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Hypoxia is a key component of the tumor microenvironment (TME) and promotes not only tumor growth and metastasis, but also negatively affects infiltrating immune cells by impairing host immunity. Dendritic cells (DCs) are the most potent antigen-presenting cells and their biology is weakened in the TME in many ways, including the modulation of their viability. RNASET2 belongs to the T2 family of extracellular ribonucleases and, besides its nuclease activity, it exerts many additional functions. Indeed, RNASET2 is involved in several human pathologies, including cancer, and it is functionally relevant in the TME. RNASET2 functions are not restricted to cancer cells and its expression could be relevant also in other cell types which are important players in the TME, including DCs. Therefore, this study aimed to unravel the effect of hypoxia (2% O2) on the expression of RNASET2 in DCs. Here, we showed that hypoxia enhanced the expression and secretion of RNASET2 in human monocyte-derived DCs. This paralleled the HIF-1α accumulation and HIF-dependent and -independent signaling, which are associated with DCs’ survival/autophagy/apoptosis. RNASET2 expression, under hypoxia, was regulated by the PI3K/AKT pathway and was almost completely abolished by TLR4 ligand, LPS. Taken together, these results highlight how hypoxia- dependent and -independent pathways shape RNASET2 expression in DCs, with new perspectives on its implication for TME and, therefore, in anti-tumor immunity.

A great bulk of recent experimental evidence suggests the key role of the complex crosstalk between the extracellular matrix (ECM) and the cellular component of tissues during morphogenesis and embryogenesis. In particular, remodeling of the ECM and of its physical interactions pattern with surrounding cells represent two crucial processes that might be involved in muscle development. However, little information is available on this topic, especially on invertebrate species. To obtain new insights on how tuning the ECM microenvironment might drive cellular fate during embryonic development, we used the invertebrate medicinal leech Hirudo verbana as a valuable experimental model, due to its simple anatomy and the recapitulation of many aspects of the basic biological processes of vertebrates. Our previous studies on leech post-embryonic development have already shown the pivotal role of ECM changes during the growth of the body wall and the role of Yes-associated protein 1 (YAP1) in mechanotransduction. Here, we suggest that the interactions between stromal cell telocytes and ECM might be crucial in driving the organization of muscle layers during embryogenesis. Furthermore, we propose a possible role of the pleiotropic enzyme HvRNASET2 as a possible modulator of collagen deposition and ECM remodeling not only during regenerative processes (as previously demonstrated) but also in embryogenesis.

Aging is often a choice between developing cancer or autoimmune disorders, often due in part to loss of self-tolerance or loss of immunological recognition of rogue-acting tumor cells. Self-tolerance and cell recognition by the immune system are processes very much dependent on the specific signatures of glycans and glycosylated factors present on the cell plasma membrane or in the stromal components of tissue. Glycosylated factors are generated in nearly innumerable variations in nature, allowing for the immensely diverse role of these factors in aging and flexibility necessary for cellular interactions in tissue functionality. In previous studies, we showed that differential expression of TMEM230, an endoplasmic reticulum (ER) protein was associated with specific signatures of enzymes regulating glycan synthesis and processing and glycosylation in rheumatoid arthritis synovial tissue using single-cell transcript sequencing. In this current study, we characterize the genes and pathways co-modulated in all cell types of the synovial tissue with the enzymes regulating glycan synthesis and processing, as well as glycosylation. Genes and biological and molecular pathways associated with hallmarks of aging were in mitochondria-dependent oxidative phosphorylation and reactive oxygen species synthesis, ER-dependent stress and unfolded protein response, DNA repair (UV response and P53 signaling pathways), and senescence, glycolysis and apoptosis regulation through PI3K-AKT-mTOR signaling have been shown to play important roles in aging or neurodegeneration (such as Parkinson’s and Alzheimer’s disease). We propose that the downregulation of TMEM230 and RNASET2 may represent a paradigm for the study of age-dependent autoimmune disorders due to their role in regulating glycosylation, unfolded protein response, and PI3K-AKT-mTOR signaling.

Ovarian cancer represents one of the most malignant gynecological cancers worldwide, with an overall 5-year survival rate, being locked in the 25–30% range in the last decade. Cancer immunotherapy is currently one of the most intensively investigated and promising therapeutic strategy and as such, is expected to provide in the incoming years significant benefits for ovarian cancer treatment as well. Here, we provide a detailed survey on the highly pleiotropic oncosuppressive roles played by the human RNASET2 gene, whose protein product has been consistently reported to establish a functional crosstalk between ovarian cancer cells and key cellular effectors of the innate immune system (the monocyte/macrophages lineage), which is in turn able to promote the recruitment to the cancer tissue of M1-polarized, antitumoral macrophages. This feature, coupled with the ability of T2 ribonucleases to negatively affect several cancer-related parameters in a cell-autonomous manner on a wide range of ovarian cancer experimental models, makes human RNASET2 a very promising candidate to develop a “multitasking” therapeutic approach for innovative future applications for ovarian cancer treatment.

