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.

Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons.

Somatosensory neurons have evolved to detect noxious stimuli and activate defensive reflexes. By sharing means of communication, nociceptor neurons also tune host defenses by controlling the activity of the immune system. The communication between these systems is mostly adaptive, helping to protect homeostasis, it can also lead to, or promote, the onset of chronic diseases. Both systems co-evolved to allow for such local interaction, as found in primary and secondary lymphoid tissues and mucosa. Recent studies have demonstrated that nociceptors directly detect and respond to foreign antigens, immune cell-derived cytokines, and microbes. Nociceptor activation not only results in pain hypersensitivity and itching, but lowers the nociceptor firing threshold, leading to the local release of neuropeptides. The peptides that are produced by, and released from, the peripheral terminals of nociceptors can block the chemotaxis and polarization of lymphocytes, controlling the localization, duration, and type of inflammation. Recent evidence shows that sensory neurons interact with innate immune cells via cell-cell contact, for example, engaging group 2D (NKG2D) receptors on natural killer (NK) cells. Given that NK cells express the cognate receptors for various nociceptor-produced mediators, it is conceivable that nociceptors use neuropeptides to control the activity of NK cells. Here, we devise a co-culture method to study nociceptor neuron-NK cell interactions in a dish. Using this approach, we found that lumbar nociceptor neurons decrease NK cell cytokine expression. Overall, such a reductionist method could be useful to study how tumor-innervating neurons control the anticancer function of NK cells and how NK cells control the elimination of injured neurons.

Promoting antigen escape from dendritic cell endosomes potentiates anti-tumoral immunity.

The cross-presenting capacity of dendritic cells (DCs) can be limited by non-specific degradation during endosome maturation. To bypass this limitation, we present in this study a new Accum-based formulation designed to promote endosome-to-cytosol escape. Treatment of primary DCs with Accum linked to the xenoantigen ovalbumin (OVA) triggers endosomal damages and enhances protein processing. Despite multiple challenges using ascending doses of tumor cells, DC prophylactic vaccination results in complete protection due to increased levels of effector CD4 and CD8 T cells as well as high production of pro-inflammatory mediators. When combined with anti-PD-1, therapeutic vaccination using both syngeneic and allogeneic Accum-OVA-pulsed DCs triggers potent anti-tumoral responses. The net outcome culminates in increased CD11c, CD8, and NK infiltration along with a high CD8/Treg ratio. These highly favorable therapeutic effects highlight the promising potential of Accum as a distinct and potent technology platform suitable for the design of next generation cell cancer vaccines.

Nanophotonics Enable Targeted Photothermal Silencing of Nociceptor Neurons.

The sensory nervous and immune systems work in concert to preserve homeostasis. While this endogenous interplay protects from danger, it may drive chronic pathologies. Currently, genetic engineering of neurons remains the primary approach to interfere selectively with this potentially deleterious interplay. However, such manipulations are not feasible in a clinical setting. Here, this work reports a nanotechnology-enabled concept to silence subsets of unmodified nociceptor neurons that exploits their ability to respond to heat via the transient receptor potential vanilloid type 1 (TRPV1) channel. This strategy uses laser stimulation of antibody-coated gold nanoparticles to heat-activate TRPV1, turning this channel into a cell-specific drug-entry port. This delivery method allows transport of a charged cationic derivative of an N-type calcium channel blocker (CNCB-2) into targeted sensory fibers. CNCB-2 delivery blocks neuronal calcium currents and neuropeptides release, resulting in targeted silencing of nociceptors. Finally, this work demonstrates the ability of the approach to probe neuro-immune crosstalk by targeting cytokine-responsive nociceptors and by successfully preventing nociceptor-induced CD8+ T-cells polarization. Overall, this work constitutes the first demonstration of targeted silencing of nociceptor neuron subsets without requiring genetic modification, establishing a strategy for interfering with deleterious neuro-immune interplays.

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.

Engineering immunoproteasome-expressing mesenchymal stromal cells: A potent cellular vaccine for lymphoma and melanoma in mice.

