Search

Activation of nociceptor sensory neurons by noxious stimuli both triggers pain and increases capillary permeability and blood flow to produce neurogenic inflammation1,2, but whether nociceptors also interact with the immune system remains poorly understood. Here we report a neurotechnology for selective epineural optogenetic neuromodulation of nociceptors and demonstrate that nociceptor activation drives both protective pain behavior and inflammation. The wireless optoelectronic system consists of sub-millimeter-scale light-emitting diodes embedded in a soft, circumneural sciatic nerve implant, powered and driven by a miniaturized head-mounted control unit. Photostimulation of axons in freely moving mice that express channelrhodopsin only in nociceptors resulted in behaviors characteristic of pain, reflecting orthodromic input to the spinal cord. It also led to immune reactions in the skin in the absence of inflammation and potentiation of established inflammation, a consequence of the antidromic activation of nociceptor peripheral terminals. These results reveal a link between nociceptors and immune cells, which might have implications for the treatment of inflammation.

Dendritic cells (DCs) are critical players in skin homeostasis. A subset of mannose receptor (CD206)–expressing monocyte-derived DCs was found in skin, and their migratory counterpart is present in skin-draining lymph nodes (sdLNs). Skin CD206+ DCs were shown to upregulate MHC class II (MHCII) progressively, raising the question of whether this feature affects their biology. In this study, we assessed the role of MHCII regulation in the development and migration of these cells in mouse models expressing differential MHCII levels. Using CD206 as a surrogate marker, we found that skin CD206+ DCs develop in an MHCII-independent manner. However, their migration to sdLNs was affected by overexpression rather than absence or lower expression of MHCII. Accordingly, B16 tumor growth was exacerbated in mice overexpressing MHCII in the absence of ubiquitination. Mechanistically, CD206+ DCs from these mice showed decreased IRF4 and CCR7 expression. LPS, which is known to promote monocyte-derived DC recruitment to sdLNs, partially improved these defects. However, GM-CSF delivery restored CD206+ DC migration by promoting IRF4 expression. Collectively, these data show that MHCII downregulation is crucial for IRF4-dependent migration of CD206+ DCs to sdLNs in health and disease.

Nociceptors, the high-threshold primary sensory neurons that trigger pain, interact with immune cells in the periphery to modulate innate immune responses. Whether they also participate in adaptive and humoral immunity is, however, not known. In this study, we probed if nociceptors have a role in distinct airway and skin models of allergic inflammation. In both models, the genetic ablation and pharmacological silencing of nociceptors substantially reduced inflammatory cell infiltration to the affected tissue. Moreover, we also found a profound and specific deficit in IgE production in these models of allergic inflammation. Mechanistically, we discovered that the nociceptor-released neuropeptide substance P helped trigger the formation of antibody-secreting cells and their release of IgE. Our findings suggest that nociceptors, in addition to their contributions to innate immunity, play a key role in modulating the adaptive immune response, particularly B cell antibody class switching to IgE.

Cell bioprinting technologies aim to fabricate tissuelike constructs by delivering biomaterials layer-by-layer. Bioprinted constructs can reduce the use of animals in drug development and hold promise for addressing the shortage of organs for transplants. Here, we sought to validate the feasibility of bioprinting primary adult sensory neurons using a newly developed laser-assisted cell bioprinting technology, known as Laser-Induced Side Transfer (LIST). We used dorsal root ganglion neurons (DRG, cell bodies of somatosensory neurons) to prepare our bioink. DRG-laden- droplets were printed on fibrin-coated coverslips and their viability, calcium kinetics, neuropeptides release, and neurite outgrowth were measured. The transcriptome of the neurons was sequenced. We found that LIST-printed neurons maintain high viability (Printed: 86%, Control: 87% on average) and their capacity to release neuropeptides (Printed CGRP: 130 pg/mL, Control CGRP: 146 pg/mL). In addition, LIST-printed neurons do not show differences in the expressed genes compared to control neurons. However, in printed neurons, we found compromised neurite outgrowth and lower sensitivity to the ligand of the TRPV1 channel, capsaicin. In conclusion, LIST-printed neurons maintain high viability and marginal functionality losses. Overall, this work paves the way for bioprinting functional 2D neuron assays.

