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Abstract Background Melanoma progression is based on a close interaction between cancer cells and immune cells in the tumor microenvironment (TME). Thus, a better understanding of the mechanisms controlling TME dynamics and composition will help improve the management of this dismal disease. Work from our and other groups has reported the requirement of an active Hedgehog-GLI (HH-GLI) signaling for melanoma growth and stemness. However, the role of the downstream GLI1 transcription factor in melanoma TME remains largely unexplored. Methods The immune-modulatory activity of GLI1 was evaluated in a syngeneic B16F10 melanoma mouse model assessing immune populations by flow cytometry. Murine polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were differentiated from bone marrow cells and their immunosuppressive ability was assessed by inhibition of T cells. Conditioned media (CM) from GLI1-overexpressing mouse melanoma cells was used to culture PMN-MDSCs, and the effects of CM were evaluated by Transwell invasion assay and T cell inhibition. Cytokine array analysis, qPCR and chromatin immunoprecipitation were performed to explore the regulation of CX3CL1 expression by GLI1. Human monocyte-derived dendritic cells (moDCs) were cultured in CM from GLI1-silenced patient-derived melanoma cells to assess their activation and recruitment. Blocking antibodies anti-CX3CL1, anti-CCL7 and anti-CXCL8 were used for in vitro functional assays. Results Melanoma cell-intrinsic activation of GLI1 promotes changes in the infiltration of immune cells, leading to accumulation of immunosuppressive PMN-MDSCs and regulatory T cells, and to decreased infiltration of dendric cells (DCs), CD8 + and CD4 + T cells in the TME. In addition, we show that ectopic expression of GLI1 in melanoma cells enables PMN-MDSC expansion and recruitment, and increases their ability to inhibit T cells. The chemokine CX3CL1, a direct transcriptional target of GLI1, contributes to PMN-MDSC expansion and recruitment. Finally, silencing of GLI1 in patient-derived melanoma cells promotes the activation of human monocyte-derived dendritic cells (moDCs), increasing cytoskeleton remodeling and invasion ability. This phenotype is partially prevented by blocking the chemokine CCL7, but not CXCL8. Conclusion Our findings highlight the relevance of tumor-derived GLI1 in promoting an immune-suppressive TME, which allows melanoma cells to evade the immune system, and pave the way for the design of new combination treatments targeting GLI1.

IntroductionNeuroinflammation is a hallmark of multiple neurodegenerative diseases, shared by all pathological processes which primarily impact on neurons, including Central Nervous System (CNS) injuries. In reactive CNS, activated glia releases extracellular vesicles (EVs), nanosized membranous particles known to play a key role in intercellular communication. EVs mediate neuroinflammatory responses and might exacerbate tissue deterioration, ultimately influencing neurodegenerative disease progression.MethodsWe treated spinal cord organotypic slices with LPS, a ligand extensively used to induce sEVs release, to mimic mild inflammatory conditions. We combine atomic force microscopy (AFM), nanoparticle tracking (NTA) and western blot (WB) analysis to validate the isolation and characterisation of sEVs. We further use immunofluorescence and confocal microscopy with live calcium imaging by GCaMP6f reporter to compare glial reactivity to treatments with sEVs when isolated from resting and LPS treated organ slices.ResultsIn our study, we focus on CNS released small EVs (sEVs) and their impact on the biology of inflammatory environment. We address sEVs local signalling within the CNS tissue, in particular their involvement in inflammation spreading mechanism(s). sEVs are harvested from mouse organotypic spinal cord cultures, an in vitro model which features 3D complexity and retains spinal cord resident cells. By confocal microscopy and live calcium imaging we monitor glial responses in naïve spinal slices when exposed to sEVs isolated from resting and LPS treated organ slices.DiscussionWe show that sEVs, only when released during LPS neuroinflammation, recruit naïve astrocytes in the neuroinflammation cycle and we propose that such recruitment be mediated by EVs hemichannel (HC) permeability.

