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The link between cancer development or progression and immune system dysregulation has long been established. Virtually every cell type belonging to both the innate and adaptive immune system has been reported to be involved in a complex interplay that might culminate into either a pro- or anti-tumorigenic response. Among the cellular components of the innate immune system, cells belonging to the monocyte/macrophage lineage have been consistently shown to play a key role in the tumorigenic process. The most advanced human tumors are reported to be strongly infiltrated with Tumor-Associated Macrophages (TAMs) endowed with the ability to contribute to tumor growth and dissemination. However, given their widely acknowledged functional plasticity, macrophages can display anti-tumor properties as well. Based on these premises, experimental approaches to promote the macrophage shift from pro-tumor to anti-tumor phenotype represent one of the most promising research field aimed at developing immune system-mediated tumor suppressive therapies. In this context, the human oncosuppressor gene has emerged as a potential tool for macrophage-mediated tumor suppression. A growing body of experimental evidence has been reported to suggest a role for this gene in the regulation of macrophage activity in both and experimental models. Moreover, several recent reports suggest a role for this gene in a broad range of cell types involved in immune response, pointing at as a putative regulator of several functional features within the immune system.

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 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.

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.

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.

In recent years, there has been a growing interest in developing innovative anticancer therapies targeting the tumor microenvironment (TME). The TME is a complex and dynamic milieu surrounding the tumor mass, consisting of various cellular and molecular components, including those from the host organism, endowed with the ability to significantly influence cancer development and progression. Processes such as angiogenesis, immune evasion, and metastasis are crucial targets in the search for novel anticancer drugs. Thus, identifying molecules with “multi-tasking” properties that can counteract cancer cell growth at multiple levels represents a relevant but still unmet clinical need. Extensive research over the past two decades has revealed a consistent anticancer activity for several members of the T2 ribonuclease family, found in evolutionarily distant species. Initially, it was believed that T2 ribonucleases mainly acted as anticancer agents in a cell-autonomous manner. However, further investigation uncovered a complex and independent mechanism of action that operates at a non-cell-autonomous level, affecting crucial processes in TME-induced tumor growth, such as angiogenesis, evasion of immune surveillance, and immune cell polarization. Here, we review and discuss the remarkable properties of ribonucleases from the T2 family in the context of “multilevel” oncosuppression acting on the TME.

Background An isochromosome of the long arm of chromosome 7, i(7)(q10), and an interstitial deletion of the long arm of chromosome 20, del(20)(q), are the most frequent anomalies in the bone marrow of patients with Shwachman-Diamond syndrome, which is caused in most cases by mutations of the SBDS gene. These clonal changes imply milder haematological symptoms and lower risk of myelodysplastic syndromes and acute myeloid leukaemia, thanks to already postulated rescue mechanisms. Results Bone marrow from fourteen patients exhibiting either the i(7)(q10) or the del(20)(q) and coming from two large cohorts of patients, were subjected to chromosome analyses, Fluorescent In Situ Hybridization with informative probes and array-Comparative Genomic Hybridization. One patient with the i(7)(q10) showed a subsequent clonal rearrangement of the normal chromosome 7 across years. Four patients carrying the del(20)(q) evolved further different del(20)(q) independent clones, within a single bone marrow sample, or across sequential samples. One patient with the del(20)(q), developed a parallel different clone with a duplication of chromosome 3 long arm. Eight patients bore the del(20)(q) as the sole chromosomal abnormality. An overall overview of patients with the del(20)(q), also including cases already reported, confirmed that all the deletions were interstitial. The loss of material varied from 1.7 to 26.9 Mb and resulted in the loss of the EIF6 gene in all patients. Conclusions Although the i(7)(q) and the del(20)(q) clones are frequent and clinically benign in Shwachman Diamond-syndrome, in the present work we show that they may rearrange, may be lost and then reconstructed de novo , or may evolve with independent clones across years. These findings unravel a striking selective pressure exerted by SBDS deficiency driving to karyotype instability and to specific clonal abnormalities.

In recent years, the role played by the stromal microenvironment has been given growing attention in order to achieve a full understanding of cancer initiation and progression. Because cancer is a tissue-based disease, the integrity of tissue architecture is a major constraint toward cancer growth. Indeed, a large contribution of the natural resistance to cancer stems from stromal microenvironment components, the dysregulation of which can facilitate cancer occurrence. For instance, recent experimental evidence has highlighted the involvement of stromal cells in ovarian carcinogenesis, as epitomized by ovarian xenografts obtained by a double KO of the murine Dicer and Pten genes. Likewise, we reported the role of an ancient extracellular RNase, called Ribonuclease T2 (RNASET2), within the ovarian stromal microenvironment. Indeed, hyperexpression of RNASET2 is able to control tumorigenesis by recruiting macrophages (mostly of the anticancer M1 subtype) at the tumor sites. We present biological data obtained by RNASET2 silencing in the poorly tumorigenetic and highly RNASET2 -expressing human OVCAR3 cell line. RNASET2 knockdown was shown to stimulate in vivo tumor growth early after microinjection of OVCAR3 cells in nude mice. Moreover, we have investigated by molecular profiling the in vivo expression signature of human and mouse cell xenografts and disclosed the activation of pathways related to activation of the innate immune response and modulation of ECM components. Finally, we provide evidence for a role of RNASET2 in triggering an in vitro chemotactic response in macrophages. These results further highlight the critical role played by the microenvironment in RNASET2 -mediated ovarian tumor suppression, which could eventually contribute to better clarify the pathogenesis of this disease.