Solid tumors are innervated by nerve fibers that arise from the autonomic and sensory peripheral nervous systems. In prostate cancer, doublecortin-expressing neural progenitors initiate autonomic adrenergic neurogenesis1 which facilitates tumor development and dissemination2, via an angiogenic switch that fuels cancer growth3,4. Similarly, a loss of TP53 drives the reprogramming of tumor-innervating sensory nerves into adrenergic neurons in head and neck tumors, which promotes tumor growth5. However, the impact of tumor neo-innervation by pain-initiating sensory neurons remains unclear. We show that melanoma cells interact with nociceptors, increasing neurite outgrowth, responsiveness to noxious ligands, and neuropeptide release. In turn, CGRP, a nociceptor-produced neuropeptide, directly increases exhaustion of cytotoxic CD8+ T-cells (PD1+Lag3+Tim3+IFNγ-), limiting their capacity to eliminate melanoma. Genetic NaV1.8 or TRPV1 lineage ablation, local pharmacological silencing or blockade of neuropeptide release from tumor-innervating nociceptors, and the antagonism of the CGRP receptor RAMP1, all blunt tumor-infiltrating leukocyte exhaustion, and tumor growth, nearly tripling survival of B16F10-inoculated mice. Inversely, CD8+ T-cell exhaustion increased following optogenetic activation of tumor-innervating NaV1.8 neurons+ and was rescued in sensory neuron depleted mice treated with recombinant CGRP. In comparison to wild-type CD8+ T-cells, RAMP1-/- CD8+ T-cells were protected from undergoing exhaustion when co-transplanted into tumor-bearing Rag1 deficient mice. Single-cell RNA sequencing of patient tumors revealed that intratumoral RAMP1-expressing CD8+ T-cells are more exhausted than their RAMP1 negative counterparts. RAMP1 expression in intratumoral CD8+ T-cells was also associated with resistance to immune checkpoint inhibitor treatment, while RAMP1 overexpression within the tumor correlated with a worse clinical prognosis. We conclude that reducing CGRP release from tumor-innervating nociceptors, by eliminating its immunomodulatory action on cytotoxic CD8+ T-cells, constitutes a useful strategy to safeguard anti-tumor immunity.

The host evolves redundant mechanisms to preserve physiological processing and homeostasis. These functions range from sensing internal and external threats, creating a memory of the insult and generating reflexes, which aim to resolve inflammation. Impairment in such functioning leads to chronic inflammatory diseases. By interacting through a common language of ligands and receptors, the immune and sensory nervous systems work in concert to accomplish such protective functions. Whilst this bidirectional communication helps to protect from danger, it can contribute to disease pathophysiology. Thus, the somatosensory nervous system is anatomically positioned within primary and secondary lymphoid tissues and mucosa to modulate immunity directly. Upstream of this interplay, neurons detect danger, which prompts the release of neuropeptides initiating (i) defensive reflexes (ranging from withdrawal response to coughing) and (ii) chemotaxis, adhesion and local infiltration of immune cells. The resulting outcome of such neuro-immune interplay is still ill-defined, but consensual findings start to emerge and support neuropeptides not only as blockers of TH 1-mediated immunity but also as drivers of TH 2 immune responses. However, the modalities detected by nociceptors revealed broader than mechanical pressure and temperature sensing and include signals as various as cytokines and pathogens to immunoglobulins and even microRNAs. Along these lines, we aggregated various dorsal root ganglion sensory neuron expression profiling datasets supporting such wide-ranging sensing capabilities to help identifying new danger detection modalities of these cells. Thus, revealing unexpected aspects of nociceptor neuron biology might prompt the identification of novel drivers of immunity, means to resolve inflammation and strategies to safeguard homeostasis.

