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This preclinical proof-of-concept study aimed to evaluate the effectiveness of secretome therapy in diabetic mice with pressure ulcers. We utilized a custom-made hyaluronic acid (HA)-based porous sponge, which was rehydrated either with normal culture medium or secretome derived from human mesenchymal stromal cells (MSCs) to achieve a hydrogel consistency. Following application onto skin ulcers, both the hydrogel-only and the hydrogel + secretome combination accelerated wound closure compared to the vehicle group. Notably, the presence of secretome significantly enhanced the healing effect of the hydrogel, as evidenced by a thicker epidermis and increased revascularization of the healed area compared to the vehicle group. Notably, molecular analysis of healed skin revealed significant downregulation of genes involved in delayed wound healing and abnormal inflammatory response in ulcers treated with the hydrogel + secretome combination, compared to those treated with the hydrogel only. Additionally, we found no significant differences in therapeutic outcomes when comparing the use of secretome from fetal dermal MSCs to that from umbilical cord MSCs. This observation is supported by the proteomic profile of the two secretomes, which suggests a shared molecular signature responsible of the observed therapeutic effects.

Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent cells that can be obtained from various tissues, such as dental pulp, adipose tissue, bone marrow and placenta. MSCs have gained importance in the field of regenerative medicine because of their promising role in cell therapy and their regulatory abilities in tissue repair and regeneration. However, a better characterization of these cells and their products is necessary to further potentiate their clinical application. In this study, we used unbiased high-resolution mass spectrometry-based proteomic analysis to investigate the impact of distinct priming strategies, such as hypoxia and IFN-γ treatment, on the composition and therapeutic functionality of the secretome produced by MSCs derived from the amniotic membrane of the human placenta (hAMSCs). Our investigation revealed that both types of priming improved the therapeutic efficacy of hAMSCs, and these improvements were related to the secretion of functional factors present in the conditioned medium (CM) and exosomes (EXOs), which play crucial roles in mediating the paracrine effects of MSCs. In particular, hypoxia was able to induce a pro-angiogenic, innate immune response-activating, and tissue-regenerative hAMSC phenotype, as highlighted by the elevated production of regulatory factors such as VEGFA, PDGFRB, ANGPTL4, ENG, GRO-γ, IL8, and GRO-α. IFN-γ priming, instead, led to an immunosuppressive profile in hAMSCs, as indicated by increased levels of TGFB1, ANXA1, THBS1, HOMER2, GRN, TOLLIP and MCP-1. Functional assays validated the increased angiogenic properties of hypoxic hAMSCs and the enhanced immunosuppressive activity of IFN-γ-treated hAMSCs. This study extends beyond the direct priming effects on hAMSCs, demonstrating that hypoxia and IFN-γ can influence the functional characteristics of hAMSC-derived secretomes, which, in turn, orchestrate the production of functional factors by peripheral blood cells. This research provides valuable insights into the optimization of MSC-based therapies by systematically assessing and comparing the priming type-specific functional features of hAMSCs. These findings highlight new strategies for enhancing the therapeutic efficacy of MSCs, particularly in the context of multifactorial diseases, paving the way for the use of hAMSC-derived products in clinical practice.

Abstract Cigarette smoking impairs the lung innate immune response making smokers more susceptible to infections and severe symptoms. Dysregulation of cell death is emerging as a key player in chronic inflammatory conditions. We have recently reported that short exposure of human monocyte-derived macrophages (hMDMs) to cigarette smoke extract (CSE) altered the TLR4-dependent response to lipopolysaccharide (LPS). CSE caused inhibition of the MyD88-dependent inflammatory response and activation of TRIF/caspase-8/caspase-1 pathway leading to Gasdermin D (GSDMD) cleavage and increased cell permeability. Herein, we tested the hypothesis that activation of caspase-8 by CSE increased pro-inflammatory cell death of LPS-stimulated macrophages. To this purpose, we measured apoptotic and pyroptotic markers as well as the expression/release of pro-inflammatory mediators in hMDMs exposed to LPS and CSE, alone or in combination, for 6 and 24 h. We show that LPS/CSE-treated hMDMs, but not cells treated with CSE or LPS alone, underwent lytic cell death (LDH release) and displayed apoptotic features (activation of caspase-8 and -3/7, nuclear condensation, and mitochondrial membrane depolarization). Moreover, the negative regulator of caspase-8, coded by CFLAR gene, was downregulated by CSE. Activation of caspase-3 led to Gasdermin E (GSDME) cleavage. Notably, lytic cell death caused the release of the damage-associated molecular patterns (DAMPs) heat shock protein-60 (HSP60) and S100A8/A9. This was accompanied by an impaired inflammatory response resulting in inhibited and delayed release of IL6 and TNF. Of note, increased cleaved caspase-3, higher levels of GSDME and altered expression of cell death-associated genes were found in alveolar macrophages of smoker subjects compared to non-smoking controls. Overall, our findings show that CSE sensitizes human macrophages to cell death by promoting pyroptotic and apoptotic pathways upon encountering LPS. We propose that while the delayed inflammatory response may result in ineffective defenses against infections, the observed cell death associated with DAMP release may contribute to establish chronic inflammation. CS exposure sensitizes human macrophages to pro-inflammatory cell death. Upon exposure to LPS, CS inhibits the TLR4/MyD88 inflammatory response, downregulating the pro-inflammatory genes TNF and IL6 and the anti-apoptotic gene CFLAR, known to counteract caspase-8 activity. CS enhances caspase-8 activation through TLR4/TRIF, with a partial involvement of RIPK1, resulting on the activation of caspase-1/GSDMD axis leading to increased cell permeability and DAMP release through gasdermin pores [19]. At later timepoints caspase-3 becomes strongly activated by caspase-8 triggering apoptotic events which are associated with mitochondrial membrane depolarization, gasdermin E cleavage and secondary necrosis with consequent massive DAMP release.

