Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

Oxidative Stress by the Mitochondrial Monoamine Oxidase B Mediates Calcium Pyrophosphate Crystal-Induced Arthritis.

OBJECTIVE: Calcium pyrophosphate (CPP) crystal deposition in the joints is associated with a heterogeneous set of debilitating syndromes characterized by inflammation and pain, for which no effective therapies are currently available. Because we found that the mitochondrial enzyme monoamine oxidase B (MAO-B) plays a fundamental role in promoting inflammatory pathways, this study aims at assessing the efficacy of two clinical-grade inhibitors (iMAO-Bs) in preclinical models of this disease to pave the way for a novel treatment. METHODS: We tested our hypothesis in two murine models of CPP-induced arthritis, by measuring cytokine and chemokine levels, along with immune cell recruitment. iMAO-Bs (rasagiline and safinamide) were administered either before or after crystal injection. To elucidate the molecular mechanism, we challenged in vitro primed macrophages with CPP crystals and assessed the impact of iMAO-Bs in dampening proinflammatory cytokines and in preserving mitochondrial function. RESULTS: Both in preventive and therapeutic in vivo protocols, iMAO-Bs blunted the release of proinflammatory cytokines (interleukin [IL]-6 and IL1-ß) and chemokines (CXCL10, CXCL1, CCL2 and CCL5) (n > 6 mice/group). Importantly, they also significantly reduced ankle swelling (50.3% vs 17.1%; P < 0.001 and 23.1%; P = 0.005 for rasagiline and safinamide, respectively). Mechanistically, iMAO-Bs dampened the burst of reactive oxygen species and the mitochondrial dysfunction triggered by CPP crystals in isolated macrophages. Moreover, iMAO-Bs blunted cytokine secretion and NLRP3 inflammasome activation through inhibition of the NF-κB and STAT3 pathways. CONCLUSION: iMAO-Bs dampen inflammation in murine models of crystal-induced arthropathy, thereby uncovering MAO-B as a promising target to treat these diseases.

RAGE engagement by SARS-CoV-2 enables monocyte infection and underlies COVID-19 severity.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has fueled the COVID-19 pandemic with its enduring medical and socioeconomic challenges because of subsequent waves and long-term consequences of great concern. Here, we chart the molecular basis of COVID-19 pathogenesis by analyzing patients' immune responses at single-cell resolution across disease course and severity. This approach confirms cell subpopulation-specific dysregulation in COVID-19 across disease course and severity and identifies a severity-associated activation of the receptor for advanced glycation endproducts (RAGE) pathway in monocytes. In vitro THP1-based experiments indicate that monocytes bind the SARS-CoV-2 S1-receptor binding domain (RBD) via RAGE, pointing to RAGE-Spike interaction enabling monocyte infection. Thus, our results demonstrate that RAGE is a functional receptor of SARS-CoV-2 contributing to COVID-19 severity.

OPA1 drives macrophage metabolism and functional commitment via p65 signaling.

Macrophages are essential players for the host response against pathogens, regulation of inflammation and tissue regeneration. The wide range of macrophage functions rely on their heterogeneity and plasticity that enable a dynamic adaptation of their responses according to the surrounding environmental cues. Recent studies suggest that metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the metabolic pathways orchestrating macrophage activation are still under scrutiny. Optic atrophy 1 (OPA1) is a mitochondria-shaping protein controlling mitochondrial fusion, cristae biogenesis and respiration; clear evidence shows that the lack or dysfunctional activity of this protein triggers the accumulation of metabolic intermediates of the TCA cycle. In this study, we show that OPA1 has a crucial role in macrophage activation. Selective Opa1 deletion in myeloid cells impairs M1-macrophage commitment. Mechanistically, Opa1 deletion leads to TCA cycle metabolite accumulation and defective NF-κB signaling activation. In an in vivo model of muscle regeneration upon injury, Opa1 knockout macrophages persist within the damaged tissue, leading to excess collagen deposition and impairment in muscle regeneration. Collectively, our data indicate that OPA1 is a key metabolic driver of macrophage functions.

Psoriasis and Cardiovascular Diseases: An Immune-Mediated Cross Talk?

Psoriasis is a chronic immune-mediated inflammatory skin disease, characterized by well-demarcated scaly, erythematous, infiltrated plaques. The cutaneous-to-systemic expansion of the inflammation in psoriasis leads to the concept of "psoriatic march" or "inflammatory skin march". Accordingly, psoriasis is thought to be a systemic inflammatory disease associated with numerous comorbidities. Indeed, it's currently considered an independent risk factor for cardiovascular diseases. Here, we discuss the current knowledge on TNF-α and IL-23/IL-17 mediated pathways linking the psoriatic plaque to the cardiovascular compartment. We further argue the possible involvement of the endothelial compartment in the psoriatic plaque- cardiovascular system crosstalk.