Summary: The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca2+ influx sarco/ER Ca2+-ATPase inhibitor thapsigargin (THP). Baseline ER Ca2+ levels measured with the ER Ca2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca2+ influx and decreased intracellular Ca2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca2+ entry regulators, with no alterations in ER Ca2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons. : Korecka, Isacson, and colleagues show that human iPSC-derived neurons with the Parkinson disease (PD) LRRK2 G2019S mutation have decreased basal ER calcium levels. Upon ER calcium influx inhibition, the LRRK2 G2019S PD patient neurons show specific and reversible neurite collapse and altered calcium uptake and buffering. These results demonstrate LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons. Key words: Parkinson disease, human iPSC-derived neurons, LRRK2 G2019S mutation, ER, calcium homeostasis, ER calcium

Background and Purpose: Many pain-triggering nociceptor neurons express TRPV1 or TRPA1, cation-selective channels with large pores that enable permeation of QX-314, a cationic analogue of lidocaine. Co-application of QX-314 with TRPV1 or TRPA1 activators can silence nociceptors. We now describe BW-031, a novel more potent cationic sodium channel inhibitor, test whether its application alone can inhibit the pain associated with tissue inflammation, and whether this strategy can also inhibit cough. Experimental Approach: We characterized BW-031 inhibition of sodium channels and tested BW-031 in three models of inflammatory pain: rat paw inflammation produced by Complete Freund’s Adjuvant injection or surgical incision and a mouse paw UV burn model. We also tested the ability of BW-031 to inhibit cough induced by inhalation of dilute citric acid in guinea pigs. Key Results: BW-031 inhibited Nav1.7 and Nav1.1 channels with ~6-fold greater potency than QX-314 when introduced inside cells and entered capsaicin-activated TRPV1 expressing sensory neurons. BW-031 inhibited inflammatory pain in all three models, producing more effective and longer-lasting inhibition of pain than QX-314 in the mouse UV burn model. BW-031 was also effective in reducing cough counts by 78-90% when applied intratracheally under isoflurane anesthesia or by aerosol inhalation in awake guinea pigs with airway inflammation produced by ovalbumin sensitization. Conclusion and Implications: BW-031 a novel cationic sodium channel inhibitor can be applied locally as a single agent to inhibit inflammatory pain and also effectively inhibits cough in a guinea pig model of nociceptor-activated cough, suggesting a new clinical approach to treating cough.

Despite impressive strides in light-enabled stimulation, approaches to silence targeted unmodified neurons subsets are yet to be devised. We will present a novel method to silence unmodified neurons via photothermal-mediated targeted drug delivery. Our approach uses laser-stimulation of antibody-coated gold nanoparticles to heat-activate TRPV1 (a noxious heat-sensing channel), turning this channel into a cell-specific drug-entry port. We will show that photothermal silencing blocks neuronal calcium currents and peptide release, resulting in targeted silencing of nociceptors. This approach can help to elucidate the contribution of nociceptor subpopulations to autoimmune pathologies and with further efforts can be translated into novel treatments in neuroscience.

Dendritic cells (DCs) excel at cross-presenting antigens, but their effectiveness as cancer vaccine is limited. Here, we describe an innovative vaccine approach using mesenchymal stromal cells (MSCs) engineered to express the immunoproteasome (IPr) complex (MSC-IPr). Such modification instilled efficient antigen cross-presenting abilities associated with enhanced major histocompatibility complex (MHC)I and CD80 expression, de novo expression of interleukin-12 along with higher chemokine secretion. In addition, the cross-presentation capacity of MSC-IPr is highly dependent on AMPK signaling, oxidative phosphorylation and production of reactive oxygen species. Compared to DCs, MSC-IPr hold the ability to cross-present a vastly different epitope repertoire, which translates into potent re-activation of T-cell immunity against EL4 lymphoma and B16 melanoma tumors. Moreover, therapeutic vaccination of animals with pre-established tumors efficiently controls cancer growth, an effect further enhanced when combined with antibodies targeting PD1, CTLA4, LAG3 or 4-1BB under both autologous and allogeneic settings. MSC-IPr constitute therefore a novel subset of non-hematopoietic antigen-presenting cells suitable for the future design of universal cell-based cancer vaccines.

