlated with poor disease‐free and overall survival across 17 cancer types, including sarcomas. Of particular interest to osteosarcoma (OS) research is the interaction of CBFβ with RUNX2, the master regulator of bone growth and differentiation, also dysregulated in aggressive OS and implicated in chemoresistance. Recent research supports a non‐canonical role of CBFβ as a regulator of protein translation initiation. In breast cancer cells, this translation‐associated activity occurs via interactions with heterogeneous nuclear ribonucleoprotein K (hnRNPK) and allows CBFβ to influence translation of hundreds of mRNA transcripts. Binding of CBFβ to hnRNPK or RUNX1 appears mutually exclusive, suggesting possible competition between these two roles. We hypothesized that CBFβ plays a role in the translation of RUNX2 and RUNX2‐target gene mRNAs in OS and that disruption of this role may result in an antitumor effect. The intertwined nature of the two roles of CBFβ hampers study of each in OS. Thus, we aimed to decouple them to identify which presents the most viable therapeutic target in OS. Using CRISPR/Cas9 we generated a CBFβ knockout (KO) U2OS cell line and evaluated RUNX2 protein and mRNA levels via western blot (WB) and qRT‐PCR, respectively. Cycloheximide chase assay and proteasome inhibition were used to evaluate RUNX2 stability and degradation, respectively. Interactions between CBFβ and RUNX2 or hnRNPK were assessed via co‐immunoprecipitation. Site‐directed mutagenesis (SDM) of CBFβ and transfection of U2OS CBFβ KO cells with FLAG tagged CBFβ mutants was used to prevent RUNX2‐CBFβ interaction. WB was used to evaluate changes in subcellular CBFβ localization. Global, as well as protein‐specific, changes in de novo protein synthesis were evaluated using the Click‐IT system. Finally, in silico modeling was used to design a peptide that mimics a 10 amino acid sequence on RUNX2 in the CBFβ binding interface, which was then evaluated in vitro using a recombinant RUNX2‐CBFβ pulldown assay. Our results demonstrate that loss of CBFβ leads to reduced RUNX2 protein expression without changes in RUNX2 mRNA levels. RUNX2 protein half‐life is not reduced in CBFβ nor does proteasome inhibition rescue RUNX2 protein levels. CBFβ interacts with hnRNPK in OS cells, and through this binding may perform a role in oncoprotein translation in OS, specifically modulating protein levels of RUNX2. SDM of CBFβ identified important residues for RUNX2 binding, which when targeted via a peptide resulted in a nearly 50% reduction in CBFβ and RUNX2 interaction. Ribosome footprinting combined with RNAseq and immunoprecipitation‐mass spectrometry utilizing WT U2OS, CBFβ mutants and peptide‐inhibited cells...