The anti-inflammatory cytokine IL-37 improves the NK cell-mediated anti-tumor response.

IL-37 is a member of the IL-1 superfamily exerting anti-inflammatory functions in a number of diseases. Extracellular IL-37 triggers the inhibitory receptor IL-1R8 that is known to regulate different NK cell pathways and functional activities including their anti-tumor effect. However, the effect of IL-37 on human NK cell functions is still to be unveiled. This study aimed to investigate the functional effect of IL-37 in human NK cells activated with IL-15. We found that IL-37 enhanced both NK cell cytotoxic activity against different tumor cell lines and cytokines production. These effects were associated with increased phosphorylation of ERK and NF-Kb. The improved NK cell activity was also strictly related to a time-dependent GSK3ß-mediated degradation of IL-1R8. The enhanced activation profile of IL-37 treated NK cells possibly due to IL-1R8 degradation was confirmed by the results with IL-1R8-silenced NK cells. Lastly, in line with these data, through the analysis of the TNM plot database of a large group of patients, IL-37 mRNA expression was found to be significantly lower in colon and skin cancers than in normal tissues. Colon adenocarcinoma and neuroblastoma patients with higher IL-37 mRNA levels had significantly higher overall survival, suggesting that the presence of IL-37 might be considered an independent positive prognostic factor for this tumor. Our results provide novel information on the mechanisms regulating IL-1R8 function in human NK cells, highlighting the IL-37-IL-1R8 axis as a potential new target to improve the anti-tumor immune response.

Analysis of the mechanisms regulating soluble PD-1 production and function in human NK cells.

NK cells represent important effectors that play a major role in innate defences against pathogens and display potent cytolytic activity against tumor cells. An array of surface receptors finely regulate their function and inhibitory checkpoints, such as PD-1, can dampen the immune response inducing an immunosuppressive state. Indeed, PD-1 expression in human NK cells correlated with impaired effector function and tumor immune evasion. Importantly, blockade of the PD-1/PD-L1 axis has been shown to reverse NK cell exhaustion and increase their cytotoxicity. Recently, soluble counterparts of checkpoint receptors, such as soluble PD-1 (sPD-1), are rising high interest due to their biological activity and ability to modulate immune responses. It has been widely demonstrated that sPD-1 can modulate T cell effector functions and tumor growth. Tumor-infiltrating T cells are considered the main source of circulating sPD-1. In addition, recently, also stimulated macrophages have been demonstrated to release sPD-1. However, no data are present on the role of sPD-1 in the context of other innate immune cell subsets and therefore this study is aimed to unveil the effect of sPD-1 on human NK cell function. We produced the recombinant sPD-1 protein and demonstrated that it binds PD-L1 and that its presence results in increased NK cell cytotoxicity. Notably, we also identified a pathway regulating endogenous sPD-1 synthesis and release in human NK cells. Secreted endogenous sPD-1, retained its biological function and could modulate NK cell effector function. Overall, these data reveal a pivotal role of sPD-1 in regulating NK-mediated innate immune responses.

IL-1R8: A molecular brake of anti-tumor and anti-viral activity of NK cells and ILC.

Interleukin-1 receptor family members (ILRs) and Toll-Like Receptors (TLRs) play pivotal role in immunity and inflammation and are expressed by most cell types including cells of both the innate and adaptive immune system. In this context, IL-1 superfamily members are also important players in regulating function and differentiation of adaptive and innate lymphoid cells. This system is tightly regulated in order to avoid uncontrolled activation, which may lead to detrimental inflammation contributing to autoimmune or allergic responses. IL-1R8 (also known as TIR8 or SIGIRR) is a member of the IL-1R family that acts as a negative regulator dampening ILR and TLR signaling and as a co-receptor for human IL-37. Human and mouse NK cells, that are key players in immune surveillance of tumors and infections, express high level of IL-1R8. In this review, we will summarize our current understanding on the structure, expression and function of IL-1R8 and we will also discuss the emerging role of IL-1R8 as an important checkpoint regulating NK cells function in pathological conditions including cancer and viral infections.

How the Immune System Responds to Allergy Immunotherapy.