Background: Dendritic cells (DCs) are usually very efficient at activating CD8 T cells. However, their capacity to cross-present soluble antigens can be limited by non-specific degradation of captured antigens during endosome maturation, which would lead to the destruction of immunogenic epitopes. To bypass this limitation, we tested a new antigen formulation using AccumTM, a technology originally designed to enhance accumulation of antibody-drug conjugate in target cells by promoting endosome-to-cytosol escape. Methods: Following chemical linking and characterization of the Accum-antigen complex, a series of in vitro antigen presentation assays were conducted to assess the T-cell activation potency of the antigen formulation. The generated cellular product was then tested in vivo for its capacity to elicit anti-tumoral responses in both prophylactic and therapeutic vaccination settings. Finally, allogeneic DCs pulsed with tumor lysate linked to AccumTM were characterized for their therapeutic potency. Results: Linking the Accum moiety to the experimental xenoantigen ovalbumin caused endosomal damages resulting in enhanced intracellular protein processing by primary DCs. As a result, production of pro-inflammatory cytokines and chemokines was enhanced by responding T cells. When evaluated for their anti-tumoral properties, Accum-ovalbumin-pulsed DCs elicited potent effector and central memory CD8 T cells, which explains the complete and long-lasting protection observed in vaccinated mice despite multiple EG.7 T-cell lymphoma challenges. When combined with the anti-PD-1 immune checkpoint inhibitor, therapeutic vaccination using both syngeneic and allogeneic DCs pulsed with Accum-linked ovalbumin triggered potent anti-tumoral responses; an observation further enhanced using Accum-linked lymphoma tumor lysate proteins. Conclusion: These highly favorable therapeutic effects highlight the promising potential of AccumTM as a distinct and potent technology platform suitable for the design of next generation cell cancer vaccines.

Although multiple diseases of the respiratory system cause cough, there are few effective treatments for this common condition. We previously developed a strategy to treat pain and itch via the long-lasting inhibition of nociceptor sensory neurons with QX-314, a cationic sodium channel blocker that selectively enters only into activated nociceptors by permeating through the endogenous TRPV1 and TRPA1 large pore ion channels they express. In this study we design and characterize BW-031, a novel cationic compound with ~6-fold greater potency than QX-314 for inhibiting sodium channels when introduced inside cells and with minimal extracellular activity. We show that inhalation of aerosolized BW-031 effectively inhibits citric acid-induced cough in an allergic inflammation guinea pig cough model. These data support the use of charged sodium channel blockers for the selective inhibition of airway sensory neurons with activated large pore channels as a novel targeted therapy for treating cough.

The prevalence of obesity skyrocketed over the past decades to become a significant public health problem. Obesity is recognized as a low-grade inflammatory disease and is linked with several comorbidities such as diabetes, circulatory disease, common neurodegenerative diseases, as well as chronic pain. Adipocytes are a major neuroendocrine organ that continually, and systemically, releases pro-inflammatory factors. While the exact mechanisms driving obesity-induced pain remain poorly defined, nociceptor hypersensitivity may result from the systemic state of inflammation characteristic of obesity as well as weight surplus-induced mechanical stress. Obesity and pain also share various genetic mutations, lifestyle risk factors, and metabolic pathways. For instance, fat pads are often found hyper-innervated and rich in immune cell types of multiple origins. These immunocytes release cytokines, amplifying nociceptor function, which, in turn, via locally released neuropeptides, sustain immunocytes' function. Here, we posit that along with mechanical stress stemming from extra weight, the local neuro-immune interplay occurring within the fat pads maintains the state of chronic low-grade inflammation and heightens sensory hypersensitivity. Overall, stopping such harmful neuro-immune crosstalk may constitute a novel pathway to prevent obesity-associated comorbidities, including neuronal hypersensitivity.

