Mast cells crosstalk with B cells in the gut and sustain IgA response in the inflamed intestine.

B lymphocytes are among the cell types whose effector functions are modulated by mast cells (MCs). The B/MC crosstalk emerged in several pathological settings, notably the colon of inflammatory bowel disease (IBD) patients is a privileged site in which MCs and IgA+ cells physically interact. Herein, by inducing conditional depletion of MCs in red MC and basophil (RMB) mice, we show that MCs control B cell distribution in the gut and IgA serum levels. Moreover, in dextran sulfate sodium (DSS)-treated RMB mice, the presence of MCs is fundamental for the enlargement of the IgA+ population in the bowel and the increase of systemic IgA production. Since both conventional B-2 and peritoneal-derived B cells populate the intestine and communicate with MCs in physiological conditions and during inflammation, we further explored this interplay through the use of co-cultures. We show that MCs finely regulate different aspects of splenic B cell biology while peritoneal B cells are unresponsive to the supporting effects provided by MCs. Interestingly, peritoneal B cells induce a pro-inflammatory skewing in MCs, characterized by increased ST2 and TNF-α expression. Altogether, this study uncovers the versatility of the B/MC liaison and highlights key aspects for the resolution of intestinal inflammation.

Combining Deep Phenotyping of Serum Proteomics and Clinical Data via Machine Learning for COVID-19 Biomarker Discovery.

The persistence of long-term coronavirus-induced disease 2019 (COVID-19) sequelae demands better insights into its natural history. Therefore, it is crucial to discover the biomarkers of disease outcome to improve clinical practice. In this study, 160 COVID-19 patients were enrolled, of whom 80 had a "non-severe" and 80 had a "severe" outcome. Sera were analyzed by proximity extension assay (PEA) to assess 274 unique proteins associated with inflammation, cardiometabolic, and neurologic diseases. The main clinical and hematochemical data associated with disease outcome were grouped with serological data to form a dataset for the supervised machine learning techniques. We identified nine proteins (i.e., CD200R1, MCP1, MCP3, IL6, LTBP2, MATN3, TRANCE, α2-MRAP, and KIT) that contributed to the correct classification of COVID-19 disease severity when combined with relative neutrophil and lymphocyte counts. By analyzing PEA, clinical and hematochemical data with statistical methods that were able to handle many variables in the presence of a relatively small sample size, we identified nine potential serum biomarkers of a "severe" outcome. Most of these were confirmed by literature data. Importantly, we found three biomarkers associated with central nervous system pathologies and protective factors, which were downregulated in the most severe cases.

Is it time for a new classification of mast cells? What do we know about mast cell heterogeneity?

Mast cells (MCs) are derived from committed precursors that leave the hematopoietic tissue, migrate in the blood, and colonize peripheral tissues where they terminally differentiate under microenvironment stimuli. They are distributed in almost all vascularized tissues where they act both as immune effectors and housekeeping cells, contributing to tissue homeostasis. Historically, MCs were classified into 2 subtypes, according to tryptic enzymes expression. However, MCs display a striking heterogeneity that reflects a complex interplay between different microenvironmental signals delivered by various tissues, and a differentiation program that decides their identity. Moreover, tissue-specific MCs show a trained memory, which contributes to shape their function in a specific microenvironment. In this review, we summarize the current state of our understanding of MC heterogeneity that reflects their different tissue experiences. We describe the discovery of unique cell molecules that can be used to distinguish specific MC subsets in vivo, and discuss how the improved ability to recognize these subsets provided new insights into the biology of MCs. These recent advances will be helpful for the understanding of the specific role of individual MC subsets in the control of tissue homeostasis, and in the regulation of pathological conditions such as infection, autoimmunity, and cancer.

Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination.

The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.

IL-10-producing B cells are characterized by a specific methylation signature.

