A Preliminary Study of Mild Heat Stress on Inflammasome Activation in Murine Macrophages.

Inflammation and mitochondrial-dependent oxidative stress are interrelated processes implicated in multiple neuroinflammatory disorders, including Alzheimer's disease (AD) and depression. Exposure to elevated temperature (hyperthermia) is proposed as a non-pharmacological, anti-inflammatory treatment for these disorders; however, the underlying mechanisms are not fully understood. Here we asked if the inflammasome, a protein complex essential for orchestrating the inflammatory response and linked to mitochondrial stress, might be modulated by elevated temperatures. To test this, in preliminary studies, immortalized bone-marrow-derived murine macrophages (iBMM) were primed with inflammatory stimuli, exposed to a range of temperatures (37-41.5 °C), and examined for markers of inflammasome and mitochondrial activity. We found that exposure to mild heat stress (39 °C for 15 min) rapidly inhibited iBMM inflammasome activity. Furthermore, heat exposure led to decreased ASC speck formation and increased numbers of polarized mitochondria. These results suggest that mild hyperthermia inhibits inflammasome activity in the iBMM, limiting potentially harmful inflammation and mitigating mitochondrial stress. Our findings suggest an additional potential mechanism by which hyperthermia may exert its beneficial effects on inflammatory diseases.

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.

Mechanistic Differences in Neuropathic Pain Modalities Revealed by Correlating Behavior with Global Expression Profiling.

Chronic neuropathic pain is a major morbidity of neural injury, yet its mechanisms are incompletely understood. Hypersensitivity to previously non-noxious stimuli (allodynia) is a common symptom. Here, we demonstrate that the onset of cold hypersensitivity precedes tactile allodynia in a model of partial nerve injury, and this temporal divergence was associated with major differences in global gene expression in innervating dorsal root ganglia. Transcripts whose expression change correlates with the onset of cold allodynia were nociceptor related, whereas those correlating with tactile hypersensitivity were immune cell centric. Ablation of TrpV1 lineage nociceptors resulted in mice that did not acquire cold allodynia but developed normal tactile hypersensitivity, whereas depletion of macrophages or T cells reduced neuropathic tactile allodynia but not cold hypersensitivity. We conclude that neuropathic pain incorporates reactive processes of sensory neurons and immune cells, each leading to distinct forms of hypersensitivity, potentially allowing drug development targeted to each pain type.

The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor.

Diverse innate lymphoid cell (ILC) subtypes have been defined on the basis of effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways that lead to ILC-lineage specification remain poorly characterized. Here we found that the transcriptional regulator TOX was required for the in vivo differentiation of common lymphoid progenitors into ILC lineage-restricted cells. In vitro modeling demonstrated that TOX deficiency resulted in early defects in the survival or proliferation of progenitor cells, as well as ILC differentiation at a later stage. In addition, comparative transcriptome analysis of bone marrow progenitors revealed that TOX-deficient cells failed to upregulate many genes of the ILC program, including genes that are targets of Notch, which indicated that TOX is a key determinant of early specification to the ILC lineage.

Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.