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 FceR1γ knockdown (TRPV1Cre::FceR1γfl/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 FceR1, the levels of which increase in OVA-sensitized mice. FceR1γ-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::FceR1γfl/fl mice and in FceR1α–/– mice into which bone marrow has been transplanted. Finally, increased in vivo circulating levels of IgE following allergen sensitization enhances the responsiveness of FceR1 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, FceR1-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, FceR1γ expressed by nociceptors.

Patients with densely innervated tumors do poorly as compared to those with sparsely innervated disease. Why some tumors heavily recruit nerves while others do not, remains unknown as does the functional contribution of tumor-infiltrating nerves to cancer. Moreover, while patients receive chemotherapeutic treatment, whether these drugs affect nerve recruitment has not been tested. Using a murine model of ovarian cancer, we show that tumor-infiltrating sensory nerves potentiate tumor growth, decrease survival, and contribute to treatment resistance. Furthermore, matched patient samples show significantly increased tumor innervation following chemotherapy.In vitroanalysis of tumor-released extracellular vesicles (sEVs) shows they harbor neurite outgrowth activity. These data suggest that chemotherapy may alter sEV cargo, endowing it with robust nerve recruiting capacity.

Tumor denervation limits cancer growth, but the mechanisms behind this are unknown. We find that malignant melanoma cells interact with pain-initiating nociceptor neurons by increasing neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. In turn, nociceptor-produced neuropeptides increase exhaustion of cytotoxic T cells (PD1+Lag3+Tim3+INFγ-), limiting their capacity to eliminate melanoma cells. Genetic TRPV1 or NaV1.8 lineage ablation, local pharmacological silencing as well as blockade of vesicle release from tumor-innervating nociceptors enhance tumor-infiltrating leukocyte (TIL) numbers and increase survival of mice subject to orthotropic melanoma inoculation, blunting tumor growth and TIL exhaustion. We conclude that reducing neuropeptide release from tumor-innervating nociceptors, by eliminating their immunomodulatory action on cytotoxic CD8 T cell, may be a useful therapeutic intervention to boost immune surveillance.

When pathogens and toxins breech the epithelial barrier, antigens are transported by the lymphatic system to lymph nodes. In previously immunized animals, antigens become trapped in the draining lymph nodes, but the underlying mechanism that controls antigen restriction is poorly understood. Here we describe the role of neurons in sensing and restricting antigen flow in lymph nodes. The antigen keyhole-limpet hemocyanin (KLH) injected into the mouse hind paw flows from the popliteal lymph node to the sciatic lymph node, continuing through the upper lymphatics to reach the systemic circulation. Re-exposure to KLH in previously immunized mice leads to decreased flow from the popliteal to the sciatic lymph node as compared with naïve mice. Administering bupivacaine into the lymph node region restores antigen flow in immunized animals. In contrast, neural activation using magnetic stimulation significantly decreases antigen trafficking in naïve animals as compared with sham controls. Ablating NaV1.8 + sensory neurons significantly reduces antigen restriction in immunized mice. Genetic deletion of FcγRI/FcεRI also reverses the antigen restriction. Colocalization of PGP9.5-expressing neurons, FcγRI receptors and labeled antigen occurs at the antigen challenge site. Together, these studies reveal that neuronal circuits modulate antigen trafficking through a pathway that requires NaV1.8 and FcγR.

Voltage-dependent sodium and calcium channels in pain-initiating nociceptor neurons are attractive targets for new analgesics. We made a permanently charged cationic derivative of an N-type calcium channel-inhibitor. Unlike cationic derivatives of local anesthetic sodium channel blockers like QX-314, this cationic compound inhibited N-type calcium channels more effectively with extracellular than intracellular application. Surprisingly, the compound is also a highly effective sodium channel inhibitor when applied extracellularly, producing more potent inhibition than lidocaine or bupivacaine. The charged inhibitor produced potent and long-lasting analgesia in mouse models of incisional wound and inflammatory pain, inhibited release of the neuropeptide calcitonin gene-related peptide (CGRP) from dorsal root ganglion neurons, and reduced inflammation in a mouse model of allergic asthma, which has a strong neurogenic component. The results show that some cationic molecules applied extracellularly can powerfully inhibit both sodium channels and calcium channels, thereby blocking both nociceptor excitability and pro-inflammatory peptide release.