Among the family of regulatory B cells, the subset able to produce interleukin-10 (IL-10) is the most studied, yet its biology is still a matter of investigation. The DNA methylation profiling of the il-10 gene locus revealed a novel epigenetic signature characterizing murine B cells ready to respond through IL-10 synthesis: a demethylated region located 4.5 kb from the transcription starting site (TSS), that we named early IL10 regulatory region (eIL10rr). This feature allows to distinguish B cells that are immediately prone and developmentally committed to IL-10 production from those that require a persistent stimulation to exert an IL-10-mediated regulatory function. These late IL-10 producers are instead characterized by a delayed IL10 regulatory region (dIL10rr), a partially demethylated DNA portion located 9 kb upstream from the TSS. A demethylated region was also found in human IL-10-producing B cells and, very interestingly, in some B-cell malignancies, such as chronic lymphocytic leukemia and mantle cell lymphoma, characterized by an immunosuppressive microenvironment. Our findings define murine and human regulatory B cells as an epigenetically controlled functional state of mature B cell subsets and open a new perspective on IL-10 regulation in B cells in homeostasis and disease.

Rheostatic Functions of Mast Cells in the Control of Innate and Adaptive Immune Responses.

Mast cells are evolutionarily ancient cells, endowed with a unique developmental, phenotypic, and functional plasticity. They are resident cells that participate in tissue homeostasis by constantly sampling the microenvironment. As a result of their large repertoire of receptors, they can respond to multiple stimuli and selectively release different types and amounts of mediator. Here, we present and discuss the recent mast cell literature, focusing on studies that demonstrate that mast cells are more than a switch that is turned 'off' when in the resting state and 'on' when in the degranulating state. We propose a new vision of mast cells in which, by operating in a 'rheostatic' manner, these cells finely modulate not only immune responses, but also the pathogenesis of several inflammatory disorders, including infection, autoimmunity, and cancer.

Mast cells are associated with the onset and progression of celiac disease.

BACKGROUND: Celiac disease (CD) is an immune-mediated disorder characterized by an accumulation of immune cells in the duodenal mucosa as a consequence of both adaptive and innate immune responses to undigested gliadin peptides. Mast cells (MCs) are innate immune cells that are a major source of costimulatory signals and inflammatory mediators in the intestinal mucosa. Although MCs have previously been associated with CD, functional studies have never been performed. OBJECTIVE: We aimed at evaluating the role of MCs in the pathogenesis of CD. METHODS: Intestinal biopsy specimens of patients with CD were scored according to the Marsh classification and characterized for leukocyte infiltration and MC distribution. Moreover, MC reactivity to gliadin and its peptides was characterized by using in vitro assays. RESULTS: Infiltrating MCs were associated with the severity of mucosal damage, and their numbers were increased in patients with higher Marsh scores. MCs were found to directly respond to nonimmunodominant gliadin fragments by releasing proinflammatory mediators. Immunohistochemical characterization of infiltrating MCs and the effects of gliadin peptides on intestinal MCs indicated an increase in proinflammatory MC function in advanced stages of the disease. This was also associated with increased neutrophil accumulation, the prevalence of M1 macrophages, and the severity of tissue damage. CONCLUSION: We provide a description of the progressive stages of CD, in which MCs are the hallmark of the inflammatory process. Thus the view of CD should be revised, and the contribution of MCs in the onset and progression of CD should be reconsidered in developing new therapeutic approaches.

CD4+CD25+ regulatory T cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction.

T regulatory (Treg) cells play a role in the suppression of immune responses, thus serving to induce tolerance and control autoimmunity. Here, we explored whether Treg cells influence the immediate hypersensitivity response of mast cells (MCs). Treg cells directly inhibited the FcvarepsilonRI-dependent MC degranulation through cell-cell contact involving OX40-OX40L interactions between Treg cells and MCs, respectively. When activated in the presence of Treg cells, MCs showed increased cyclic adenosine monophosphate (cAMP) concentrations and reduced Ca(2+) influx, independently of phospholipase C (PLC)-gamma2 or Ca(2+) release from intracellular stores. Antagonism of cAMP in MCs reversed the inhibitory effects of Treg cells, restoring normal Ca(2+) responses and degranulation. Importantly, the in vivo depletion or inactivation of Treg cells caused enhancement of the anaphylactic response. The demonstrated crosstalk between Treg cells and MCs defines a previously unrecognized mechanism controlling MC degranulation. Loss of this interaction may contribute to the severity of allergic responses.