COVID-19 Vaccination Limits Systemic Danger Signals in SARS-CoV-2 Infected Patients.

Vaccination with an mRNA COVID-19 vaccine determines not only a consistent reduction in the risk of SARS-CoV-2 infection but also contributes to disease attenuation in infected people. Of note, hyperinflammation and damage-associated molecular patterns (DAMPs) have been clearly associated with severe illness and poor prognosis in COVID-19 patients. In this report, we revealed a significant reduction in the levels of IL-1ß and DAMPs molecules, as S100A8 and High Mobility Group Protein B1 (HMGB1), in vaccinated patients as compared to non-vaccinated ones. COVID-19 vaccination indeed prevents severe clinical manifestations in patients and limits the release of systemic danger signals in SARS-CoV-2 infected people.

IL1ß Promotes TMPRSS2 Expression and SARS-CoV-2 Cell Entry Through the p38 MAPK-GATA2 Axis.

After the outburst of the SARS-CoV-2 pandemic, a worldwide research effort has led to the uncovering of many aspects of the COVID-19, among which we can count the outstanding role played by inflammatory cytokine milieu in the disease progression. Despite that, molecular mechanisms that regulate SARS-CoV-2 pathogenesis are still almost unidentified. In this study, we investigated whether the pro-inflammatory milieu of the host affects the susceptibility of SARS-CoV-2 infection by modulating ACE2 and TMPRSS2 expression. Our results indicated that the host inflammatory milieu favors SARS-CoV-2 infection by directly increasing TMPRSS2 expression. We unveiled the molecular mechanism that regulates this process and that can be therapeutically advantageously targeted.

The J2-Immortalized Murine Macrophage Cell Line Displays Phenotypical and Metabolic Features of Primary BMDMs in Their M1 and M2 Polarization State.

Macrophages are immune cells that are important for the development of the defensive front line of the innate immune system. Following signal recognition, macrophages undergo activation toward specific functional states, consisting not only in the acquisition of specific features but also of peculiar metabolic programs associated with each function. For these reasons, macrophages are often isolated from mice to perform cellular assays to study the mechanisms mediating immune cell activation. This requires expensive and time-consuming breeding and housing of mice strains. To overcome this issue, we analyzed an in-house J2-generated immortalized macrophage cell line from BMDMs, both from a functional and metabolic point of view. By assaying the intracellular and extracellular metabolism coupled with the phenotypic features of immortalized versus primary BMDMs, we concluded that classically and alternatively immortalized macrophages display similar phenotypical, metabolic and functional features compared to primary cells polarized in the same way. Our study validates the use of this immortalized cell line as a suitable model with which to evaluate in vitro how perturbations can influence the phenotypical and functional features of murine macrophages.

Arrhythmogenic Cardiomyopathy Is a Multicellular Disease Affecting Cardiac and Bone Marrow Mesenchymal Stromal Cells.

Arrhythmogenic cardiomyopathy (AC) is a familial cardiac disorder at high risk of arrhythmic sudden death in the young and athletes. AC is hallmarked by myocardial replacement with fibro-fatty tissue, favoring life-threatening cardiac arrhythmias and contractile dysfunction. The AC pathogenesis is unclear, and the disease urgently needs mechanism-driven therapies. Current AC research is mainly focused on 'desmosome-carrying' cardiomyocytes, but desmosomal proteins are also expressed by non-myocyte cells, which also harbor AC variants, including mesenchymal stromal cells (MSCs). Consistently, cardiac-MSCs contribute to adipose tissue in human AC hearts. We thus approached AC as a multicellular disorder, hypothesizing that it also affects extra-cardiac bone marrow (BM)-MSCs. Our results show changes in the desmosomal protein profile of both cardiac- and BM- MSCs, from desmoglein-2 (Dsg2)-mutant mice, accompanied with profound alterations in cytoskeletal organization, which are directly caused by AC-linked DSG2 downregulation. In addition, AC BM-MSCs display increased proliferation rate, both in vitro and in vivo, and, by using the principle of the competition homing assay, we demonstrated that mutant circulating BM-MSCs have increased propensity to migrate to the AC heart. Taken altogether, our results indicate that cardiac- and BM- MSCs are additional cell types affected in Dsg2-linked AC, warranting the novel classification of AC as a multicellular and multiorgan disease.

