Intracellular osteopontin promotes the release of TNF 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 TRAMP spontaneous prostate cancer model, in which the genetic depletion of either mast cells (MCs) 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 (iOPN), so far neglected compared to the secreted isoform. Mechanistically, we unraveled that iOPN promotes TNF production in MCs via the TLR2/TLR4-MyD88 axis, specifically triggered by the encounter with NEPC cells. We found that MC-derived TNF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 provdied by in silco 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.

Relationship between cytokine release and stress hyperglycemia in patients hospitalized with COVID-19 infection.

Introduction: Stress hyperglycemia is a frequent finding in patients with COVID-19 infection and could affect the outcome of disease. Cytokines released in response to infection could have adverse effects on insulin sensitivity and pancreatic beta-cell function. The aim of the study was to examine the relationships of stress hyperglycemia with cytokines and clinical outcomes in hospitalized patients with COVID-19. Methods: In a cross-sectional analysis of 150 patients hospitalized for COVID-19 infection who were included in the GIRA-COVID database, we identified patients with stress hyperglycemia by calculation of the Stress Hyperglycemia Ratio (SHR) and use of a cut-off of 1.14. Plasma levels of cytokines principally involved in COVID-19 infection-related cytokine storm were measured. Outcome variables were use of mechanical ventilation and death within 60 days from hospital admission. Results: Patients with SHR > 1.14 had significantly higher plasma insulin, HOMA-index, and levels of interleukin-10 (IL-10), interleukin-10/tumor necrosis factor-a ratio (IL-10/TNF-α), and CXC motif chemokine ligand 10 (CXCL10) than patients with SHR ≤ 1.14. IL-10, IL-10/TNF-α ratio, CXCL10, and IFN-γ were significantly and directly related with SHR in univariate analysis and multivariate logistic regression models showed that IL-10, IL-10/TNF-α ratio, and CXCL10 were independently associated with SHR>1.14. In a multivariate logistic model, stress hyperglycemia predicted use of mechanical ventilation (OR 2.453; CI 1.078-6.012) and death (OR 2.281; CI 1.049-7.369) independently of diabetes and other major confounders. Conclusions: In patients hospitalized for COVID-19 infection, stress hyperglycemia is associated with worse clinical outcomes and is independently related to levels of cytokines that might impair glucose homeostasis.

Selected recent advances in understanding the role of human mast cells in health and disease.

Mast cells are highly granular tissue-resident cells and key drivers of inflammation, particularly in allergies as well as in other inflammatory diseases. Most mast cell research was initially conducted in rodents but has increasingly shifted to the human system, with the advancement of research technologies and methodologies. Today we can analyze primary human cells including rare subpopulations, we can produce and maintain mast cells isolated from human tissues, and there are several human mast cell lines. These tools have substantially facilitated our understanding of their role and function in different organs in both health and disease. We can now define more clearly where human mast cells originate from, how they develop, which mediators they store, produce de novo, and release, how they are activated and by which receptors, and which neighboring cells they interact with and by which mechanisms. Considerable progress has also been made regarding the potential contribution of mast cells to disease, which, in turn, has led to the development of novel approaches for preventing key pathogenic effects of mast cells, heralding the era of mast cell-targeted therapeutics. In this review, we present and discuss a selection of some of the most significant advancements and remaining gaps in our understanding of human mast cells during the last 25 years, with a focus on clinical relevance.

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.

Integrated multi-omics analyses on patient-derived CRC organoids highlight altered molecular pathways in colorectal cancer progression involving PTEN.

BACKGROUND: Colorectal cancer (CRC) represents the fourth leading cause of cancer-related deaths. The heterogeneity of CRC identity limits the usage of cell lines to study this type of tumor because of the limited representation of multiple features of the original malignancy. Patient-derived colon organoids (PDCOs) are a promising 3D-cell model to study tumor identity for personalized medicine, although this approach still lacks detailed characterization regarding molecular stability during culturing conditions. Correlation analysis that considers genomic, transcriptomic, and proteomic data, as well as thawing, timing, and culturing conditions, is missing. METHODS: Through integrated multi-omics strategies, we characterized PDCOs under different growing and timing conditions, to define their ability to recapitulate the original tumor. RESULTS: Whole Exome Sequencing allowed detecting temporal acquisition of somatic variants, in a patient-specific manner, having deleterious effects on driver genes CRC-associated. Moreover, the targeted NGS approach confirmed that organoids faithfully recapitulated patients' tumor tissue. Using RNA-seq experiments, we identified 5125 differentially expressed transcripts in tumor versus normal organoids at different time points, in which the PTEN pathway resulted of particular interest, as also confirmed by further phospho-proteomics analysis. Interestingly, we identified the PTEN c.806_817dup (NM_000314) mutation, which has never been reported previously and is predicted to be deleterious according to the American College of Medical Genetics and Genomics (ACMG) classification. CONCLUSION: The crosstalk of genomic, transcriptomic and phosphoproteomic data allowed to observe that PDCOs recapitulate, at the molecular level, the tumor of origin, accumulating mutations over time that potentially mimic the evolution of the patient's tumor, underlining relevant potentialities of this 3D model.