Mast cells control the expansion and differentiation of IL-10-competent B cells.

The discovery of B cell subsets with regulatory properties, dependent on IL-10 production, has expanded our view on the mechanisms that control inflammation. Regulatory B cells acquire the ability to produce IL-10 in a stepwise process: first, they become IL-10 competent, a poised state in which B cells are sensitive to trigger signals but do not actually express the Il-10 gene; then, when exposed to appropriate stimuli, they start producing IL-10. Even if the existence of IL-10-competent B cells is now well established, it is not yet known how different immune cell types cross talk with B cells and affect IL-10-competent B cell differentiation and expansion. Mast cells (MCs) contribute to the differentiation and influence the effector functions of various immune cells, including B lymphocytes. In this study, we explored whether MCs could play a role in the expansion of IL-10-competent B cells and addressed the in vivo relevance of MC deficiency on the generation of these cells. We show that MCs can expand IL-10-competent B cells, but they do not directly induce IL-10 production; moreover, the absence of MCs negatively affects IL-10-competent B cell differentiation. Noteworthy, our findings reveal that the CD40L/CD40 axis plays a significant role in MC-driven expansion of IL-10-competent B cells in vitro and highlight the importance of MC CD40L signaling in the colon.

Mast cell activation: a complex interplay of positive and negative signaling pathways.

Mast cells regulate the immunological responses causing allergy and autoimmunity, and contribute to the tumor microenvironment through generation and secretion of a broad array of preformed, granule-stored and de novo synthesized bioactive compounds. The release and production of mast cell mediators is the result of a coordinated signaling machinery, followed by the FcεRI and FcγR antigen ligation. In this review, we present the latest understanding of FcεRI and FcγR signaling, required for the canonical mast cell activation during allergic responses and anaphylaxis. We then describe the cooperation between the signaling of FcR and other recently characterized membrane-bound receptors (i.e., IL-33R and thymic stromal lymphopoietin receptor) and their role in the chronic settings, where mast cell activation is crucial for the development and the sustainment of chronic diseases, such as asthma or airway inflammation. Finally, we report how the FcR activation could be used as a therapeutic approach to treat allergic and atopic diseases by mast cell inactivation. Understanding the magnitude and the complexity of mast cell signaling is necessary to identify the mechanisms underlying the potential effector and regulatory roles of mast cells in the biology and pathology of those disease settings in which mast cells are activated.

The aryl hydrocarbon receptor modulates acute and late mast cell responses.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor whose activity is modulated by xenobiotics as well as physiological ligands. These compounds may modulate inflammatory responses and contribute to the rising prevalence of allergic diseases observed in industrialized countries. Mast cells (MCs), located within tissues at the boundary of the external environment, represent a potential target of AhR ligands. In this study, we report that murine and human MCs constitutively express AhR, and its activation by the high-affinity ligand 6-formylindolo[3,2-b]carbazole (FICZ) determines a boost in degranulation. On the contrary, repeated exposure to FICZ inhibits MC degranulation. Accordingly, histamine release, in an in vivo passive systemic anaphylactic model, is exacerbated by a single dose and is attenuated by repetitive stimulation of AhR. FICZ-exposed MCs produce reactive oxygen species and IL-6 in response to cAMP-dependent signals. Moreover, AhR-activated MCs produce IL-17, a critical player in chronic inflammation and autoimmunity, suggesting a novel pathway for MC activation in the pathogenesis of these diseases. Indeed, histological analysis of patients with chronic obstructive pulmonary disease revealed an enrichment in AhR/IL-6 and AhR/IL-17 double-positive MCs within bronchial lamina propria. Thus, tissue-resident MCs could translate external chemical challenges through AhR by modulating allergic responses and contributing to the generation of inflammation-related diseases.