TGF-ß in Cancer: Metabolic Driver of the Tolerogenic Crosstalk in the Tumor Microenvironment.

Overcoming tumor immunosuppression still represents one ambitious achievement for cancer immunotherapy. Of note, the cytokine TGF-ß contributes to immune evasion in multiple cancer types, by feeding the establishment of a tolerogenic environment in the host. Indeed, it fosters the expansion and accumulation of immunosuppressive regulatory cell populations within the tumor microenvironment (TME), where it also activates resident stromal cells and enhances angiogenesis programs. More recently, TGF-ß has also turned out as a key metabolic adjuster in tumors orchestrating metabolic pathways in the TME. In this review, we will scrutinize TGF-ß-mediated immune and stromal cell crosstalk within the TME, with a primary focus on metabolic programs.

Administration of Human MSC-Derived Extracellular Vesicles for the Treatment of Primary Sclerosing Cholangitis: Preclinical Data in MDR2 Knockout Mice.

Primary Sclerosing Cholangitis (PSC) is a progressive liver disease for which there is no effective medical therapy. PSC belongs to the family of immune-mediated biliary disorders and it is characterized by persistent biliary inflammation and fibrosis. Here, we explored the possibility of using extracellular vesicles (EVs) derived from human, bone marrow mesenchymal stromal cells (MSCs) to target liver inflammation and reduce fibrosis in a mouse model of PSC. Five-week-old male FVB.129P2-Abcb4tm1Bor mice were intraperitoneally injected with either 100 µL of EVs (± 9.1 × 109 particles/mL) or PBS, once a week, for three consecutive weeks. One week after the last injection, mice were sacrificed and liver and blood collected for flow cytometry analysis and transaminase quantification. In FVB.129P2-Abcb4tm1Bor mice, EV administration resulted in reduced serum levels of alkaline phosphatase (ALP), bile acid (BA), and alanine aminotransferase (ALT), as well as in decreased liver fibrosis. Mechanistically, we observed that EVs reduce liver accumulation of both granulocytes and T cells and dampen VCAM-1 expression. Further analysis revealed that the therapeutic effect of EVs is accompanied by the inhibition of NFkB activation in proximity of the portal triad. Our pre-clinical experiments suggest that EVs isolated from MSCs may represent an effective therapeutic strategy to treat patients suffering from PSC.

Age-severity matched cytokine profiling reveals specific signatures in Covid-19 patients.

A global effort is currently undertaken to restrain the COVID-19 pandemic. Host immunity has come out as a determinant for COVID-19 clinical outcomes, and several studies investigated the immune profiling of SARS-CoV-2 infected people to properly direct the clinical management of the disease. Thus, lymphopenia, T-cell exhaustion, and the increased levels of inflammatory mediators have been described in COVID-19 patients, in particular in severe cases1. Age represents a key factor in COVID-19 morbidity and mortality2. Understanding age-associated immune signatures of patients are therefore important to identify preventive and therapeutic strategies. In this study, we investigated the immune profile of COVID-19 hospitalized patients identifying a distinctive age-dependent immune signature associated with disease severity. Indeed, defined circulating factors - CXCL8, IL-10, IL-15, IL-27, and TNF-α - positively correlate with older age, longer hospitalization, and a more severe form of the disease and may thus represent the leading signature in critical COVID-19 patients.

CD73+ extracellular vesicles inhibit angiogenesis through adenosine A2B receptor signalling.

Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A2B adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression in vivo. Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis.

Correction: Marcuzzi, E., et al. Chemokines and Chemokine Receptors: Orchestrating Tumor Metastasization. Int. J. Mol. Sci. 2019, 20, 96.

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Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis.

Extracellular ATP is a signaling molecule exploited by the immune cells for both autocrine regulation and paracrine communication. By performing live calcium imaging experiments, we show that triggered mouse macrophages are able to propagate calcium signals to resting bystander cells by releasing ATP. ATP-based intercellular communication is mediated by P2X4 and P2X7 receptors and is a feature of pro-inflammatory macrophages. In terms of functional significance, ATP signaling is required for efficient phagocytosis of pathogen-derived molecules and apoptotic cells and may represent a target for macrophage regulation by CD39-expressing cells. These results highlight a cell-to-cell communication mechanism tuning innate immunity.

Chemokines and Chemokine Receptors: Orchestrating Tumor Metastasization.