IgE-mediated diseases represent a highly diversified and multifactorial group of disorders that can deeply impact the patients' quality of life. Currently, allergy immunotherapy (AIT) still remains the gold standard for the management of such pathologies. In this review, we comprehensively examine and discuss how AIT can affect both the innate and the adaptive immune responses at different cell levels and propose timing-scheduled alterations induced by AIT by hypothesizing five sequential phases: after the desensitization of effector non-lymphoid cells and a transient increase of IgE (phase 1), high doses of allergen given by AIT stimulate the shift from type 2/type 3 towards type 1 response (phase 2), which is progressively potentiated by the increase of IFN-γ that promotes the chronic activation of APCs, progressively leading to the hyperexpression of Notch1L (Delta4) and the secretion of IL-12 and IL-27, which are essential to activate IL-10 gene in Th1 and ILC1 cells. As consequence, an expansion of circulating memory Th1/Tr1 cells and ILC-reg characterizes the third phase addressed to antagonize/balance the excess of type 1 response (phase 3). The progressive increase of IL-10 triggers a number of regulatory circuits sustained by innate and adaptive immune cells and favoring T-cell tolerance (phase 4), which may also be maintained for a long period after AIT interruption (phase 5). Different administration approaches of AIT have shown a similar tailoring of the immune responses and can be monitored by timely, optimized biomarkers. The clinical failure of this treatment can occur, and many genetic/epigenetic polymorphisms/mutations involving several immunological mechanisms, such as the plasticity of immune responses and the induction/maintenance of regulatory circuits, have been described. The knowledge of how AIT can shape the immune system and its responses is a key tool to develop novel AIT strategies including the engineering of allergen or their epitopes. We now have the potential to understand the precise causes of AIT failure and to establish the best biomarkers of AIT efficacy in each phase of the treatment.

Activation of CEACAM1 with an agonistic monoclonal antibody results in inhibition of melanoma cells.

Inhibitory receptors (IRs), such as the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), are cell surface molecules expressed on both normal epithelial, endothelial, and hematopoietic cells and on neoplastic cells. IRs are usually used by cancer cells to inhibit immune cell functions. Thus, CEACAM1 positive tumor cells can interact homophilically with CEACAM1 expressed on T and NK cells to inhibit their antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we investigated the effect of agonistic/activating anti-CEACAM1 monoclonal antibody (mAb) on melanoma cell lines in vitro and in vivo, following our hypothesis that activation of CEACAM1 on melanoma cells by distinct mAbs may induce inhibition of cancer cell proliferation and/or their death. To address this, we established an activating anti-CEACAM1 mAb (CCM5.01) and characterized its binding to the CEACAM1 receptor. Using this mAb, we assessed the expression of CEACAM1 on four different human melanoma cell lines by western blot and flow cytometry and determined its effect on cell viability in vitro by MTT assay. Furthermore, we evaluated the mAb mechanism of action and found that binding of CEACAM1 with CCM5.01 induced SHP1 phosphorylation and p53 activation resulting in melanoma cell apoptosis. For in vivo studies, a xenograft model of melanoma was performed by injection of Mel-14 cells subcutaneously (s.c.) in SCID/Beige mice followed by intraperitoneal (i.p.) injection of CCM5.01 or of IgG1 isotype control every other day. CCM5.01 treated mice showed a slight but not significant decrease in tumor weight in comparison to the control group. Based on the obtained data, we suggest that activating CEACAM1 on melanoma cells might be a promising novel approach to fight cancers expressing this IR.

IL-1R8 silencing improves the anti-tumor function of freshly isolated human NK cells.

The inhibitory receptor interleukin-1 receptor 8 (IL-1R8) has been recently recognized to be expressed also by human natural killer (NK) cells. This study was aimed to design and optimize IL-1R8 silencing conditions in human NK cells to precisely establish the activity of such receptor in these cells. Electroporation of freshly isolated or IL-2-cultured NK cells with small interfering RNA (siRNA), resulted in a marked, even though variable, IL-1R8-silencing. Although the expression profile revealed downregulation of most genes involved in several intracellular pathways, some genes related to proliferation, expression of some chemokine receptors, antibody-dependent cell cytotoxicity and cytotoxic activity were upregulated in IL-1R8-silenced NK cells. Furthermore, upon IL-15 activation, the majority of genes involved in NK cell function were upregulated in IL-1R8-siRNA-compared with control-siRNA-transfected NK cells. More importantly, in agreement with these findings, the reduction of IL-1R8 gene expression levels resulted in enhanced expression of NK cell activation markers, production of cytokines and chemokines, and cytotoxic activity against several NK cell targets with different susceptibility to NK-mediated lysis. Similar results were obtained following stimulation with IL-18. All together these data, deeply impacting on the main effector functions of human NK cells, can lead to a better understanding of IL-1R8-mediated regulation on these cells and to the design of new strategies for improving NK cell-mediated anti-tumor responses.

