Nociceptor neurons affect cancer immunosurveillance.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.

FcεR1-expressing nociceptors trigger allergic airway inflammation.

BACKGROUND: Lung nociceptor neurons amplify immune cell activity and mucus metaplasia in response to an inhaled allergen challenge in sensitized mice. OBJECTIVE: We sought to identify the cellular mechanisms by which these sensory neurons are activated subsequent to allergen exposure. METHODS: We used calcium microscopy and electrophysiologic recording to assess whether vagal neurons directly respond to the model allergen ovalbumin (OVA). Next, we generated the first nociceptor-specific FcεR1γ knockdown (TRPV1Cre::FcεR1γfl/fl) mice to assess whether this targeted invalidation would affect the severity of allergic inflammation in response to allergen challenges. RESULTS: Lung-innervating jugular nodose complex ganglion neurons express the high-affinity IgE receptor FcεR1, the levels of which increase in OVA-sensitized mice. FcεR1γ-expressing vagal nociceptor neurons respond directly to OVA complexed with IgE with depolarization, action potential firing, calcium influx, and neuropeptide release. Activation of vagal neurons by IgE-allergen immune complexes, through the release of substance P from their peripheral terminals, directly amplifies TH2 cell influx and polarization in the airways. Allergic airway inflammation is decreased in TRPV1Cre::FcεR1γfl/fl mice and in FcεR1α-/- mice into which bone marrow has been transplanted. Finally, increased in vivo circulating levels of IgE following allergen sensitization enhances the responsiveness of FcεR1 to immune complexes in both mouse jugular nodose complex ganglion neurons and human induced pluripotent stem cell-derived nociceptors. CONCLUSIONS: Allergen sensitization triggers a feedforward inflammatory loop between IgE-producing plasma cells, FcεR1-expressing vagal sensory neurons, and TH2 cells, which helps to both initiate and amplify allergic airway inflammation. These data highlight a novel target for reducing allergy, namely, FcεR1γ expressed by nociceptors.

IL-33-Stimulated Murine Mast Cells Polarize Alternatively Activated Macrophages, Which Suppress T Cells That Mediate Experimental Autoimmune Encephalomyelitis.

IL-33 is known to promote type 2 immune responses through ST2, a component of the IL-33R complex, expressed primarily on mast cells, Th2 cells, group 2 innate lymphoid cells and regulatory T cells, and to a lesser extent, on NK cells and Th1 cells. Consistent with previous studies, we found that IL-33 polarized alternatively activated macrophages (AAMΦ) in vivo. However, in vitro stimulation of murine bone marrow-derived or peritoneal macrophages with IL-33 failed to promote arginase activity or expression of YM-1 or Retnla, markers of AAMΦ. Furthermore, macrophages have low/no basal expression of ST2. This suggested that alternative activation of macrophages may involve an IL-33-responsive third-party cell. Because mast cells have the highest expression of ST2 relative to other leukocytes, we focused on this cell type. Coculture experiments showed that IL-33-stimulated mast cells polarized AAMΦ through production of soluble factors. IL-33-stimulated mast cells produced a range of cytokines, including IL-6 and IL-13. Mast cell-derived IL-13 was required for induction of AAMΦ, whereas mast cell-derived IL-6 enhanced macrophage responsiveness to IL-13 via upregulation of the IL-4Rα receptor. Furthermore, we found that AAMΦ polarized by IL-33-stimulated mast cells could suppress proliferation and IL-17 and IFN-γ production by T cells. Finally, we show that AAMΦ polarized by IL-33-stimulated mast cells attenuated the encephalitogenic function of T cells in the experimental autoimmune encephalomyelitis model. Our findings reveal that IL-33 can promote immunosuppressive responses by polarizing AAMΦ via mast cell-derived IL-6 and IL-13.

Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Cell differentiation is directed by extracellular cues and intrinsic epigenetic modifications, which control chromatin organization and transcriptional activation. Central to this process is PRC2, which modulates the di- and trimethylation of lysine 27 on histone 3; however, little is known concerning the direction of PRC2 to specific loci. Here, we have investigated the physical interactome of EZH2, the enzymatic core of PRC2, during retinoic acid-mediated differentiation of neuroepithelial, pluripotent NT2 cells and the dedifferentiation of neuroretinal epithelial ARPE19 cells in response to TGF-ß. We identified Smad3 as an EZH2 interactor in both contexts. Co-occupation of the CDH1 promoter by Smad3 and EZH2 and the cooperative, functional nature of the interaction were established. We propose that the interaction between Smad3 and EZH2 targets the core polycomb assembly to defined regions of the genome to regulate transcriptional repression and forms a molecular switch that controls promoter access through epigenetic mechanisms leading to gene silencing.-Andrews, D., Oliviero, G., De Chiara, L., Watson, A., Rochford, E., Wynne, K., Kennedy, C., Clerkin, S., Doyle, B., Godson, C., Connell, P., O'Brien, C., Cagney, G., Crean, J. Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

Dynamic Protein Interactions of the Polycomb Repressive Complex 2 during Differentiation of Pluripotent Cells.

Polycomb proteins assemble to form complexes with important roles in epigenetic regulation. The Polycomb Repressive Complex 2 (PRC2) modulates the di- and tri-methylation of lysine 27 on histone H3, each of which are associated with gene repression. Although three subunits, EZH1/2, SUZ12, and EED, form the catalytic core of PRC2, a wider group of proteins associate with low stoichiometry. This raises the question of whether dynamic variation of the PRC2 interactome results in alternative forms of the complex during differentiation. Here we compared the physical interactions of PRC2 in undifferentiated and differentiated states of NTERA2 pluripotent embryonic carcinoma cells. Label-free quantitative proteomics was used to assess endogenous immunoprecipitation of the EZH2 and SUZ12 subunits of PRC2. A high stringency data set reflecting the endogenous state of PRC2 was produced that included all previously reported core and associated PRC2 components, and several novel interacting proteins. Comparison of the interactomes obtained in undifferentiated and differentiated cells revealed candidate proteins that were enriched in complexes isolated from one of the two states. For example, SALL4 and ZNF281 associate with PRC2 in pluripotent cells, whereas PCL1 and SMAD3 preferentially associate with PRC2 in differentiating cells. Analysis of the mRNA and protein levels of these factors revealed that their association with PRC2 correlated with their cell state-specific expression. Taken together, we propose that dynamic changes to the PRC2 interactome during differentiation may contribute to directing its activity during cell fate transitions.