Astrocytes, the most abundant glia subtype, exert a wide range of functions, many of which are essential for maintaining neuronal homeostasis. A variety of neurotransmitter receptors are expressed on astrocytes allowing them to sense extracellular signals and respond by releasing neuroactive mediators. Among them, a wide variety of G protein-coupled receptors have been detected, including those for dopamine (DA), known to play a major role in modulating astrocytic activity. Evidence that astrocytic DA D2 receptors (D2R) increase the release of trophic factors and suppress neuroinflammation has been provided. Thus, DA signaling in astrocytes may be crucially involved in the mechanisms underlying the degeneration of DA neurons in Parkinson's disease (PD). In this study, human astrocytes were generated from induced pluripotent stem cell (iPSC) lines derived from two PD patients bearing G2019S LRRK2 kinase activating mutation. The effect of the PD-related mutation in astrocytes was analyzed, focusing on DA receptor's expression and localization. As expected, astrocytes carrying G2019S mutation in LRRK2 displayed a reactive phenotype with increased secretion of inflammatory cytokines and reduced ability to support DA neurons' trophism in astrocytes/neurons co-culture experiments. Intriguingly, PD astrocytes exhibited reduced membrane expression of D2R. Inhibiting the abnormally increased kinase activity was able to revert the PD astrocytes' reactive phenotype and to rescue the D2R membrane localization. We thus provide new insights into how G2019S mutation in LRRK2, by disrupting the astrocytic physiological localization of D2R, may impair protective DA signaling, resulting in increased neuroinflammation and neuronal damage.

Abstract Background MicroRNAs (miRNAs) are short regulatory RNAs that can be released in extracellular vesicles and, under pathological conditions such as autoimmunity, activate innate immune cells through Toll-like receptor (TLR) 7 and 8. This mechanism may sustain chronic inflammation. Psoriasis is an immune-mediated skin disease where the role and activation pathways of Natural Killer (NK) cells remain incompletely understood. We investigated whether miRNAs upregulated in psoriatic lesions contribute to NK cell activation. Methods A pool of psoriasis-associated miRNAs (pso-miR) was generated and used to stimulate either purified NK cells or peripheral blood mononuclear cells (PBMCs). NK cell activation was assessed in terms of cytokine secretion and target cell killing. Inhibitor experiments were performed to demonstrate TLR activation by pso-miR. Results Pso-miR did not directly activate NK cells, which lack TLR7/8, but triggered NK effector functions, including IFN-γ secretion and cytotoxicity, within PBMCs, indicating the involvement of accessory cells. Mechanistically, pso-miR engaged TLR7/8-expressing plasmacytoid dendritic cells and monocytes, leading to the secretion of IFN-α, IL-12, and IL-18. These cytokines, in turn, drove full NK cell activation. We also identified a previously overlooked subset of CD56dim NK cells in psoriatic skin, representing mature cytotoxic NKs. Moreover, pso-miR stimulation of PBMCs induced IFN-γ-producing CD8+ T cells, further amplifying tissue-damaging responses. Conclusions Altogether, these findings reveal that psoriatic miRNAs activate an innate immune loop, which indirectly drives NK cell and CD8+ T cell effector functions via TLR7/8-dependent cytokine signalling, representing a novel pathogenic mechanism of psoriatic inflammation and keratinocyte damage. Of note, such miRNA-mediated crosstalk is abrogated by the dual TLR7/8 antagonist Enpatoran, highlighting a therapeutic avenue for modulating immune activation in psoriasis.