Toll-like receptor 8 agonists improve NK-cell function primarily targeting CD56brightCD16- subset.

BACKGROUND: Toll-like receptors (TLRs) are pattern-recognition sensors mainly expressed in innate immune cells that directly recognize conserved pathogen structures (pathogen-associated molecular patterns-PAMPs). Natural killer (NK) cells have been described to express different endosomal TLRs triggered by RNA and DNA sequences derived from both viruses and bacteria. This study was addressed to establish which endosomal TLR could directly mediate NK activation and function after proper stimuli. It was also important to establish the most suitable TLR agonist to be used as adjuvant in tumor vaccines or in combined cancer immunotherapies. METHODS: We assessed endosomal TLR expression in total NK cells by using RT-qPCR and western blotting technique. In some experiments, we purified CD56brightCD16- and CD56dimCD16+ cells subsets by using NK Cell Isolation Kit Activation marker, cytokine production, CD107a expression and cytotoxicity assay were evaluated by flow cytometry. Cytokine release was quantified by ELISA. NK cells obtained from ovarian ascites underwent the same analyses. RESULTS: Although the four endosomal TLRs (TLR3, TLR7/8, and TLR9) were uniformly expressed on CD56brightCD16- and CD56dimCD16+ cell subsets, the TLR7/8 (R848), TLR3 (polyinosinic-polycytidylic acid, Poly I:C) and TLR9 (ODN2395) ligands promoted NK-cell function only in the presence of suboptimal doses of cytokines, including interleukin (IL)-2, IL-12, IL-15, and IL-18, produced in vivo by other environmental cells. We showed that R848 rather than TLR3 and TLR9 agonists primarily activated CD56brightCD16- NK cells by increasing their proliferation, cytokine production and cytotoxic activity. Moreover, we demonstrated that R848, which usually triggers TLR7 and TLR8 on dendritic cells, macrophages and neutrophils cells, activated CD56brightCD16- NK-cell subset only via TLR8. Indeed, specific TLR8 but not TLR7 agonists increased cytokine production and cytotoxic activity of CD56brightCD16- NK cells. Importantly, these activities were also observed in peritoneal NK cells from patients with metastatic ovarian carcinoma, prevalently belonging to the CD56brightCD16- subset. CONCLUSION: These data highlight the potential value of TLR8 in NK cells as a new target for immunotherapy in patients with cancer.

Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.

Activation of Siglec-7 results in inhibition of in vitro and in vivo growth of human mast cell leukemia cells.

Advanced systemic mastocytosis is a rare and still untreatable disease. Blocking antibodies against inhibitory receptors, also known as "immune checkpoints", have revolutionized anti-cancer treatment. Inhibitory receptors are expressed not only on normal immune cells, including mast cells but also on neoplastic cells. Whether activation of inhibitory receptors through monoclonal antibodies can lead to tumor growth inhibition remains mostly unknown. Here we show that the inhibitory receptor Siglec-7 is expressed by primary neoplastic mast cells in patients with systemic mastocytosis and by mast cell leukemia cell lines. Activation of Siglec-7 by anti-Siglec-7 monoclonal antibody caused phosphorylation of Src homology region 2 domain-containing phosphatase-1 (SHP-1), reduced phosphorylation of KIT and induced growth inhibition in mast cell lines. In SCID-beige mice injected with either the human mast cell line HMC-1.1 and HMC-1.2 or with Siglec-7 transduced B cell lymphoma cells, anti-Siglec-7 monoclonal antibody reduced tumor growth by a mechanism involving Siglec-7 cytoplasmic domains in "preventive" and "treatment" settings. These data demonstrate that activation of Siglec-7 on mast cell lines can inhibit their growth in vitro and in vivo. This might pave the way to additional treatment strategies for mastocytosis.

Siglec-7 on peripheral blood eosinophils: Surface expression and function.