SARS-CoV-2, which causes COVID-19, has altered human activities all over the world and has become a global hazard to public health. Despite considerable advancements in pandemic containment techniques, in which vaccination played a key role, COVID-19 remains a global threat, particularly for frail patients and unvaccinated individuals, who may be more susceptible to developing ARDS. Several studies reported that patients with COVID-19-related ARDS who were treated with ECMO had a similar survival rate to those with COVID-19-unrelated ARDS. In order to shed light on the potential mechanisms underlying the COVID-19 infection, we conducted this proof-of-concept study using single-cell V(D)J and gene expression sequencing of B cells to examine the dynamic changes in the transcriptomic BCR repertoire present in patients with COVID-19 at various stages. We compared a recovered and a deceased COVID-19 patient supported by ECMO with one COVID-19-recovered patient who did not receive ECMO treatment and one healthy subject who had never been infected previously. Our analysis revealed a downregulation of FXYD, HLA-DRB1, and RPS20 in memory B cells; MTATP8 and HLA-DQA1 in naïve cells; RPS4Y1 in activated B cells; and IGHV3-73 in plasma cells in COVID-19 patients. We further described an increased ratio of IgA + IgG to IgD + IgM, suggestive of an intensive memory antibody response, in the COVID ECMO D patient. Finally, we assessed a V(D)J rearrangement of heavy chain IgHV3, IGHJ4, and IGHD3/IGHD2 families in COVID-19 patients regardless of the severity of the disease.

Mesenchymal stromal/stem cells (MSCs) have emerged as a therapeutic tool in regenerative medicine. Recent studies have shown that exosome (EXO)-derived microRNAs (miRNAs) play a crucial role in mediating MSC functions. Additionally, intracellular miRNAs have been found to regulate MSC therapeutic capacities. However, the molecular mechanisms underlying miRNA-mediated MSC effects are not fully understood. We used 3D culture and IFN-γ to prime/enhance the MSC therapeutic effects in terms of functional miRNAs. After priming, our analysis revealed stable variations in intracellular miRNA among the MSC biological replicates. Conversely, a significant variability of miRNA was observed among EXOs released from biological replicates of the priming treatment. For each priming, we observed distinct miRNA expression profiles between the MSCs and their EXOs. Moreover, in both types of priming, gene ontology (GO) analysis of deregulated miRNAs highlighted their involvement in tissue repair/regeneration pathways. In particular, the 3D culture enhanced angiogenic properties in both MSCs and EXOs, while IFN-γ treatment enriched miRNAs associated with immunomodulatory pathways. These findings suggest that 3D culture and IFN-γ treatment are promising strategies for enhancing the therapeutic potential of MSCs by modulating miRNA expression. Additionally, the identified miRNAs may contribute to understanding the molecular mechanisms underlying the miRNA-mediated therapeutic effects of MSCs.

Since its identification, HCV has been considered one of the main causes of hepatitis and liver cancer. Currently, the molecular mechanisms of HCC development induced by HCV infection have not been sufficiently clarified. The recent discovery of novel treatments that inhibit HCV replication gave rise to new questions concerning HCC mechanisms. In particular, the HCV eradication mediated by new direct-acting antiviral (DAAs) drugs does not exclude the possibility of de novo HCC development; this finding opened more questions on the interplay between liver cells and the virus. Different groups have investigated the pathways leading to cancer recurrence in patients treated with DAAs. For this reason, we tried to gain molecular insights into the changes induced by HCV infection in the target liver cells. In particular, we observed an increase in microRNA34a (miR34a) expression following HCV infection of HCC cell line Huh7.5. In addition, Huh7.5 treated with extracellular vesicles (EVs) from the previously HCV-infected Huh7.5 underwent apoptosis. Since miR34 expression was increased in Huh7.5 EVs, we hypothesized a paracrine mechanism of viral infection mediated by miR34a cargo of EVs. The balance between viral infection and cell transformation may raise some questions on the possible use of antiviral drugs in association with antineoplastic treatment.

Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and can be activated by several cytokines, including interleukin-2 (IL-2), IL-15, and interferon-alpha (IFN-α). By exploiting the Huh7.5 hepatoma cell line infected with the HCV JFH1 genome, we provide novel insights into the antiviral effector functions of human primary NK cells after cytokine stimulation. NK cells activated with IFN-α (IFNα-NKs) had enhanced contact-dependent and -independent responses as compared with NK cells activated with IL-2/IL-15 (IL2/IL15-NKs) and could inhibit HCV replication both in vitro and in vivo. Importantly, IFN-α, but not IL-2/IL-15, protected NK cells from the functional inhibition exerted by HCV. By performing flow cytometry, multiplex cytokine profiling, and mass-spectrometry-based proteomics, we discovered that IFNα-NKs secreted high levels of galectin-9 and interferon-gamma (IFN-γ), and by conducting neutralization assays, we confirmed the major role of these molecules in HCV suppression. We speculated that galectin-9 might act extracellularly to inhibit HCV binding to host cells and downstream infection. In silico approaches predicted the binding of HCV envelope protein E2 to galectin-9 carbohydrate-recognition domains, and co-immunoprecipitation assays confirmed physical interaction. IFN-γ, on the other hand, triggered the intracellular expressions of two antiviral gate-keepers in target cells, namely, myxovirus-1 (MX1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Collectively, our data add more complexity to the antiviral innate response mediated by NK cells and highlight galectin-9 as a key molecule that might be exploited to neutralize productive viral infection.

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma.

Human mesenchymal stromal/stem cells (MSCs), being immunoprivileged and having immunomodulatory ability, represent a promising tool to be applied in the field of regenerative medicine. Based on numerous in vitro evidences, the immunological effects of MSCs on immune cells could depend on different mechanisms as cell-to-cell contact and paracrine signals. Furthermore, recent studies have shown that the immunomodulatory activity of MSCs is initiated by activated immune cells; thus, their interaction represents a potential homeostatic mechanism by which MSCs regulate the immune response. MSCs also release exosomes able to give different effects, in a paracrine manner, by influencing inflammatory processes. In this study, we aimed to establish the potential role of human amnion-derived MSCs (hAMSCs), in immunomodulation. We found that the immunosuppressive properties of hAMSCs are not constitutive, but require “supportive signals” capable of promoting these properties. Indeed, we observed that hAMSCs alone are not able to produce an adequate amount of soluble immunomodulatory factors. Here, we studied, in depth, the strong immunomodulatory licensing signal deriving from the direct interaction between hAMSCs and stimulated peripheral blood mononuclear cells. We found that the immunomodulatory effect of hAMSCs also depends on cell-to-cell contact through the contribution of the PDL-1/PD-1 axis. We then investigated the IFN-γ priming of hAMSCs (γ-hAMSCs), which induce the increase of PDL-1 expression, high production of IDO, and upregulation of different immunomodulatory exosome-derived miRNAs. Our miRNA–target network analysis revealed that nine of the deregulated miRNAs are involved in the regulation of key proteins that control both T cell activation/anergy and monocyte differentiation pathways. Finally, we observed that γ-hAMSCs induce in monocytes both M2-like phenotype and the increase of IL-10 production. The extensive implications of MSCs in modulating different aspects of the immune system make these cells attractive candidates to be employed in therapeutic application in immune-based diseases. For these reasons, we aimed, with this study, to shed light on the potential of hAMSCs, and how they could become a useful tool for treating different inflammatory diseases, including end-stage pathologies or adverse effects in transplanted patients.