Intracellular Osteopontin Promotes the Release of TNFα by Mast Cells to Restrain Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive form of prostate cancer that emerges as tumors become resistant to hormone therapies or, rarely, arises de novo in treatment-naïve patients. The urgent need for effective therapies against NEPC is hampered by the limited knowledge of the biology governing this lethal disease. Based on our prior observations in the transgenic adenocarcinoma of the mouse prostate (TRAMP) spontaneous prostate cancer model, in which the genetic depletion of either mast cells (MC) or the matricellular protein osteopontin (OPN) increases NEPC frequency, we tested the hypothesis that MCs can restrain NEPC through OPN production, using in vitro co-cultures between murine or human tumor cell lines and MCs, and in vivo experiments. We unveiled a role for the intracellular isoform of OPN, so far neglected compared with the secreted isoform. Mechanistically, we unraveled that the intracellular isoform of OPN promotes TNFα production in MCs via the TLR2/TLR4-MyD88 axis, specifically triggered by the encounter with NEPC cells. We found that MC-derived TNFα, in turn, hampered the growth of NEPC. We then identified the protein syndecan-1 (SDC1) as the NEPC-specific TLR2/TLR4 ligand that triggered this pathway. Interrogating published single-cell RNA-sequencing data, we validated this mechanism in a different mouse model. Translational relevance of the results was provided by in silico analyses of available human NEPC datasets and by immunofluorescence on patient-derived adenocarcinoma and NEPC lesions. Overall, our results show that MCs actively inhibit NEPC, paving the way for innovative MC-based therapies for this fatal tumor. We also highlight SDC1 as a potential biomarker for incipient NEPC.

Modulation of FcεRI-dependent mast cell response by OX40L via Fyn, PI3K, and RhoA.

BACKGROUND: The interaction of mast cells (MCs) with regulatory T cells through the OX40 ligand (OX40L):OX40 axis downregulates FcεRI-dependent immediate hypersensitivity responses both in vitro and in vivo. Little is known on OX40L-mediated intracellular signaling or on the mechanism by which OX40L engagement suppresses MC degranulation. OBJECTIVE: We explored the role of OX40L engagement on IgE/antigen-triggered MCs both in vitro and in vivo. METHODS: The soluble form of OX40 molecule was used to selectively trigger OX40L on MCs in vitro and was used to dissect OX40L contribution in an in vivo model of systemic anaphylaxis. RESULTS: OX40L:OX40 interaction led to the recruitment of C-terminal src kinase into lipid rafts, causing a preferential suppression of Fyn kinase activity and subsequent reduction in the phosphorylation of Gab2, the phosphatidylinositol 3-OH kinase regulatory subunit p85, and Akt, without affecting the Lyn pathway. Dampening of Fyn kinase activity also inhibited RhoA activation and microtubule nucleation, key regulators of MC degranulation. The in vivo administration of a blocking antibody to OX40L in wild-type mice caused enhanced immediate hypersensitivity, whereas the administration of soluble OX40 to regulatory T-cell-depleted or OX40-deficient mice reduced MC degranulation. CONCLUSIONS: The engagement of OX40L selectively suppresses Fyn-initiated signals required for MC degranulation and serves to limit immediate hypersensitivity. Our data suggest that soluble OX40 can restore the aberrant or absent regulatory T-cell activity, revealing a previously unappreciated homeostatic role for OX40L in setting the basal threshold of MC response.

Single-cell dynamics of mast cell-CD4+ CD25+ regulatory T cell interactions.

The biological behavior of immune cells is determined by their intrinsic properties and interactions with other cell populations within their microenvironment. Several studies have confirmed the existence of tight spatial interactions between mast cells (MCs) and Tregs in different settings. For instance, we have recently identified the functional cross-talk between MCs and Tregs, through the OX40L-OX40 axis, as a new mechanism of reciprocal influence. However, there is scant information regarding the single-cell dynamics of this process. In this study, time-lapse video microscopy revealed direct interactions between Tregs and MCs in both murine and human cell co-cultures, resulting in the inhibition of the MC degranulation response. MCs incubated with WT, but not OX40-deficient, Tregs mediated numerous and long-lasting interactions and displayed different morphological features lacking the classical signs of exocytosis. MC degranulation and Ca2+ mobilization upon activation were inhibited by Tregs on a single-cell basis, without affecting overall cytokine secretion. Transmission electron microscopy showed ultrastructural evidence of vesicle-mediated secretion reconcilable with the morphological pattern of piecemeal degranulation. Our results suggest that MC morphological and functional changes following MC-Treg interactions can be ascribed to cell-cell contact and represent a transversal, non-species-specific mechanism of immune response regulation. Further research, looking at the molecular composition of this interaction will broaden our understanding of its contribution to immunity.