Testicular germ cell tumors are the most common solid malignancy in young adult males with non-seminomatous representing a clinically aggressive subtype. Although cisplatin (CP)-based chemotherapy is highly effective, a subset of patients develop resistance. This study explored a potential new treatment by targeting WEE1, a key cell-cycle regulator, using the drug adavosertib in non-seminoma cell models, aiming to overcome CP resistance. Two non-seminoma cell lines, NCCIT and NT2/D1, and their CP-resistant subclones (NCCIT-R and NT2/D1-R) were used. The effects of adavosertib and CP, alone or in combination, on cell viability, cell-cycle progression, apoptosis, and DNA damage markers was assessed. WEE1 was expressed in non-seminoma cell models. Adavosertib reduced cell viability in a dose-dependent manner across all cell models in both 2D and 3D cultures. IC50 values were in low micromolar range (NCCIT: 0.550 µM; NCCIT-R: 0.630 µM; NT2/D1: 0.415 µM; NT2/D1-R: 0.630 µM). Adavosertib altered cell-cycle distribution, increasing S-phase population. Western blot analysis revealed that inhibiting WEE1 increases CDK1 activity and mitotic marker pH3, indicating disrupted cell cycle control. This leads to replication stress and forces cells with DNA damage into early mitosis, causing mitotic catastrophe. The treatment also triggered higher levels of DNA damage and cell death, as shown by increased caspase activity and apoptosis markers (cleaved-PARP and cleaved-Caspase 3). In combination treatments, adavosertib enhanced CP efficacy across all models, including resistant lines. Overall, our results provide compelling preclinical evidence supporting the combination of WEE1 inhibition with standard chemotherapy as a promising strategy to overcome CP-resistance in non-seminoma testicular cancer.

Supplementary Figures 1-7 with legends

ObjectivesMonocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation.MethodsHuman monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors.ResultsEx-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts.ConclusionsThis work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.

Extracellular vesicles (EVs) are nanosized heat-stable vesicles released by virtually all cells in the body, including tumor cells and tumor-infiltrating dendritic cells (DCs). By carrying molecules from originating cells, EVs work as cell-to-cell communicators in both homeostasis and cancer but may also represent valuable therapeutic and diagnostic tools. This review focuses on the role of tumor-derived EVs (TEVs) in the modulation of DC functions and on the therapeutic potential of both tumor- and DC-derived EVs in the context of immunotherapy and DC-based vaccine design. TEVs were originally characterized for their capability to transfer tumor antigens to DCs but are currently regarded as mainly immunosuppressive because of the expression of DC-inhibiting molecules such as PD-L1, HLA-G, PGE2 and others. However, TEVs may still represent a privileged system to deliver antigenic material to DCs upon appropriate engineering to reduce their immunosuppressive cargo or increase immunogenicity. DC-derived EVs are more promising than tumor-derived EVs since they expose antigen-loaded MHC, costimulatory molecules and NK cell-activating ligands in the absence of an immunosuppressive cargo. Moreover, DC-derived EVs possess several advantages as compared to cell-based drugs such as a higher antigen/MHC concentration and ease of manipulation and a lower sensitivity to immunosuppressive microenvironments. Preclinical models showed that DC-derived EVs efficiently activate tumor-specific NK and T cell responses either directly or indirectly by transferring antigens to tumor-infiltrating DCs. By contrast, however, phase I and II trials showed a limited clinical efficacy of EV-based anticancer vaccines. We discuss that the future of EV-based therapy depends on our capability to overcome major challenges such as a still incomplete understanding of their biology and pharmacokinetic and the lack of standardized methods for high-throughput isolation and purification. Despite this, EVs remain in the limelight as candidates for cancer immunotherapy which may outmatch cell-based strategies in the fullness of their time.

Chemerin is a distant member of the cystatin protein family, initially discovered as a chemotactic factor and subsequently also reported to act as adipokine and angiogenetic factor. The biological activity of chemerin is regulated at different levels, such as gene expression, protein processing, and interaction with both signaling and nonsignaling receptors. Chemerin is mostly produced by stromal cells, such as adipocytes, fibroblasts, and epithelial and endothelial cells, and circulates in almost all human tissues as a zymogen that needs to be proteolytically activated to exert its biological functions. At the receptor level, chemerin binds a G protein-coupled 7-transmembrane domain receptor Chemerin1 (also named ChemR23 and CMKLR1), mostly expressed by innate immune cells, such as macrophages, dendritic cells, and natural killer cells, and by border cells. In addition, chemerin may bind GPR1, a weak signaling receptor, and CCRL2, a nonsignaling receptor expressed by barrier cells, such as endothelial and epithelial cells, able to regulate leukocytes' migration by multiple mechanisms. The aim of this review is to summarize the contribution of chemerin in the regulation of immune responses.