Abstract Extracellular vesicles (EVs) are lipid-bilayered particles, containing various biomolecules, including nucleic acids, lipids, and proteins, released by cells from all the domains of life and performing multiple communication functions. Evidence suggests that the interaction between host immune cells and fungal EVs induces modulation of the immune system. Most of the studies on fungal EVs have been conducted in the context of fungal infections; therefore, there is a knowledge gap in what concerns the production of EVs by yeasts in other contexts rather than infection and that may affect human health. In this work, we characterized EVs obtained by Saccharomyces cerevisiae and Pichia fermentans strains isolated from a fermented milk product with probiotic properties. The immunomodulation abilities of EVs produced by these strains have been studied in vitro through immune assays after internalization from human monocyte-derived dendritic cells. Results showed a significant reduction in antigen presentation activity of dendritic cells treated with the fermented milk EVs. The small RNA fraction of EVs contained mainly yeast mRNA sequences, with a few molecular functions enriched in strains of two different species isolated from the fermented milk. Our results suggest that one of the mechanisms behind the anti-inflammatory properties of probiotic foods could be mediated by the interactions of human immune cells with yeast EVs.

Abstract The central nervous system (CNS) is finely protected by the blood–brain barrier (BBB). Immune soluble factors such as cytokines (CKs) are normally produced in the CNS, contributing to physiological immunosurveillance and homeostatic synaptic scaling. CKs are peptide, pleiotropic molecules involved in a broad range of cellular functions, with a pivotal role in resolving the inflammation and promoting tissue healing. However, pro-inflammatory CKs can exert a detrimental effect in pathological conditions, spreading the damage. In the inflamed CNS, CKs recruit immune cells, stimulate the local production of other inflammatory mediators, and promote synaptic dysfunction. Our understanding of neuroinflammation in humans owes much to the study of multiple sclerosis (MS), the most common autoimmune and demyelinating disease, in which autoreactive T cells migrate from the periphery to the CNS after the encounter with a still unknown antigen. CNS-infiltrating T cells produce pro-inflammatory CKs that aggravate local demyelination and neurodegeneration. This review aims to recapitulate the state of the art about CKs role in the healthy and inflamed CNS, with focus on recent advances bridging the study of adaptive immune system and neurophysiology.

Abstract Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 β) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ventral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments.

Relapsing-remitting multiple sclerosis (RRMS) is a demyelinating disease in which pathogenesis T cells have a major role. Despite the unknown etiology, several risk factors have been described, including a strong association with human leukocyte antigen (HLA) genes. Recent findings showed that HLA class I-G (HLA-G) may be tolerogenic in MS, but further insights are required. To deepen the HLA-G role in MS inflammation, we measured soluble HLA-G (sHLA-G) and cytokines serum level in 27 patients with RRMS at baseline and after 12 and 24 months of natalizumab (NTZ) treatment. Patients were divided into high (sHLA-G>20 ng/ml), medium (sHLA-G between 10 and 20 ng/ml), and low (sHLA-G

Food contamination can be a serious concern for public health because it can be related to the severe spreading of pathogens. This is a main issue, especially in the case of fresh fruits and vegetables; indeed, they have often been associated with gastrointestinal outbreak events, due to contamination with pathogenic bacteria. However, little is known about the physiological adaptation and bacterial response to stresses encountered in the host plant. Thus, this work aimed to investigate the adaptation of a commensal E. coli strain while growing in tomato pericarp. Pre-adapted and non-adapted cells were compared and used to contaminate tomatoes, demonstrating that pre-adaptation boosted cell proliferation. DNA extracted from pre-adapted and non-adapted cells was sequenced, and their methylation profiles were compared. Hence, genes involved in cell adhesion and resistance against toxic compounds were identified as genes involved in adaptation, and their expression was compared in these two experimental conditions. Finally, pre-adapted and non-adapted E. coli were tested for their ability to resist the presence of toxic compounds, demonstrating that adaptation exerted a protective effect. In conclusion, this work provides new information about the physiological adaptation of bacteria colonizing the tomato fruit pericarp.

