Objective: This study aimed to examine the impact of high-fat-diet(HFD)-induced obesity on pain sensitivity and tetrahydrobiopterin (BH4) levels. The effect of moderate-intensity physical exercise, an anti-inflammatory non-pharmacological intervention, on pain scores was also investigated. Methods: Adult male C57BL/J6 mice were fed standard or an HFD for eight weeks. Their total body weight, food intake, locomotor and motivational behavior, and pain reflexes were measured. A subgroup of animals underwent physical exercise for five days per week over six weeks. Blood was collected for glucose tolerance testing and levels of lactate. Urine samples were collected to measure BH4 levels. Results: We showed that the HFD increased weight gain, epididymal white adipose tissue, and the percentage of body weight as epididymal fat. These anthropometric alterations were characterized by impaired glucose tolerance at four and eight weeks of the dietary intervention. It was also observed that reduced locomotor activity and higher pain scores in the HFD-fed mice were prevented by a physical exercise intervention. The HFD also induced an increase in urinary BH4 levels at four and eight weeks of intervention. Conclusions: The HFD increased scores of chemical and mechanical hyperalgesias, as well as urinary BH4 levels. Urinary BH4 can be proposed as a potential easy-to-access, sensitive, and reliable biomarker of pain development, and a promising target for the control of pain hypersensitivity in obesity.

Abstract Cancer is the top cause of death in the USA, responsible for 30% of deaths. A cure remains undiscovered. Therapy effectiveness varies by patient and tumor type and can have negative side effects. The immune system, designed to eliminate abnormal cells, including cancer cells, is often impaired. For example, in skin cancer, extended activation of certain receptors can exhaust immune cells, reducing their cancer-fighting ability. Our lab found that cancer cells trigger nociceptor neurons in tumors, leading to neuropeptide release that exhausts CD8+ T cells. To explore their interactions, we conducted single-cell RNA sequencing on tumor-related sensory neurons, leukocytes, and cancer cells. We found a distinct group of tumor-related neurons expressingATF3, Sox10, Sprr1a, and galanin. In our study, mice with injury-resistant nociceptor neurons (NaV1.8ΔATF3) developed smaller melanoma tumors and had reduced galanin levels compared to controls. These mice also showed stronger anti-tumor immune responses, especially in cytotoxic CD8+ T-cells. Restoring galanin in mice with ATF3-deficient nociceptor neurons resulted in tumor growth similar to normal mice. We observed that galanin increases exhaustion, reduces the cytotoxic capacity of CD8+ T-cells, and leads the cells to a state of immunoparalysis. Higher galanin levels in melanoma patients were linked to worse outcomes. Using single-cell RNA sequencing from melanoma patients, we also found that cytotoxic CD8+ T cells with the galanin receptor 1(GALR1) were more exhausted than those without it. In summary, ATF3-modifiedtumor-related sensory neurons that release galanin can weaken anti-tumor immunity, suggesting cancer cells may use this aspect of neuro-immunity to promote tumor growth. Citation Format: Tuany Eichwald, Mohammad Balood, Maryam Ahmadi, Karine Roversi, Dylan Wong, Judith Mandl, Moutih Rafei, Nader Ghasemlou, Paola Vermeer, Moran Amit, Sébastien Talbot. Nociceptor neurons control anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY31-02.

Evolution has created complex mechanisms to sense environmental danger and protect tissues, with the nervous and immune systems playing pivotal roles. These systems work together, coordinating local and systemic reflexes to restore homeostasis in response to tissue injury and infection. By sharing receptors and ligands, they influence the pathogenesis of various diseases. Recently, a less-explored aspect of neuroimmune communication has emerged: the release of neuropeptides from immune cells and cytokines/chemokines from sensory neurons. This article reviews evidence of this unique neuroimmune interplay and its impact on the development of allergy, inflammation, itch, and pain. We highlight the effects of this neuroimmune signaling on vital processes such as host defense, tissue repair, and inflammation resolution, providing avenues for exploration of the underlying mechanisms and therapeutic potential of this signaling.