Metastasis still represents the primary cause of cancer morbidity and mortality worldwide. Chemokine signalling contributes to the overall process of cancer growth and metastasis, and their expression in both primary tumors and metastatic lesions correlate with prognosis. Chemokines promote tumor metastasization by directly supporting cancer cell survival and invasion, angiogenesis, and by indirectly shaping the pre-metastatic niches and antitumor immunity. Here, we will focus on the relevant chemokine/chemokine receptor axes that have been described to drive the metastatic process. We elaborate on their role in the regulation of tumor angiogenesis and immune cell recruitment at both the primary tumor lesions and the pre-metastatic foci. Furthermore, we also discuss the advantages and limits of current pharmacological strategies developed to target chemokine networks for cancer therapy.

Proteomic analysis of the secretome of human bone marrow-derived mesenchymal stem cells primed by pro-inflammatory cytokines.

Mesenchymal stem cells (MSC) represent an impressive opportunity in term of regenerative medicine and immunosuppressive therapy. Although it is clear that upon transplantation MSC exert most of their therapeutic effects through the secretion of bioactive molecules, the effects of a pro-inflammatory recipient environment on MSC secretome have not been characterized. In this study, we used a label free mass spectrometry based quantitative proteomic approach to analyze how pro-inflammatory cytokines modulate the composition of the human MSC secretome. We found that pro-inflammatory cytokines have a strong impact on the secretome of human bone marrow-derived MSC and that the large majority of cytokine-induced proteins are involved in inflammation and/or angiogenesis. Comparative analyses with results recently obtained on mouse MSC secretome stimulated under the same conditions reveals both analogies and differences in the effect of pro-inflammatory cytokines on MSC secretome in the two organisms. In particular, functional analyses confirmed that tissue inhibitor of metalloproteinase-1 (TIMP1) is a key effector molecule responsible for the anti-angiogenic properties of both human and mouse MSC within an inflammatory microenvironment. Mass spectrometry data are available via ProteomeXchange with identifier PXD005746 SIGNIFICANCE: The secretion of a broad range of bioactive molecules is believed to be the main mechanism by which MSC exert specific therapeutic effects. MSC are very versatile and respond to specific environments by producing and releasing a variety of effector molecules. To the best of our knowledge this is the first study aimed at describing the secretome of human MSC primed using a mixture of cytokines, to mimic pro-inflammatory conditions encountered in vivo, by a quantitative high-resolution mass spectrometry based approach. The main output of the study concerns the identification of a list of specific proteins involved in inflammation and angiogenesis which are overrepresented in stimulated MSC secretome. The data complement a previous study on the secretome of mouse MSC stimulated under the same conditions. Comparative analyses reveal analogies and differences in the biological processes affected by overrepresented proteins in the two organisms. In particular, the key role of TIMP-1 for the anti-angiogenic properties of stimulated MSC secretome already observed in mouse is confirmed in human. Overall, these studies represent key steps necessary to characterize the different biology of MSC in the two organisms and design successful pre-clinical experiments as well as clinical trials.

Tailored chemokine receptor modification improves homing of adoptive therapy T cells in a spontaneous tumor model.

In recent years, tumor Adoptive Cell Therapy (ACT), using administration of ex vivo-enhanced T cells from the cancer patient, has become a promising therapeutic strategy. However, efficient homing of the anti-tumoral T cells to the tumor or metastatic site still remains a substantial hurdle. Yet the tumor site itself attracts both tumor-promoting and anti-tumoral immune cell populations through the secretion of chemokines. We attempted to identify these chemokines in a model of spontaneous metastasis, in order to "hijack" their function by expressing matching chemokine receptors on the cytotoxic T cells used in ACT, thus allowing us to enhance the recruitment of these therapeutic cells. Here we show that this enabled the modified T cells to preferentially home into spontaneous lymph node metastases in the TRAMP model, as well as in an inducible tumor model, E.G7-OVA. Due to the improved homing, the modified CD8+ T cells displayed an enhanced in vivo protective effect, as seen by a significant delay in E.G7-OVA tumor growth. These results offer a proof of principle for the tailored application of chemokine receptor modification as a means of improving T cell homing to the target tumor, thus enhancing ACT efficacy. Surprisingly, we also uncover that the formation of the peri-tumoral fibrotic capsule, which has been shown to impede T cell access to tumor, is partially dependent on host T cell presence. This finding, which would be impossible to observe in immunodeficient model studies, highlights possible conflicting roles that T cells may play in a therapeutic context.