The use of cell secreted factors in clinical settings could be an alternative to conventional cell therapy, with the advantage of limiting concerns generally associated with traditional cell transplantation, such as tumorigenicity, immunoreactivity, and carrying of infections. Based on our published data, we predict a potential role for extracellular vesicles (EVs) in contributing to the proangiogenic activity of human fetal dermal cell secretome. Depletion of nanosized EVs from secretome significantly impaired its ability to induce formation of mesh-like structures in vitro. The isolated EVs were characterized for size and concentration by nanoparticle tracking analysis, and for protein markers (Rab5+, Alix+, CD63+, and calnexin-). The microRNA profile of EVs revealed 87 microRNAs significantly upregulated (≥15-fold increase) in fetal compared to adult dermal cell-derived EVs. Interestingly, these upregulated microRNAs included microRNAs with a validated role in angiogenesis according to literature. Moreover, the DIANA-TarBase v7.0 analysis confirmed enrichment in the KEGG signaling pathways associated with angiogenesis and wound healing, with the identification of putative target genes including thrombospondin 1. To validate the in silico data, EVs were also characterized for total protein contents. When tested in in vitro angiogenesis, fetal dermal cell-derived EVs were more effective than their adult counterpart in inducing formation of complete mesh-like structures. Furthermore, treatment of fibroblasts with fetal dermal-derived EVs determined a 4-fold increase of thrombospondin 1 protein amounts compared with the untreated fibroblasts. Finally, visualization of CSFE-labeled EVs in the cytosol of target cells suggested a successful uptake of these particles at 4-8 hours of incubation. We conclude that EVs are important contributors of the proangiogenic effect of fetal dermal cell secretome. Hence, EVs could also serve as vehicle for a successful delivery of microRNAs or other molecules of therapeutic interest to target cells.

Ischemia/reperfusion injury (IRI) represents one of the leading causes of primary non-function acute liver transplantation failure. IRI, generated by an interruption of organ blood flow and the subsequent restoration upon transplant, i.e., reperfusion, generates the activation of an inflammatory cascade from the resident Kupffer cells, leading first to neutrophils recruitment and second to apoptosis of the parenchyma. Recently, human mesenchymal stromal/stem cells (hMSCs) and derivatives have been implemented for reducing the damage induced by IRI. Interestingly, sparse data in the literature have described the use of human amnion-derived MSCs (hAMSCs) and, more importantly, no evidence regarding hMSCs priming on liver IRI have been described yet. Thus, our study focused on the definition of an in vitro model of liver IRI to test the effect of primed hAMSCs to reduce IRI damage on immune and hepatic cells. We found that the IFNγ pre-treatment and 3D culture of hAMSCs strongly reduced inflammation induced by M1-differentiated macrophages. Furthermore, primed hAMSCs significantly inhibited parenchymal apoptosis at early timepoints of reperfusion by blocking the activation of caspase 3/7. All together, these data demonstrate that hAMSCs priming significantly overcomes IRI effects in vitro by engaging the possibility of defining the molecular pathways involved in this process.

The use of extracorporeal membrane oxygenation (ECMO) for acute respiratory distress syndrome (ARDS) has increased in the last decade. However, mortality remains high, and the complexity of ECMO requires individualized treatment. There are some biomarkers to monitor progression and predict clinical outcomes of ARDS. This project aims to advance the management of ARDS patients treated with ECMO by exploring miRNA expression in whole blood. The analysis was conducted on two groups with different length of ECMO: Group A (longer runs) and group B (shorter runs). We analyzed miRNAs before ECMO cannulation, and at 7 and 14 days of ECMO support. Our results showed that in the group B patients, 11 deregulated miRNAs were identified, and showed an opposite trend of expression compared to the group A patients. In silico analysis revealed that these 11 miRNAs were related to processes involved in the pathogenesis and evolution of ARDS. This scenario could represent homeostatic mechanisms by which, in ECMO responsive patients, pathways activated during ARDS progression are switched-off. Circulating miRNAs could represent promising biomarkers to monitor the evolution of ARDS under ECMO support. Further studies may shed light on this topic to improve a personalized approach in such a complex setting of patients.

Currently, Klebsiella pneumoniae is a pathogen of clinical relevance due to its plastic ability of acquiring resistance genes to multiple antibiotics. During K. pneumoniae infections, lipopolysaccharides (LPS) play an ambiguous role as they both activate immune responses but can also play a role in immune evasion. The LPS O2a and LPS O2afg serotypes are prevalent in most multidrug resistant K. pneumoniae strains. Thus, we sought to understand if those two particular LPS serotypes were involved in a mechanism of immune evasion. We have extracted LPS (serotypes O1, O2a and O2afg) from K. pneumoniae strains and, using human monocytes ex vivo, we assessed the ability of those LPS antigens to induce the production of pro-inflammatory cytokines and chemokines. We observed that, when human monocytes are incubated with LPS serotypes O1, O2a or O2afg strains, O2afg and, to a lesser extent, O2a but not O1 failed to elicit the production of pro-inflammatory cytokines and chemokines, which suggests a role in immune evasion. Our preliminary data also shows that nuclear translocation of NF-κB, a process which regulates an immune response against infections, occurs in monocytes incubated with LPS O1 and, to a smaller extent, with LPS O2a, but not with the LPS serotype O2afg. Our results indicate that multidrug resistant K. pneumoniae expressing LPS O2afg serotypes avoid an initial inflammatory immune response and, consequently, are able to systematically spread inside the host unharmed, which results in the several pathologies associated with this bacterium.