The immune and nervous systems have co-evolved complex mechanisms to sense environmental dangers and orchestrate a concerted response to safeguard tissue and mobilize host defenses. This sophisticated interplay, marked by a shared repertoire of receptors and ligands, influences disease pathogenesis. Neuro-immune interactions in allergic diseases are pivotal for symptom development, from anaphylaxis to chronic conditions like asthma and atopic dermatitis. This review explores the neuro-immune interplay within the atopic march, emphasizing its role in host defense, inflammation resolution, and tissue repair. We delve into the multifaceted functions of nociceptors in orchestrating type 2 immune responses and the progression of allergic disorders, focusing on key regulators such as CGRP-RAMP1 and SP-MRGPRB2/A2. Additionally, we discuss the potential of nociceptor neuron-blocking drugs to target neuro-immunity, offering the possibility of reversing the progression of the atopic march. Altogether, we underscore the need for targeted interventions to disrupt the pathological processes and enhance therapeutic outcomes at various stages of the atopic march.

Nociceptor neurons impact tumor immunity. Removing nociceptor neurons reduced myeloid-derived suppressor cell (MDSCs) tumor infiltration in mouse models of head and neck carcinoma and melanoma. Carcinoma-released small extracellular vesicles (sEVs) attract nociceptive nerves to tumors. sEV-deficient tumors fail to develop in mice lacking nociceptor neurons. Exposure of dorsal root ganglia (DRG) neurons to cancer sEVs elevated expression of Substance P, IL-6 and injury-related neuronal markers while treatment with cancer sEVs and cytotoxic CD8 T-cells induced an immunosuppressive state (increased exhaustion ligands and cytokines). Cancer patient sEVs enhanced DRG responses to capsaicin, indicating increased nociceptor sensitivity. Conditioned media from DRG and cancer cell co-cultures promoted expression of MDSC markers in primary bone marrow cells while DRG conditioned media together with cancer sEVs induced checkpoint expression on T-cells. Our findings indicate that nociceptor neurons facilitate CD8+ T cell exhaustion and enhance MDSC infiltration. Targeting nociceptor-released IL-6 emerges as a novel strategy to disrupt harmful neuro-immune interactions in cancer and enhance anti-tumor immunity.

The immune and sensory nervous systems communicate to maintain homeostasis. Wu et al. recently demonstrated that sensory neurons innervate the mouse spleen. These neurons promote calcitonin gene-related peptide (CGRP)-dependent responses in splenic B cell germinal centers (GCs) and antigen-specific antibody production. Dietary capsaicin activates these neurons to enhance humoral immunity against influenza virus infection.

Evidence has shown that caffeine administration reduces pro-inflammatory biomarkers, delaying fatigue and improving endurance performance. This study examined the effects of caffeine administration on the expression of inflammatory-, adenosine receptor- (the targets of caffeine), epigenetic-, and oxidative metabolism-linked genes in the vastus lateralis muscle of mice submitted to lipopolysaccharide (LPS)-induced inflammation. We showed that caffeine pre-treatment before LPS administration reduced the expression of Il1b, Il6, and Tnfa, and increased Il10 and Il13. The negative modulation of the inflammatory response induced by caffeine involved the reduction of inflammasome components, Asc and Casp1, promoting an anti-inflammatory scenario. Caffeine treatment per se promoted the upregulation of adenosinergic receptors, Adora1 and Adora2A, an effect that was counterbalanced by LPS. Moreover, there was observed a marked Adora2A promoter hypermethylation, which could represent a compensatory response towards the increased Adora2A expression. Though caffeine administration did not alter DNA methylation patterns, the expression of DNA demethylating enzymes, Tet1 and Tet2, was increased in mice receiving Caffeine+LPS, when compared with the basal condition. Finally, caffeine administration attenuated the LPS-induced catabolic state, by rescuing basal levels of Ampk expression. Altogether, the anti-inflammatory effects of caffeine in the muscle can be mediated by modifications on the epigenetic landscape.