Dendritic cells (DCs) are critical players in skin homeostasis. A subset of mannose receptor (CD206)-expressing monocyte-derived DCs was found in skin, and their migratory counterpart is present in skin-draining lymph nodes (sdLNs). Skin CD206

summary Airway sensory neuron-produced Substance P heightens allergy-induced goblet cell hyperplasia and hypersecretion of Muc5AC, electrically silencing these overreactive neurons reduced these components of lung type 2 allergic inflammatory response.

Diabetes is a common condition characterized by persistent hyperglycemia. High blood sugar primarily affects cells that have a limited capacity to regulate their glucose intake. These cells include capillary endothelial cells in the retina, mesangial cells in the renal glomerulus, Schwann cells, and neurons of the peripheral and central nervous systems. As a result, hyperglycemia leads to largely intractable complications such as retinopathy, nephropathy, hypertension, and neuropathy. Diabetic pain neuropathy is a complex and multifactorial disease that has been associated with poor glycemic control, longer diabetes duration, hypertension, advanced age, smoking status, hypoinsulinemia, and dyslipidemia. While many of the driving factors involved in diabetic pain are still being investigated, they can be broadly classified as either neuron -intrinsic or -extrinsic. In neurons, hyperglycemia impairs the polyol pathway, leading to an overproduction of reactive oxygen species and reactive nitrogen species, an enhanced formation of advanced glycation end products, and a disruption in Na+/K+ ATPase pump function. In terms of the extrinsic pathway, hyperglycemia leads to the generation of both overactive microglia and microangiopathy. The former incites a feed-forward inflammatory loop that hypersensitizes nociceptor neurons, as observed at the onset of diabetic pain neuropathy. The latter reduces neurons' access to oxygen, glucose and nutrients, prompting reductions in nociceptor terminal expression and losses in sensation, as observed in the later stages of diabetic pain neuropathy. Overall, microglia can be seen as potent and long-lasting amplifiers of nociceptor neuron activity, and may therefore constitute a potential therapeutic target in the treatment of diabetic pain neuropathy.

SummaryLung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8+ sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4+ and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma.

The gastrin-releasing peptide (GRP) and its receptor (GRPR) are important components of itch transmission. Upstream, but not downstream, aspects of GRPR signaling have been investigated extensively. We hypothesize that GRPR signals in part through the PI3Kγ/Akt pathway. We used pharmacological, electrophysiological, and behavioral approaches to further evaluate GRPR downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization (∼20 mV). GRPR activation also induces Akt phosphorylation, a proxy for PI3Kγ activity, inex vivonaive mouse spinal cords and in GRPR transiently expressing HEK293 cells. The intrathecal injection of GRP led to intense scratching, an effect largely reduced by either GRPR antagonists or PI3Kγ inhibitor. Scratching behavior was also induced by the intrathecal injection of an Akt activator. In a dry skin model of itch, we show that GRPR blockade or PI3Kγ inhibition reversed the scratching behavior. Altogether, these findings are highly suggestive that GRPR is expressed by the central terminals of DRG nociceptive afferents, which transmit itch via the PI3Kγ/Akt pathway.SIGNIFICANCE STATEMENTItch is the most common symptom of the skin and is related to noncutaneous diseases. It severely impairs patients' quality of life when it becomes chronic and there is no specific or effective available therapy, mainly because itch pathophysiology is not completely elucidated. Our findings indicate that the enzyme PI3Kγ is a key central mediator of itch transmission. Therefore, we suggest PI3Kγ as an attractive target for the development of new anti-pruritic drugs. With this study, we take a step forward in our understanding of the mechanisms underlying the central transmission of itch sensation.