T-cell receptor (TCR) repertoire diversity is a determining factor for the immune system capability in fighting infections and preventing autoimmunity. During life, the TCR repertoire diversity progressively declines as a physiological aging progress. The investigation of TCR repertoire dynamics over life represents a powerful tool unraveling the impact of immunosenescence in health and disease. Multiple Sclerosis (MS) is a demyelinating, inflammatory, T-cell mediated autoimmune disease of the Central Nervous System in which age is crucial: it is the most widespread neurological disease among young adults and, furthermore, patients age may impact on MS progression and treatments outcome. Crossing knowledge on the TCR repertoire dynamics over MS patients’ life is fundamental to investigate disease mechanisms, and the advent of high- throughput sequencing (HTS) has significantly increased our knowledge on the topic. Here we report an overview of current literature about the impact of immunosenescence and age-related TCR dynamics variation in autoimmunity, including MS.

Background: T cells play a key role in the pathogenesis of multiple sclerosis (MS), a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). Although several studies recently investigated the T-cell receptor (TCR) repertoire in cerebrospinal fluid (CSF) of MS patients by high-throughput sequencing (HTS), a deep analysis on repertoire similarities and differences among compartments is still missing. Methods: We performed comprehensive bioinformatics on high-dimensional TCR Vβ sequencing data from published and unpublished MS and healthy donors (HD) studies. We evaluated repertoire polarization, clone distribution, shared CDR3 amino acid sequences (CDR3s-a.a.) across repertoires, clone overlap with public databases, and TCR similarity architecture. Findings: CSF repertoires showed a significantly higher public clones percentage and sequence similarity compared to peripheral blood (PB). On the other hand, we failed to reject the null hypothesis that the repertoire polarization is the same between CSF and PB. One Primary-Progressive MS (PPMS) CSF repertoire differed from the others in terms of TCR similarity architecture. Cluster analysis splits MS from HD. Interpretation: In MS patients, the presence of a physiological barrier, the blood-brain barrier, does not impact clone prevalence and distribution, but impacts public clones, indicating CSF as a more private site. We reported a high Vβ sequence similarity in the CSF-TCR architecture in one PPMS. If confirmed it may be an interesting insight into MS progressive inflammatory mechanisms. The clustering of MS repertoires from HD suggests that disease shapes the TCR Vβ clonal profile. Funding: This study was partly financially supported by the Italian Multiple Sclerosis Foundation (FISM), that contributed to Ballerini-DB data collection (grant #2015 R02).

Fatty acid ethanolamides acting on proliferator-activated receptor (PPAR)-α are among the endogenous lipid molecules that attenuate inflammatory processes and pain sensitivity. Whereas these properties are well-known for palmitoylethanolamide (PEA), the efficacy of oleoylethanolamide (OEA, first described as a satiety hormone synthesized in the jejunum) has been overlooked. In this study, we aimed to evaluate the effect of OEA administration in a mouse model of colitis. C57BL/6J mice were exposed to 2.5% dextran sodium sulphate (DSS) in drinking water for 5 days. Daily i.p. administration of 10 mg/kg OEA started 3 days before DSS and lasted for 12 days. The DSS-untreated control group received only ultrapure water. DSS mice treated with OEA had a significant improvement of disease score. OEA restored mRNA transcription of PPAR-α, of tight junctions and protective factors of colon integrity disrupted by DSS. The improvement correlated with significant decrease of colonic and systemic levels of pro-inflammatory cytokines compared to the DSS group. OEA antiinflammatory effects were mediated by the selective targeting of the TLR4 axis causing a downstream inhibition of nuclear factor kappa B (NF-κB)- MyD88-dependent and NLRP3 inflammation pathways. OEA treatment also inhibited DSS-induced increase of inflammatory cytokines levels in the mesenteric lymph nodes. Conclusions and implications: These results underscore the validity of OEA as a potent protective and anti-inflammatory agent in ulcerative colitis that may be exploited to broaden the pharmacological strategies against inflammatory bowel disease.

Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the post-treatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naïve and memory CD4+ and CD8+) across 15 RRMS patients before and after two years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multi-dimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis and treatment regimes. Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing–remitting multiple sclerosis (RRMS), an autoimmune T-cell–driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the posttreatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naive and memory CD4+ and CD8+) across 15 RRMS patients before and after 2 years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRβ sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRβ repertoire dynamics with respect to clonal expansion, clonal diversity, and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multidimensional computational immunology to a TCRβ dataset of treated MS patients, we show that qualitative changes of TCRβ repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis, and treatment regimens.