LPS Guides Distinct Patterns of Training and Tolerance in Mast Cells.

Mast cells (MCs) are tissue-resident, long lived innate immune cells with important effector and immunomodulatory functions. They are equipped with an eclectic variety of receptors that enable them to sense multiple stimuli and to generate specific responses according on the type, strength and duration of the stimulation. Several studies demonstrated that myeloid cells can retain immunological memory of their encounters - a process termed 'trained immunity' or 'innate immune memory'. As MCs are among the one of first cells to come into contact with the external environment, it is possible that such mechanisms of innate immune memory might help shaping their phenotype and effector functions; however, studies on this aspect of MC biology are still scarce. In this manuscript, we investigated the ability of MCs primed with different stimuli to respond to a second stimulation with the same or different ligands, and determined the molecular and epigenetic drivers of these responses. Our results showed that, while the stimulation with IgE and ß-glucan failed to induce either tolerant or trained phenotypes, LPS conditioning was able to induce a profound and long-lasting remodeling of the signaling pathways involved in the response against LPS or fungal pathogens. On one side, LPS induced a strong state of unresponsiveness to secondary LPS stimulation due to the impairment of the PI3K-AKT signaling pathway, which resulted in the reduced activation of NF-κB and the decreased release of TNF-α and IL-6, compared to naïve MCs. On the other side, LPS primed MCs showed an increased release of TNF-α upon fungal infection with live Candida albicans, thus suggesting a dual role of LPS in inducing both tolerance and training phenotypes depending on the secondary challenge. Interestingly, the inhibition of HDAC during LPS stimulation partially restored the response of LPS-primed MCs to a secondary challenge with LPS, but failed to revert the increased cytokine production of these cells in response to C. albicans. These data indicate that MCs, as other innate immune cells, can develop innate immune memory, and that different stimulatory environments can shape and direct MC specific responses towards the dampening or the propagation of the local inflammatory response.

Mast cells counteract regulatory T-cell suppression through interleukin-6 and OX40/OX40L axis toward Th17-cell differentiation.

The development of inflammatory diseases implies inactivation of regulatory T (Treg) cells through mechanisms that still are largely unknown. Here we showed that mast cells (MCs), an early source of inflammatory mediators, are able to counteract Treg inhibition over effector T cells. To gain insight into the molecules involved in their interplay, we set up an in vitro system in which all 3 cellular components were put in contact. Reversal of Treg suppression required T cell-derived interleukin-6 (IL-6) and the OX40/OX40L axis. In the presence of activated MCs, concomitant abundance of IL-6 and paucity of Th1/Th2 cytokines skewed Tregs and effector T cells into IL-17-producing T cells (Th17). In vivo analysis of lymph nodes hosting T-cell priming in experimental autoimmune encephalomyelitis revealed activated MCs, Tregs, and Th17 cells displaying tight spatial interactions, further supporting the occurrence of an MC-mediated inhibition of Treg suppression in the establishment of Th17-mediated inflammatory responses.

High-performance metabolic marker assessment in breast cancer tissue by mass spectrometry.