The challenge of translating findings from animal models to the clinic is well known. An example of this challenge is the striking effectiveness of neurokinin-1 receptor (NK-1R) antagonists in mouse models of inflammation coupled with their equally striking failure in clinical investigations in humans. Here, we provide an explanation for this dichotomy: Mas-related GPCRs (Mrgprs) mediate some aspects of inflammation that had been considered mediated by NK-1R. In support of this explanation, we show that conventional NK-1R antagonists have off-target activity on the mouse receptor MrgprB2 but not on the homologous human receptor MRGPRX2. An unrelated tripeptide NK-1R antagonist has dual activity on MRGPRX2. This tripeptide both suppresses itch in mice and inhibits degranulation from the LAD-2 human mast cell line elicited by basic secretagogue activation of MRGPRX2. Antagonists of Mrgprs may fill the void left by the failure of NK-1R antagonists.

Kinins are the endogenous ligands of the constitutive B2 receptor (B2R) and the inducible B1 receptor (B1R). Whereas B2R prevents insulin resistance, B1R is involved in insulin resistance and metabolic syndrome. However, the contribution of B1R in type 2 diabetes associated with obesity remains uncertain. The aim of the present study was to examine the impact of 1-week treatment with a selective B1R antagonist (SSR240612, 10 mg/kg per day, by gavage) on hyperglycemia, hyperinsulinemia, leptinemia, body mass gain, and abnormal plasma fatty acids in obese Zucker diabetic fatty (ZDF) rats. Treatment with SSR240612 abolished the body mass gain and reduced polyphagia, polydipsia, and plasma fatty acid alterations in ZDF rats without affecting hyperglycemia, hyperinsulinemia, and hyperleptinemia. The present study suggests that the upregulated B1R plays a role in body mass gain and circulating fatty acid alterations in ZDF rats. However, mechanisms other than B1R induction would be implicated in glucose metabolism disorder in ZDF rats, based on the finding that SSR240612 did not reverse hyperglycemia and hyperinsulinemia.

Evolution has yielded multiple complex and complementary mechanisms to detect environmental danger and protect tissues from damage. The nervous system rapidly processes information and coordinates complex defense behaviors, and the immune system eliminates diverse threats by virtue of mobile, specialized cell populations. The two systems are tightly integrated, cooperating in local and systemic reflexes that restore homeostasis in response to tissue injury and infection. They further share a broad common language of cytokines, growth factors, and neuropeptides that enables bidirectional communication. However, this reciprocal cross talk permits amplification of maladaptive feedforward inflammatory loops that contribute to the development of allergy, autoimmunity, itch, and pain. Appreciating the immune and nervous systems as a holistic, coordinated defense system provides both new insights into inflammation and exciting opportunities for managing acute and chronic inflammatory diseases.

Using a rat lung slice model, this study compared the stress responses induced by cigarette whole smoke (WS) to that induced by the vapor phase (VP) of the smoke. Following a 3-day exposure, lung slices exposed to 4, 10 and 20% WS retained 85, 42 and 16% relative survival respectively in comparison to the air-exposed ones. Consistently, histological observations revealed concentration-related alveolar damages in the lung slices. Expression of 5 stress–response genes was examined following a single 30 min exposure to 4% WS or VP. WS exposure resulted in 4, 11 and 50-fold induction of IL-1β, kinin type I receptor (B 1 R) and CYP1A1 genes, respectively, while CYP1B1 and TNF-α genes expression was found only two times higher in comparison to VP group. Since cigarette WS consists of particulate and vapor phases, these results highlight the preferential or synergistic role of the particulate phase in the induction of IL-1β, B 1 R and CYP1A1 genes and that VP did not have comparable effects on expression of these genes. However, both phases fairly contributed to the induction of CYP1B1 and TNF-α genes. VP was the fraction responsible for the toxic effect since WS did not produce further toxicity. The 4% whole smoke deposited about 7.1 μg/cm 2 of total particulate matter (TPM) to the exposure chamber which may account for observed differential stress responses in the lung slices.