The molecular and functional contributions of intratumoral nerves to disease remain largely unknown. We localized synaptic markers within tumors suggesting that these nerves form functional connections. Consistent with this, electrophysiological analysis shows that malignancies harbor significantly higher electrical activity than benign disease or normal tissues. We also demonstrate pharmacologic silencing of tumoral electrical activity. Tumors implanted in transgenic animals lacking nociceptor neurons show reduced electrical activity. These data suggest that intratumoral nerves remain functional at the tumor bed. Immunohistochemical staining demonstrates the presence of the neuropeptide, Substance P (SP), within the tumor space. We show that tumor cells express the SP receptor, NK1R, and that ligand/receptor engagement promotes cellular proliferation and migration. Our findings identify a mechanism whereby intratumoral nerves promote cancer progression.

Tetrahydrobiopterin (BH4) is an endogenous cofactor for some enzymatic conversions of essential biomolecules, including nitric oxide, and monoamine neurotransmitters, and for the metabolism of phenylalanine and lipid esters. Over the last decade, BH4 metabolism has emerged as a promising metabolic target for negatively modulating toxic pathways that may result in cell death. Strong preclinical evidence has shown that BH4 metabolism has multiple biological roles beyond its traditional cofactor activity. We have shown that BH4 supports essential pathways, e.g., to generate energy, to enhance the antioxidant resistance of cells against stressful conditions, and to protect from sustained inflammation, among others. Therefore, BH4 should not be understood solely as an enzyme cofactor, but should instead be depicted as a cytoprotective pathway that is finely regulated by the interaction of three different metabolic pathways, thus assuring specific intracellular concentrations. Here, we bring state-of-the-art information about the dependency of mitochondrial activity upon the availability of BH4, as well as the cytoprotective pathways that are enhanced after BH4 exposure. We also bring evidence about the potential use of BH4 as a new pharmacological option for diseases in which mitochondrial disfunction has been implicated, including chronic metabolic disorders, neurodegenerative diseases, and primary mitochondriopathies.

Lead (Pb), a common environmental contaminant, and ethanol (EtOH), a widely available drug of abuse, are well-known neurotoxicants. In vivo, experimental evidence indicates that Pb exposure affects oxidative EtOH metabolism with a high impact on living organisms. On these bases, we evaluated the consequences of combined Pb and EtOH exposure on aldehyde dehydrogenase 2 (ALDH2) functionality. In vitro exposure to 10 µM Pb, 200 mM EtOH, or their combination for 24 h reduced ALDH2 activity and content in SH-SY5Y human neuroblastoma cells. In this scenario, we observed mitochondrial dysfunction characterized by reduced mass and membrane potential, decreased maximal respiration, and spare capacity. We also evaluated the oxidative balance in these cells finding a significant increase in reactive oxygen species (ROS) production and lipid peroxidation products under all treatments accompanied by an increase in catalase (CAT) activity and content. These data suggest that ALDH2 inhibition induces the activation of converging cytotoxic mechanisms resulting in an interplay between mitochondrial dysfunction and oxidative stress. Notably, NAD+ (1 mM for 24 h) restored ALDH2 activity in all groups, while an ALDH2 enhancer (Alda-1, 20 µM for 24 h) also reversed some of the deleterious effects resulting from impaired ALDH2 function. Overall, these results reveal the crucial role of this enzyme on the Pb and EtOH interaction and the potential of activators such as Alda-1 as therapeutic approaches against several conditions involving aldehydes accumulation.

