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.

A growing body of evidence links the gastrointestinal tract to Parkinson's disease (PD) development. The research by Braak and colleagues, based on analyzing post-mortem samples from PD patients, suggests that the pathology begins in the gut and progresses to the brain. Our recent work shows that PD brains and PD PBMCs display reduced DNA-binding activity of the NF-κB/c-Rel subunit, a neuroprotective transcription factor that promotes brain expression of mitochondrial regulators such as B-cell lymphoma-extra-large (Bcl-xL), uncoupling protein 4 (UCP4), and manganese superoxide dismutase (MnSOD). Previous studies have also shown that male mice lacking the NF-κB/c-Rel subunit (c-rel-/- mice) develop age-related parkinsonian neuropathology linked to both non-motor and motor symptoms. They exhibit a Braak pattern of α-synuclein (α-syn) deposition, beginning with early gut involvement marked by constipation and α-syn accumulation in the distal colon from 2 months old. Here, we further examine the progression of intestinal pathology in the proximal colon of c-rel-/- male mice which is connected to the brain via the vagus nerve, using microscopy, biochemistry, and multispectral optoacoustic tomography. At 2 months, α-syn and phosphorylated α-syn accumulation was detected in the myenteric plexus of c-rel-/- mice, with significantly greater accumulation at 10 months. Intestinal synucleinopathy was accompanied by notable oxidative stress, evidenced by increased NADPH oxidase activity and higher levels of 3-nitrotyrosine-modified proteins in the proximal colon. By 10 months, c-rel-/- mice showed gut inflammation with elevated interleukin-6 expression, infiltration of CD11b+Ly6G+ neutrophils, and increased oxygenated hemoglobin in the proximal colon, along with colon shortening and loss of TH-positive neurons. These findings, in addition to supporting the role of NF-κB/c-Rel dysregulation in PD pathophysiology, show that c-rel-/- mice develop progressive PD-like pathology in the proximal colon and provide an invaluable model for studying the potential gut-to-brain spread of endogenous α-syn via the vagus.

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.

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.