BACKGROUND: The identification of reliable markers for diagnosis of breast cancer has been thoroughly addressed by metabolic profiling using nuclear magnetic resonance (NMR) spectroscopy or imaging. Several clear diagnostic indicators have emerged using either in vitro analysis of tissue extracts, ex vivo analysis of biopsies or in vivo direct spectral observations. Most of the breast cancer characteristic metabolites could be assayed by mass spectrometry (MS) to exploit the superior sensitivity of this technique and therefore reduce the traumatic impact of current biopsy procedures. METHODS: Following extraction, aqueous metabolite mixtures were obtained that were submitted to liquid-chromatography, electrospray-ionization, mass spectrometry (LC/ESI-MS) analysis to estimate the content of choline (Cho) and its phosphorylated derivatives, phosphocholine (PCho) and glycerophosphocholine (GPCho). The determinations were performed using 10 samples from breast tissue biopsies, surgical specimens and one single sample of a hepatic metastasis. In addition, some measurements were also repeated using high-resolution ¹H NMR spectroscopy to complement the mass spectrometry results. RESULTS: The contents of Cho, PCho and GPCho in breast tissue extracts were estimated by LC/ESI-MS based on standard compound calibration curves. Sharply increased ratios of phosphorylated-to-unphosphorylated metabolites, PCho/ Cho and (PCho+GPCho)/Cho, were observed in all tumor samples, although without discrimination between benign and malignant lesions, contrary to samples from healthy individuals and from those with fibrocystic disease. CONCLUSIONS: The assessment of breast cancer markers by LC/ESI-MS is feasible and diagnostically valuable. In addition to high sensitivity, the approach also shows a resolution advantage for assaying choline derivatives compared to NMR, and could complement the latter.

Purification of Murine and Human IL-10-Producing B Cells from Different Anatomical Compartments.

IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).

il-10 Gene Locus DNA Methylation in Regulatory B Cells.

Epigenetic studies are becoming increasingly common in the immunology field thanks to the support of cutting edge technology and to their potential of providing a large amount of data at the single cell level. Moreover, epigenetic modifications were shown to play a role in autoimmune/inflammatory disorders, paving the way for the possibility of using the results of epigenetic studies for therapeutic purposes. In recent years, epigenetic marks such as DNA methylation, histone modifications and nucleosome positioning were shown to regulate B cell fate and function during an immune response, but very little has been done in the context of one of the most recently discovered B cell subsets, that is regulatory B cells. Although no consensus has yet been found on the identity of these immunosuppressive B cells, the role of the IL-10 cytokine is consolidated, both in the murine and human setting. In this chapter we will focus on the analysis of the methylation profile of a gene of interest and we will specifically describe cloning and pyrosequencing bisulphite sequencing PCR (BSP). Given the specific context, we will provide tips and tricks for the analysis of the il-10 gene locus. Nonetheless, the methods presented are valid for the study of any gene of interest.

Repurposing of the Antiepileptic Drug Levetiracetam to Restrain Neuroendocrine Prostate Cancer and Inhibit Mast Cell Support to Adenocarcinoma.

A relevant fraction of castration-resistant prostate cancers (CRPC) evolve into fatal neuroendocrine (NEPC) tumors in resistance to androgen deprivation and/or inhibitors of androgen receptor pathway. Therefore, effective drugs against both CRPC and NEPC are needed. We have previously described a dual role of mast cells (MCs) in prostate cancer, being capable to promote adenocarcinoma but also to restrain NEPC. This finding suggests that a molecule targeting both MCs and NEPC cells could be effective against prostate cancer. Using an in silico drug repurposing approach, here we identify the antiepileptic drug levetiracetam as a potential candidate for this purpose. We found that the protein target of levetiracetam, SV2A, is highly expressed by both NEPC cells and MCs infiltrating prostate adenocarcinoma, while it is low or negligible in adenocarcinoma cells. In vitro, levetiracetam inhibited the proliferation of NEPC cells and the degranulation of MCs. In mice bearing subcutaneous tumors levetiracetam was partially active on both NEPC and adenocarcinoma, the latter effect due to the inhibition of MMP9 release by MCs. Notably, in TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice subjected to surgical castration to mimic androgen deprivation therapy, levetiracetam reduced onset and frequency of both high grade prostatic intraepithelial neoplasia, adenocarcinoma and NEPC, thus increasing the number of cured mice showing only signs of tumor regression. Our results demonstrate that levetiracetam can directly restrain NEPC development after androgen deprivation, and that it can also block adenocarcinoma progression through the inhibition of some MCs functions. These findings open the possibility of further testing levetiracetam for the therapy of prostate cancer or of MC-mediated diseases.