Neutrophils (PMNs) are key players of innate immune responses through the release of cytoplasmic granule content and the formation of neutrophil extracellular traps (NETs). RNASET2 is an acidic ribonuclease, recently proposed as an alarmin signal associated with inflammatory responses. Here we show that, along the neutrophil maturation cascade, RNASET2 is expressed in segmented and mature PMNs. In human PMNs, RNASET2 colocalized with primary and tertiary granules and was found to be associated with NETs following PMA or Nigericin stimulation. Similarly, activation of PMNs by soluble immune complexes, a hallmark of several autoimmune diseases, also induced RNASET2-associated NETs. Genome-wide association studies recently identified RNASET2 among a cluster of genes associated with increased susceptibility to develop autoimmune diseases, including rheumatoid arthritis (RA). RNASET2 was found expressed by PMNs and macrophages infiltrating inflamed joints in a murine model of RA (K/BxN Serum-Transfer-Induced Arthritis, STIA), by immunostaining. Similar results were found in synovial biopsies of RA patients with active disease. In addition, we demonstrate that RNASET2 circulating levels correlated with the onset and the severity of disease in two mouse models of inflammatory arthritis, STIA and CIA (Collagen-Induced Arthritis) and in serum of RA patients. These results show that PMNs are an important source of RNASET2 and that its circulating levels are associated with RA development suggesting a role for RNASET2 in the pathogenesis of immune-mediated diseases.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

The physiological relevance of cell-to-cell communication mediated by small extracellular vesicle-encapsulated microRNAs (sEV-miRNAs) remains debated because of the limiting representativity of specific miRNAs within the extracellular pool. We hypothesize that sEV-miRNA non-canonical function consisting of the stimulation of Toll-like receptor 7 (TLR7) may rely on a global shift of the sEV cargo rather than on the induction of one or few specific miRNAs. Psoriasis represents an ideal model to test such hypothesis as it is driven by overt activation of TLR7-expressing plasmacytoid dendritic cells (pDCs) following keratinocyte damage. To mimic the onset of psoriasis, keratinocytes were treated with a cocktail of psoriatic cytokines or UV-irradiated. SmallRNA sequencing was performed on sEVs released by healthy and UV-treated keratinocytes. sEV-miRNAs were analyzed for nucleotide composition as well as for the presence of putative TLR7-binding triplets. Primary human pDCs where stimulated with sEVs +/- inhibitors of TLR7 (Enpatoran), of sEV release (GW4869 + manumycin) and of TLR7-mediated pDC activation (anti-BDCA-2 antibody). Secretion of type I IFNs and activation of CD8+T cells were used as readouts. qPCR on psoriatic and healthy skin biopsies was conducted to identify induced miRNAs. sEV-miRNAs released by damaged keratinocytes revealed a significantly higher content of TLR7-activating sequences than healthy cells. As expected, differential expression analysis confirmed the presence of miRNAs upregulated in psoriatic skin, including miR203a. More importantly, 76.5

Plasmacytoid dendritic cells (pDCs) are the major producers of type I interferons (IFNs), which are essential to mount antiviral and antitumoral immune responses. To avoid exaggerated levels of type I IFNs, which pave the way to immune dysregulation and autoimmunity, pDC activation is strictly regulated by a variety of inhibitory receptors (IRs). In tumors, pDCs display an exhausted phenotype and correlate with an unfavorable prognosis, which largely depends on the accumulation of immunosuppressive cytokines and oncometabolites. This review explores the hypothesis that tumor microenvironment may reduce the release of type I IFNs also by a more pDC-specific mechanism, namely the engagement of IRs. Literature shows that many cancer types express de novo, or overexpress, IR ligands (such as BST2, PCNA, CAECAM-1 and modified surface carbohydrates) which often represent a strong predictor of poor outcome and metastasis. In line with this, tumor cells expressing ligands engaging IRs such as BDCA-2, ILT7, TIM3 and CD44 block pDC activation, while this blocking is prevented when IR engagement or signaling is inhibited. Based on this evidence, we propose that the regulation of IFN secretion by IRs may be regarded as an “innate checkpoint”, reminiscent of the function of “classical” adaptive immune checkpoints, like PD1 expressed in CD8+ T cells, which restrain autoimmunity and immunopathology but favor chronic infections and tumors. However, we also point out that further work is needed to fully unravel the biology of tumor-associated pDCs, the neat contribution of pDC exhaustion in tumor growth following the engagement of IRs, especially those expressed also by other leukocytes, and their therapeutic potential as targets of combined immune checkpoint blockade in cancer immunotherapy.