The study aims to evaluate the impact of genetic, demographic and clinical data on various measures of outcome of anticoagulation quality in patients.The study consisted of 310 patients receiving long-term oral anticoagulation therapy in our hospital. Apart from demographic and clinical variables, 21 SNPs (in 7 genes) were analyzed and compared with the outcomes of anticoagulation therapy. Various outcomes that were measured are; supra therapeutic INRs (INR >3, >6), anticoagulation stabilization, time taken to stabilize and proportion of INRs within (2–3), above (>3) and below (<2) therapeutic range.Supra therapeutic INRs were influenced by CYP2C9*2, *3, CYP4F2 rs2108622, VKORC1-1639G>A, 1173C>T, rs55894764 along with concomitant drugs, smoking, body weight and height. Persistently fluctuating INRs/absolute instability correlated with VKORC1-1639G>A, gender, height and body mass index. The time taken to stabilize was associated with CYP4F2 rs2108622, CYP2C9*14, smoking, clinical indication and concomitant drugs. The overall distribution of INR was influenced by variants in CYP4F2 rs2108622, CYP2C9*3, rs9332230, VKORC1 1173C>T, −1639G>A, rs55894764, ABCB1 rs2032582, rs1128503, rs1045642 and F5 rs6025, age, smoking and concomitant drugs.Knowledge of factors influencing the quality of long term anticoagulation can help clinicians to customize therapy either by dose variation, therapy with alternate choice of drug, concurrent heparin therapy and/or frequent INR monitoring.

Manganese (Mn) is an essential metal for many functions in the body. However, in excess, it can be neurotoxic and cause a Parkinson-like syndrome, known as manganism. Here, we aimed to identify a protective effect of probucol, a lipid-lowering agent with anti-inflammatory and antioxidant properties, against Mn-induced toxicity in human neuroblastoma (SH-SY5Y) and glioblastoma (C6) cell lines. The cells were incubated with increasing concentrations of Mn followed by probucol addition 1, 3, 6, and/or 24 h to assess the metal toxic doses and measure the protective effect of probucol against Mn-induced oxidative damage. Longer exposition to Mn showed decreased SH-SY5Y cellular viability in concentrations higher than 100 µM, and probucol was able to prevent this effect. The C6 cells were more sensitive to the Mn deleterious actions, decreasing the cell viability after 6 h of 500 µM Mn exposure. In addition, probucol prevents the complex I and II of the mitochondrial respiratory chain (MRC) inhibition caused by Mn and decreased the intracellular ROS production. Taken together, our results showed that Mn toxicity affects differently both cell lines and probucol has a protective effect against the oxidative imbalance in the central nervous system.

Anaphylaxis is a life-threatening systemic allergic reaction affecting ∼5% of the population. It can be secondary to medications, food, or venom exposure and is generally believed to be immunoglobulin E (IgE)-mediated. Our work in neuro-immunology has shown that allergen-sensitized vagal sensory neurons overexpress FcεR1 and, through their interplay with IgE-producing plasma cells, these neurons can detect antigen-antibody complexes. Sensory neurons intact or genetically ablated (TRPV1creDTAfl/wt) mice were sensitized with the allergen ovalbumin and the adjuvant aluminum hydroxide. Survival, as well as serum immunoglobulins levels, were measured upon acute intravenous allergen challenge. Sensory neurons ablated male mice were more susceptible to life-threatening anaphylaxis than those with intact neurons. We found no difference in IgE levels between ablated and intact neuron mice. Sensory neurons protect the host from undergoing life-threatening anaphylaxis when challenged with an intravenous allergen. These data suggest the existence of a nociceptor neurons anti-inflammatory feedback loop independent of IgE.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems 1 – 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.

Half of the severe asthma patients suffer from uncontrolled exacerbations. Our work in neuro-immunology has shown that, in the context of asthma, vagal nociceptor neurons drive a feed-forward inflammatory loop with lung immune cells, and that silencing these neurons reverses allergic airway inflammation. Here, we aim to expend these findings to a clinically relevant model of pollution-exacerbated asthma.

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.