BACKGROUND: Siglec-7 is an inhibitory receptor (IR) expressed on human blood eosinophils. Whereas activation of other IRs, including Siglec-8 and CD300a, has been shown to downregulate eosinophil function, little is known about the role of Siglec-7 on human eosinophils. OBJECTIVE: To examine Siglec-7 expression and function in eosinophils from normal (ND) and eosinophilic (EO) donors. METHODS: Eosinophil expression of Siglec-7 was quantified by flow cytometry and quantitative PCR. Soluble Siglec-7 (sSiglec-7) levels were measured by ELISA in serum. The effect of Siglec-7 on eosinophil viability and degranulation was assessed in vitro by AnnexinV-FITC/7-AAD staining and by measuring GM-CSF-induced mediator release in culture supernatants. Signal transduction was studied by Western blot. RESULTS: Siglec-7 was expressed ex vivo on blood eosinophils from all eosinophilic and normal individuals studied. Siglec-7 surface, but not SIGLEC-7mRNA expression, was correlated with absolute eosinophil count (AEC). Siglec-7 was upregulated on purified eosinophils after in vitro stimulation with GM-CSF or IL-5. Serum sSiglec-7 was detectable in 133/144 subjects tested and correlated with AEC. Siglec-7 cross-linking inhibited GM-CSF-induced release of eosinophil peroxidase, TNF-α, and IL-8 (n = 7-8) but did not promote eosinophil apoptosis (n = 5). Finally, Siglec-7 cross-linking on GM-CSF-activated eosinophils induced phosphorylation of SHP-1 and de-phosphorylation of ERK1/2 and p38. CONCLUSIONS: Siglec-7 is constitutively expressed on human eosinophils and downmodulates eosinophil activation. Targeting of Siglec-7 on eosinophils might enhance treatment efficacy in eosinophil-driven disorders. Conversely, therapeutic interventions that inhibit Siglec-7 could have unanticipated consequences and promote eosinophilic inflammation.

Pyruvate dehydrogenase has a major role in mast cell function, and its activity is regulated by mitochondrial microphthalmia transcription factor.

BACKGROUND: We have recently observed that oxidative phosphorylation-mediated ATP production is essential for mast cell function. Pyruvate dehydrogenase (PDH) is the main regulator of the Krebs cycle and is located upstream of the electron transport chain. However, the role of PDH in mast cell function has not been described. Microphthalmia transcription factor (MITF) regulates the development, number, and function of mast cells. Localization of MITF to the mitochondria and its interaction with mitochondrial proteins has not been explored. OBJECTIVE: We sought to explore the role played by PDH in mast cell exocytosis and to determine whether MITF is localized in the mitochondria and involved in regulation of PDH activity. METHODS: Experiments were performed in vitro by using human and mouse mast cells, as well as rat basophil leukemia cells, and in vivo in mice. The effect of PDH inhibition on mast cell function was examined. PDH interaction with MITF was measured before and after immunologic activation. Furthermore, mitochondrial localization of MITF and its effect on PDH activity were determined. RESULTS: PDH is essential for immunologically mediated degranulation of mast cells. After activation, PDH is serine dephosphorylated. In addition, for the first time, we show that MITF is partially located in the mitochondria and interacts with PDH. This interaction is dependent on the phosphorylation state of PDH. Furthermore, mitochondrial MITF regulates PDH activity. CONCLUSION: The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases.

Mast cells' integrated actions with eosinophils and fibroblasts in allergic inflammation: implications for therapy.

Mast cells (MCs) and eosinophils (Eos) are the key players in the development of allergic inflammation (AI). Their cross-talk, named the Allergic Effector Unit (AEU), takes place through an array of soluble mediators and ligands/receptors interactions that enhance the functions of both the cells. One of the salient features of the AEU is the CD48/2B4 receptor/ligand binding complex. Furthermore, MCs and Eos have been demonstrated to play a role not only in AI but also in the modulation of its consequence, i.e., fibrosis/tissue remodeling, by directly influencing fibroblasts (FBs), the main target cells of these processes. In turn, FBs can regulate the survival, activity, and phenotype of both MCs and Eos. Therefore, a complex three players, MCs/Eos/FBs interaction, can take place in various stages of AI. The characterization of the soluble and physical mediated cross talk among these three cells might lead to the identification of both better and novel targets for the treatment of allergy and its tissue remodeling consequences.

Eosinophils as a pharmacological target for the treatment of allergic diseases.

Eosinophils are innate immune cells and active players in inflammatory responses. Their activation and increased levels in the blood and at specific sites are associated with parasitic infections and several inflammatory conditions, notably allergic diseases in which eosinophils are considered to be damaging cells. Intervention targeting eosinophils is thought to prevent and/or limit irreversible organ damage and other eosinophil-associated disorders like hypereosinophilic syndromes, some cancers and autoimmune diseases. Several eosinophil-targeted therapeutic agents which block specific steps in eosinophil differentiation, migration and activation have recently been developed, showing encouraging results and new insights into their specific role in allergy. Here, we review some potentially effective drug compounds, their drawbacks and future prospective focusing on allergic diseases.