Dendritic cells (DCs) excel at cross-presenting antigens, but their effectiveness as cancer vaccine is limited. Here, we describe a vaccination approach using mesenchymal stromal cells (MSCs) engineered to express the immunoproteasome complex (MSC-IPr). Such modification instills efficient antigen cross-presentation abilities associated with enhanced major histocompatibility complex class I and CD80 expression, de novo production of interleukin-12, and higher chemokine secretion. This cross-presentation capacity of MSC-IPr is highly dependent on their metabolic activity. Compared with 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 and A20 lymphomas and B16 melanoma tumors. Moreover, therapeutic vaccination of mice with pre-established tumors efficiently controls cancer growth, an effect further enhanced when combined with antibodies targeting PD-1, CTLA4, LAG3, or 4-1BB under both autologous and allogeneic settings. Therefore, MSC-IPr constitute a promising subset of non-hematopoietic antigen-presenting cells suitable for designing universal cell-based cancer vaccines.

Lack of the E3 Ubiquitin Ligase March1 Affects CD8 T Cell Fate and Exacerbates Insulin Resistance in Obese Mice.

Obesity is a major risk factor for the development of insulin resistance and type 2 diabetes. However, the mechanisms that trigger the underlying adipose tissues inflammation are not completely understood. Here, we show that the E3 ubiquitin ligase March1 controls the phenotypic and functional properties of CD8+ T cells in mice white adipose tissue. In a diet-induced obesity model, mice lacking March1 [March1 knockout (KO)] show increased insulin resistance compared to their WT counterparts. Also, in obese March1 KO mice, the proportions of effector/memory (Tem) and resident/memory (Trm) CD8+ T cells were higher in the visceral adipose tissue, but not in the spleen. The effect of March1 on insulin resistance and on the phenotype of adipose tissue CD8+ T cells was independent of major histocompatibility complex class II ubiquitination. Interestingly, we adoptively transferred either WT or March1 KO splenic CD8+ T cells into obese WT chimeras that had been reconstituted with Rag1-deficient bone marrow. We observed an enrichment of Tem and Trm cells and exacerbated insulin resistance in mice that received March1 KO CD8 T cells. Mechanistically, we found that March1 deficiency alters the metabolic activity of CD8+ T cells. Our results provide additional evidence of the involvement of CD8+ T cells in adipose tissue inflammation and suggest that March1 controls the metabolic reprogramming of these cells.

A Novel Sulfonyl-Based Small Molecule Exhibiting Anti-cancer Properties.

Phenotypic screening is an ideal strategy for the discovery of novel bioactive molecules. Using a customized high-throughput screening (HTS) assay employing primary T lymphocytes, we screened a small library of 4,398 compounds with unknown biological function/target to identify compounds eliciting immunomodulatory properties and discovered a sulfonyl-containing hit, we named InhiTinib. This compound inhibited interferon (IFN)-gamma production and proliferation of primary CD3+ T cells without inducing cell death. In contrast, InhiTinib triggered apoptosis in several murine and human cancer cell lines. Besides, the compound was well tolerated by immunocompetent mice, triggered tumor regression in animals with pre-established EL4 T-cell lymphomas, and prolonged the overall survival of mice harboring advanced tumors. Altogether, our data demonstrate the anti-cancer properties of InhiTinib, which can henceforth bridge to wider-scale biochemical and clinical tests following further in-depth pharmacodynamic studies.

Downregulation of MHC Class II by Ubiquitination Is Required for the Migration of CD206+ Dendritic Cells to Skin-Draining Lymph Nodes.

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.

Neurons and Microglia; A Sickly-Sweet Duo in Diabetic Pain Neuropathy.

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.

Endogenous T Cell Receptor Rearrangement Represses Aggressive Central Nervous System Autoimmunity in a TcR-Transgenic Model on the Non-Obese Diabetic Background.