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.

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).

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.

Inhibition of APE1-endonuclease activity affects cell metabolism in colon cancer cells via a p53-dependent pathway.

The pathogenesis of colorectal cancer (CRC) involves different mechanisms, such as genomic and microsatellite instabilities. Recently, a contribution of the base excision repair (BER) pathway in CRC pathology has been emerged. In this context, the involvement of APE1 in the BER pathway and in the transcriptional regulation of genes implicated in tumor progression strongly correlates with chemoresistance in CRC and in more aggressive cancers. In addition, the APE1 interactome is emerging as an important player in tumor progression, as demonstrated by its interaction with Nucleophosmin (NPM1). For these reasons, APE1 is becoming a promising target in cancer therapy and a powerful prognostic and predictive factor in several cancer types. Thus, specific APE1 inhibitors have been developed targeting: i) the endonuclease activity; ii) the redox function and iii) the APE1-NPM1 interaction. Furthermore, mutated p53 is a common feature of advanced CRC. The relationship between APE1 inhibition and p53 is still completely unknown. Here, we demonstrated that the inhibition of the endonuclease activity of APE1 triggers p53-mediated effects on cell metabolism in HCT-116 colon cancer cell line. In particular, the inhibition of the endonuclease activity, but not of the redox function or of the interaction with NPM1, promotes p53 activation in parallel to sensitization of p53-expressing HCT-116 cell line to genotoxic treatment. Moreover, the endonuclease inhibitor affects mitochondrial activity in a p53-dependent manner. Finally, we demonstrated that 3D organoids derived from CRC patients are susceptible to APE1-endonuclease inhibition in a p53-status correlated manner, recapitulating data obtained with HCT-116 isogenic cell lines. These findings suggest the importance of further studies aimed at testing the possibility to target the endonuclease activity of APE1 in CRC.

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.

Frontline Science: Mast cells regulate neutrophil homeostasis by influencing macrophage clearance activity.

The receptor tyrosine kinase cKit and its ligand stem cell factor are essential for mast cells (MC) development and survival. Strains with mutations affecting the Kit gene display a profound MC deficiency in all tissues and have been extensively used to investigate the role of MC in both physiologic and pathologic conditions. However, these mice present a variety of abnormalities in other immune cell populations that can affect the interpretation of MC-related responses. C57BL/6 KitW-sh are characterized by an aberrant extramedullary myelopoiesis and systemic neutrophilia. MC deficiency in KitW-sh mice can be selectively repaired by engraftment with in vitro-differentiated MC to validate MC-specific functions. Nevertheless, the impact of MC reconstitution on other immune populations has never been evaluated in detail. Here, we specifically investigated the neutrophil compartment in primary and secondary lymphoid organs of C57BL/6 KitW-sh mice before and after MC reconstitution. We found that, albeit not apparently affecting neutrophils phenotype or maturation, MC reconstitution of KitW-sh mice restored the number of neutrophils at a level similar to that of wild-type C57BL/6 mice. In vitro and ex vivo experiments indicated that MC can influence neutrophil clearance by increasing macrophages' phagocytic activity. Furthermore, the G-CSF/IL-17 axis was also influenced by the presence or absence of MC in KitW-sh mice. These data suggest that MC play a role in the control of neutrophil homeostasis and that this aspect should be taken into account in the interpretation of results obtained using KitW-sh mice.

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.

Mast Cells Respond to Candida albicans Infections and Modulate Macrophages Phagocytosis of the Fungus.

Mast cells (MCs) are long-lived immune cells widely distributed at mucosal surfaces and are among the first immune cell type that can get in contact with the external environment. This study aims to unravel the mechanisms of reciprocal influence between mucosal MCs and Candida albicans as commensal/opportunistic pathogen species in humans. Stimulation of bone marrow-derived mast cells (BMMCs) with live forms of C. albicans induced the release of TNF-α, IL-6, IL-13, and IL-4. Quite interestingly, BMMCs were able to engulf C. albicans hyphae, rearranging their α-tubulin cytoskeleton and accumulating LAMP1+ vesicles at the phagocytic synapse with the fungus. Candida-infected MCs increased macrophage crawling ability and promoted their chemotaxis against the infection. On the other side, resting MCs inhibited macrophage phagocytosis of C. albicans in a contact-dependent manner. Taken together, these results indicate that MCs play a key role in the maintenance of the equilibrium between the host and the commensal fungus C. albicans, limiting pathological fungal growth and modulating the response of resident macrophages during infections.