The identification of molecules that make cancer cells detectable by the immune system represents a major challenge in tumor immunology. Alarmins, endogenous, stress-induced molecules, serve as early warning signals triggering immune responses. The human RNASET2 protein has demonstrated both oncosuppressive and immunoregulatory functions across various cancer types, yet its role as an oncosuppressor or alarmin-like molecule in prostate cancer (PCa) is unexplored. Here, we investigated the effects of the human RNASET2 alarmin in two different human PCa cell lines focusing on cell proliferation, colony formation, adhesion, migration rates, and release of soluble immune-modulatory factors. In vivo studies were also carried out on nude mice to assess the immune regulatory impact. Our findings indicate that RNASET2 overexpression reduced cell proliferation and colony formation in 22Rv1 cells, through downregulation of cyclin D1. RNASET2 overexpression in 22Rv1 cells was also associated with decreased levels of TWIST , CTNNB1 , YAP , and MMP-9 . By contrast, PC-3 cells were largely unresponsive to RNASET2. RNASET2 overexpression also promoted the release of soluble factors related to monocyte/macrophage recruitment/activation and cytokines/chemokines, linked to immune cell-mediated anti-tumor responses. This effect was more pronounced in RNASET2-overexpressing 22Rv1 cells and involved both innate (NK cells, dendritic cells) and adaptive (T cells) immune activation, compared to PC-3 cells. RNASET2 overexpression also affected the cytoskeletal organization in both PCa models. RNASET2 overexpression in vivo induced a shift toward M1-like macrophage polarization pattern, while decreasing the M2-like polarization in mice challenged with 22Rv1 cells, indicating a potential tumor-suppressive role in PCa. Finally, silencing of RNASET2 in THP-1 macrophages unveiled their phagocytic activities against PCa cells. Our findings underscore the RNASET2’s dual functionality, acting through both cell-autonomous and non-cell autonomous mechanisms in PCa i n vitro and in vivo models and suggest its potential as a therapeutic target in a subset of PCa.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

Background RNASET2 has been identified as an oncogene with anti-angiogenic and immunomodulatory effects in a variety of cancers, but its function in clear cell renal cell carcinoma (ccRCC) is still not well understood. Methods The RNASET2 expression matrix was extracted from the The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets and analyzed for diagnostic and prognostic value. RNASET2 mRNA expression was detected by quantitative polymerase chain reaction (qPCR) in ccRCC patients and renal cancer cell lines. Wound healing assay, transwell assay, western blotting, and tube formation assays were used to evaluate the function of RNASET2 in renal cancer in vitro. In addition, transcriptome sequencing was performed on knockdown RNASET2 kidney cancer cells to analyze their potential signaling pathways. Moreover, the immune microenvironment and mutational status were evaluated to predict the potential mechanisms of RNASET2 involvement in renal cancer progression. Sensitivity to common chemotherapeutic and targeted agents was assessed according to the Genomics of Drug Sensitivity in Cancer (GDSC) database. Results RNASET2 expression was significantly upregulated in ccRCC tissues and renal cancer cell lines, predicting poor prognosis for patients. In vitro experiments showed that silencing RNASET2 inhibited the migration and pro-angiogenic ability of renal cancer cells. Transcriptome sequencing suggested its possible involvement in the remodeling of the immune microenvironment in renal cell carcinoma. Furthermore, bioinformatics analysis and immunohistochemical staining showed that RNASET2 was positively correlated with the infiltration abundance of regulatory T cells. Finally, we mapped the mutational landscape of RNASET2 in ccRCC and found its predictive value for drug sensitivity. Conclusions Our results suggest that RNASET2 is a promising biomarker and therapeutic target in ccRCC.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein ( RNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, RNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of RNASET2 was also evaluated on living cells by means of light, transmission, and scanning electron microscopy analysis. RNASET2 injection triggered the formation of clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released RNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area.

The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (r Hv RNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following r Hv RNASET2 injection in the leech body wall further supported the occurrence of r Hv RNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by r Hv RNASET2 injection. Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization.

Autoimmune and autoinflammatory diseases are rare but often devastating disorders, underpinned by abnormal immune function. While some autoimmune disorders are thought to be triggered by a burden of infection throughout life, others are thought to be genetic in origin. Among these heritable disorders are the type I interferonopathies, including the rare Mendelian childhood-onset encephalitis Aicardi-Goutières syndrome. Patients with Aicardi Goutières syndrome are born with defects in enzymes responsible for nucleic acid metabolism and develop devastating white matter abnormalities resembling congenital cytomegalovirus brain infection. In some cases, common infections preceded the onset of the disease, suggesting immune stimulation as a potential trigger. Thus, the antiviral immune response has been actively studied in an attempt to provide clues on the pathological mechanisms and inform on the development of therapies. Animal models have been fundamental in deciphering biological mechanisms in human health and disease. Multiple rodent and zebrafish models are available to study type I interferonopathies, which have advanced our understanding of the human disease by identifying key pathological pathways and cellular drivers. However, striking differences in phenotype have also emerged between these vertebrate models, with zebrafish models recapitulating key features of the human neuropathology often lacking in rodents. In this review, we compare rodent and zebrafish models, and summarize how they have advanced our understanding of the pathological mechanisms in Aicardi Goutières syndrome and similar disorders. We highlight recent discoveries on the impact of laboratory environments on immune stimulation and how this may inform the differences in pathological severity between mouse and zebrafish models of type I interferonopathies. Understanding how these differences arise will inform the improvement of animal disease modeling to accelerate progress in the development of therapies for these devastating childhood disorders.