The T cell response to central nervous system (CNS) antigen in experimental autoimmune encephalomyelitis (EAE) permits one to model the immune aspects of multiple sclerosis. 1C6 transgenic mice on the non-obese diabetic (NOD) background possess a class II-restricted T cell receptor (TcR; Vα5-Vß7) specific for the encephalitogenic peptide myelin oligodendrocyte glycoprotein (MOG)[35-55]. It remains to be determined what role is played by allelic inclusion in shaping the TcR repertoire of these mice. Here, we show that 1C6 T cells display substantial promiscuity in their expression of non-transgenically derived Vα chains. Further, enforced expression of the transgenic TcR in 1C6 × Rag1-/- mice profoundly disrupted thymic negative selection and led to a sharp decrease in the number of mature peripheral T cells. 1C6 × Rag1-/- mice developed spontaneous EAE at a significant frequency and rapidly developed fatal EAE upon immunization with myelin oligodendrocyte glycoprotein (MOG)[35-55]. Passive transfer of 1C6 × Rag1+/+ CD4+ T cells, but not CD8+ T cells or B cells, partially rescued 1C6 × Rag1-/- mice from severe EAE. FoxP3+ CD4+ Treg cells were present in the CNS of immunized 1C6 mice, as well as immunized 1C6 × Rag1-/- that had been supplemented with 1C6 CD4+ T cells. However, they were not observed in 1C6 × Rag1-/- that did not receive Rag1-sufficient 1C6 CD4+. Further, in vivo blockade of Treg accelerated the onset of symptoms in 1C6 mice immunized with MOG[35-55], indicating the pertinence of Treg-mediated control of autoimmune inflammation in this model. Thus, TcR allelic inclusion is crucial to the generation of FoxP3+ CD4+ T cells necessary for the suppression of severe CNS autoimmunity.

C/EBP homologous protein investigation in the serum and cerebro-spinal fluid of relapsing-remitting multiple sclerosis patients.

The exact determination of endoplasmic reticulum (ER) stress-associated proteins is not completely elucidated in the multiple sclerosis (MS) patients. We measured CHOP concentrations in the serum and cerebro-spinal fluid (CSF) of relapsing-remitting MS (RRMS) patients (n = 20) in comparison with the non-MS control group (n = 20) to determine whether this marker could be detected in the body fluids of RRMS patients. CHOP marker was not detectable in all harvested CSF samples. However, its levels were detectable in all serums harvested from both non-MS and RRMS patients and its levels in the latter group were not significantly higher than those of the non-MS control group (P value = 0.265). CHOP was not detectable in the CSF of RRMS patients in spite of the recent reports on the RRMS autopsies. Additionally, there were not any significant correlations (Spearman's correlation) between both of EDSS score and age with CHOP serum concentrations in all subjects.

Soluble CD40 ligand derived from serum is not correlated with early MS.

BACKGROUND: Soluble CD40 ligand (sCD154) is a proinflammatory and prothrombotic ligand belonging to the tumor necrosis factor family. It has been shown by a variety of studies that sCD154 is elevated in the serum of patients afflicted with system autoimmune diseases. The aim of our study was to address whether sCD154 is increased in disease affected by Multiple Sclerosis (MS). METHODS: Twenty MS patients who have been newly diagnosed as clinically definite multiple sclerosis (CDMS) along with twenty age and sex matched healthy individuals were recruited for this study. Serum cCD154 was measured by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The results showed no statistically meaningful difference between newly diagnosed MS patients (3.07ng/ul±0.66) and control group (2.95ng/ul±0.79; p=0.62) CONCLUSION: Regarding our study, it seems that soluble CD40 ligand derived from serum is not correlated with early stage of MS disease.

Decreased concentration of Klotho in the cerebrospinal fluid of patients with relapsing-remitting multiple sclerosis.

Recent investigations support that an anti-aging protein, namely Klotho, protects neurons against the oxidative stress and demyelination. We evaluated the protein concentration of Klotho and total anti-oxidant capacity (TAC) in the cerebrospinal fluid (CSF) of patients with relapsing-remitting multiple sclerosis (RRMS). Klotho concentration and TAC were significantly lower in patients as compared to controls. Klotho values showed a significant negative correlation with expanded disability status scale (EDSS). Moreover, a significantly positive correlation between TAC levels and Klotho concentrations was detected. Klotho may play an important role in the pathogenesis of MS, at least in part, through the regulation of redox system.