IL-9 and Mast Cells Are Key Players of Candida albicans Commensalism and Pathogenesis in the Gut.

Candida albicans is implicated in intestinal diseases. Identifying host signatures that discriminate between the pathogenic versus commensal nature of this human commensal is clinically relevant. In the present study, we identify IL-9 and mast cells (MCs) as key players of Candida commensalism and pathogenicity. By inducing TGF-ß in stromal MCs, IL-9 pivotally contributes to mucosal immune tolerance via the indoleamine 2,3-dioxygenase enzyme. However, Candida-driven IL-9 and mucosal MCs also contribute to barrier function loss, dissemination, and inflammation in experimental leaky gut models and are upregulated in patients with celiac disease. Inflammatory dysbiosis occurs with IL-9 and MC deficiency, indicating that the activity of IL-9 and MCs may go beyond host immunity to include regulation of the microbiota. Thus, the output of the IL-9/MC axis is highly contextual during Candida colonization and reveals how host immunity and the microbiota finely tune Candida behavior in the gut.

Cross-Talk between Myeloid-Derived Suppressor Cells and Mast Cells Mediates Tumor-Specific Immunosuppression in Prostate Cancer.

Immunotherapy, including the use of checkpoint inhibitors, is a potent therapeutic approach for some cancers, but has limited success with prostate tumors, in which immune suppression is instigated by the tumor. The immunosuppressive capacity of mast cells, which promote adenocarcinoma development in the prostate, prompted our investigation on whether mast cells promote tolerance to SV40 Large-T antigen, the transforming oncogene in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. The incidence of adenocarcinoma was reduced in the offspring of a cross between TRAMP mice and mast cell-deficient KitWsh mice. TRAMP mice are tolerant to the SV40 Large T antigen, which is otherwise immunogenic in normal syngeneic B6 mice. Genetic ablation of mast cells in TRAMP mice restored their ability to mount a tumor-specific cytotoxic T-cell response. In KitWsh-TRAMP mice, the restored T-cell immunity correlated with the reduced activity of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC), along with their reduced expression of Arg1, Nos2, and Stat3 Having found that CD40L-expressing mast cells can interact in vivo with CD40-expressing PMN-MDSC, we then determined that only KitWsh-TRAMP mice reconstituted with mast cells expressing CD40L could restore PMN-MDSCs suppressive functions, T-cell unresponsiveness and adenocarcinoma development. Thus, mast cells have an immunoregulatory effect on PMN-MDSCs activity through CD40L-CD40 interaction, favoring immunosuppression and tumor onset. In prostate cancer patients, in silico analyses correlated poor clinical outcomes with high expression of genes related to mast cells and PMN-MDSCs. Cancer Immunol Res; 6(5); 552-65. ©2018 AACR.

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.

Reciprocal influence of B cells and tumor macro and microenvironments in the ApcMin/+ model of colorectal cancer.

One of the most fascinating aspects of the immune system is its dynamism, meant as the ability to change and readapt according to the organism needs. Following an insult, we assist to the spontaneous organization of different immune cells which cooperate, locally and at distance, to build up an appropriate response. Throughout tumor progression, adaptations within the systemic tumor environment, or macroenvironment, result in the promotion of tumor growth, tumor invasion and metastasis to distal organs, but also to dramatic changes in the activity and composition of the immune system. In this work, we show the changes of the B-cell arm of the immune system following tumor progression in the ApcMin/+ model of colorectal cancer. Tumor macroenvironment leads to an increased proportion of total and IL-10-competent B cells in draining LNs while activates a differentiation route that leads to the expansion of IgA+ lymphocytes in the spleen and peritoneum. Importantly, serum IgA levels were significantly higher in ApcMin/+ than Wt mice. The peculiar involvement of IgA response in the adenomatous transformation had correlates in the gut-mucosal compartment where IgA-positive elements increased from normal mucosa to areas of low grade dysplasia while decreasing upon overt carcinomatous transformation. Altogether, our findings provide a snapshot of the tumor education of B lymphocytes in the ApcMin/+ model of colorectal cancer. Understanding how tumor macroenvironment affects the differentiation, function and distribution of B lymphocytes is pivotal to the generation of specific therapies, targeted to switching B cells to an anti-, rather than pro-, tumoral phenotype.

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.