Background Circulating biomarkers with diagnostic and prognostic value in early Rheumatoid Arthritis (RA) are still an unmet need, especially for the subgroup of seronegative RA [e.g., negative for anti-citrullinated peptides autoantibodies (ACPA) and/or rheumatoid factors (RF)]. In a recent gene-based genome-wide association analysis (GWAS) study, RNASET2 was identified as a potential susceptibility gene [1-3]. Furthermore, the extracellular ribonuclease RNASET2 was demonstrated to be involved in the regulation of several immune processes [4]. Objectives The aim of this study was to analyse RNASET2 serum levels in a cohort of RA patients upon their disease activity, in comparison with healthy controls (HC). Methods Forty consecutive patients with RA (75% female, 55% seropositive) with a median age (25th-75th percentile) of 55 years (45-68) and a CRP-DAS28 index of 3.09 (2.50-3.70), and 25 sex and age-matched HC were enrolled in the study. RNASET2 serum levels were assessed through a commercial hELISA test (WuHan Fine Biotech Co., China). Results Serum levels of RNASET2 were higher in RA patients than in HC [29.42 (17.73 – 69.82) vs 18.15 (12.15 – 28.35) ng/mL; p= 0.001]. Among patients with RA, there were no differences between RNASET2 levels in seropositive or seronegative ones [33.14 (16.79 – 74.89) vs 29.01 (17.83 – 39.85) ng/mL; p=0.308]. Furthermore, there were no differences in RNASET2 serum levels between patients in treatment with corticosteroids or not [42.09 (18.11 – 78.78) vs 23.42 (18.29 – 43.87) ng/mL; p=0.239]. RNASET2 serum levels were significantly different across 3 groups of RA patients identified upon the CRP-DAS28 score (high plus moderate disease activity –group A-; low disease activity –group B-; remission –group C-) (p= 0.024). RNaseT2 levels were higher in both group A and B as compared to group C [56.73 (30.22 – 79.67) - 83.72 (52.68 – 113.89) vs 25.43 (20.15 – 39.85) ng/mL; p= 0.005 & p= 0.007]. No difference was found between group A & B. When combining groups A & B, RNASET2 levels were significantly higher than in group C [67.14 (36.07 – 86.68) vs 25.43 (20.15 – 39.85) ng/mL; p= 0.013]. No significant correlation was found between RNASET2 serum levels and CRP-DAS28 (r= 0.36; p= 0.055). Conclusion This is the first description of circulating serum levels of RNASET2 as a potential diagnostic biomarker in RA. Higher levels of RNASET2 clustered in RA patients as compared to HC. Low levels of RNASET2 seem to characterize the remission phase of RA. These preliminary results deserve to be expanded in larger cohorts of RA patients, patients with other form of chronic arthritis and other pathological and healthy controls. References [1]Zhu et al. PLOSone 2016; [2]Walsh et al. Genome Biology 2016; [3]Muaaz Aslam et al. Disease Markers 2020; [4]Acquati et al. Frontiers Immunol 2019 Acknowledgements This work was supported by the Italian Ministry for University and Research (Prin 2017NTK4HY). Disclosure of Interests None Declared.

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies.

Inhibitors of phosphodiesterase-4 (PDE4) are small-molecule drugs that, by increasing the intracellular levels of cAMP in immune cells, elicit a broad spectrum of anti-inflammatory effects. As such, PDE4 inhibitors are actively studied as therapeutic options in a variety of human diseases characterized by an underlying inflammatory pathogenesis. Dendritic cells (DCs) are checkpoints of the inflammatory and immune responses, being responsible for both activation and dampening depending on their activation status. This review shows evidence that PDE4 inhibitors modulate inflammatory DC activation by decreasing the secretion of inflammatory and Th1/Th17-polarizing cytokines, although preserving the expression of costimulatory molecules and the CD4+ T cell-activating potential. In addition, DCs activated in the presence of PDE4 inhibitors induce a preferential Th2 skewing of effector T cells, retain the secretion of Th2-attracting chemokines and increase the production of T cell regulatory mediators, such as IDO1, TSP-1, VEGF-A and Amphiregulin. Finally, PDE4 inhibitors selectively induce the expression of the surface molecule CD141/Thrombomodulin/BDCA-3. The result of such fine-tuning is immunomodulatory DCs that are distinct from those induced by classical anti-inflammatory drugs, such as corticosteroids. The possible implications for the treatment of respiratory disorders (such as COPD, asthma and COVID-19) by PDE4 inhibitors will be discussed.