Increased autotaxin activity in multiple sclerosis.

Autotaxin (ATX) is an enzyme producing lysophosphatidic acid (LPA) from lysophosphatidyl choline (LPC) and it is up-regulated in inflammatory conditions such as various cancers, arthritis and multiple sclerosis (MS). Numerous studies have shown that the LPA signaling gives rise to angiogenesis, mitosis, cell proliferation and cytokine secretion. On the one hand, an increasing body of evidence suggests that blockade of ATX has anti-inflammatory properties in a variety of diseases. The aim of this study was to measure the enzyme activity of ATX in cerebrospinal fluid (CSF) and serum of patients with MS using an enzymatic photometric method. Twenty definite relapsing remitting MS patients along with 20 patients with other neurological diseases (OND) were recruited. The results showed that ATX activity was significantly higher (p value<0.0001) in MS patients than those patients diagnosed with OND. It is possible that inhibition of the ATX may decrease the rate of MS relapses/progression.

Elevated serum levels of lysophosphatidic acid in patients with multiple sclerosis.

A plethora of studies have shown that lysophosphatidic acid (LPA) is involved both in inflammation and T cell apoptosis evasion. The aim of this study was to measure the concentrations of LPA in serum of patients with multiple sclerosis (MS). Twenty MS patients along with 20age-sex matched healthy individuals were recruited for this investigation. By employment of ELISA method, we demonstrated that MS patients had higher levels of LPA in serum than control group (P=0.006). This study is the first report of LPA elevation in MS disease.

Inhibitor IκBα promoter functional polymorphisms in patients with multiple sclerosis.

The aim of this study is to investigate the association of IκBα promoter polymorphisms with the development of Multiple Sclerosis (MS) disease in Iranian population. One hundred and fifty patients with MS along with 150 unrelated healthy controls were enrolled in this study. The IκBα -881A/G (rs3138053), -826C/T (rs2233406) and -519C/T (rs2233408) polymorphisms were determined by the polymerase chain reaction/restriction fragment length polymorphism method. This study demonstrated that the genotype frequencies of IκBα -881A/G and -826T/T, and allele frequencies of IκBα-881G were significantly higher in patients with MS with respect to as compared to the controls. We also found that the estimated haplotype frequency of IκBα promoter -881G-826T-519C was significantly increased in the patient with MS in comparison with that of the healthy individuals. This study reveals that polymorphisms in the IκBα promoter (-881 A/G, -826 C/T) are strongly associated with the susceptibility of Iranian MS patients.

Secretory phospholipase A2 activity in serum and cerebrospinal fluid of patients with relapsing-remitting multiple sclerosis.

An increasing body of evidence suggests that patients afflicted with multiple sclerosis (MS) show alterations in immunologic markers including increases in proinflammatory cytokine activity and inflammation. Secretory Phospholipase A2 (sPLA2) is one of the key molecules contributing to the production of inflammatory lipid mediators, mainly eicosanoids. They are considered proinflammatory enzymes and their inhibition has long been recognized as a desirable therapeutic target. The aim of this study was to measure the enzyme activity of sPLA2 both in serum and cerebrospinal fluid (CSF) of MS patients. Twenty MS patients accompanied with 20 age-sex matched controls were recruited. The results showed that the enzyme activity of serum sPLA2 was 0.007±0.021 (µmol/min/ml) in MS patients vs. 0.007±0.016 (µmol/min/ml) in patients with other neurological diseases as a control group (P=0.5). Our findings also indicated that there is no correlation (P=0.6) between CSF sPLA2 enzyme activity and MS disease when the results of two groups were compared (0.072±0.020 in cases vs. 0.071±0.01 in control group). The results suggest that the enzyme activity of sPLA2 is not altered during the disease course.