Doxorubicin-Induced Cardiac Senescence Is Alleviated Following Treatment with Combined Polyphenols and Micronutrients through Enhancement in Mitophagy.

Oxidative stress and impaired mitophagy are the hallmarks of cardiomyocyte senescence. Specifically, a decrease in mitophagic flux leads to the accumulation of damaged mitochondria and the development of senescence through increased ROS and other mediators. In this study, we describe the preventive role of A5+, a mix of polyphenols and other micronutrients, in doxorubicin (DOXO)-induced senescence of H9C2 cells. Specifically, H9C2 cells exposed to DOXO showed an increase in the protein expression proteins of senescence-associated genes, p21 and p16, and a decrease in the telomere binding factors TRF1 and TRF2, indicative of senescence induction. Nevertheless, A5+ pre-treatment attenuated the senescent-like cell phenotype, as evidenced by inhibition of all senescent markers and a decrease in SA-ß-gal staining in DOXO-treated H9C2 cells. Importantly, A5+ restored the LC3 II/LC3 I ratio, Parkin and BNIP3 expression, therefore rescuing mitophagy, and decreased ROS production. Further, A5+ pre-treatment determined a ripolarization of the mitochondrial membrane and improved basal respiration. A5+-mediated protective effects might be related to its ability to activate mitochondrial SIRT3 in synergy with other micronutrients, but in contrast with SIRT4 activation. Accordingly, SIRT4 knockdown in H9C2 cells further increased MnSOD activity, enhanced mitophagy, and reduced ROS generation following A5+ pre-treatment and DOXO exposure compared to WT cells. Indeed, we demonstrated that A5+ protects H9C2 cells from DOXO-induced senescence, establishing a new specific role for A5+ in controlling mitochondrial quality control by restoring SIRT3 activity and mitophagy, which provided a molecular basis for the development of therapeutic strategies against cardiomyocyte senescence.

The Shigella flexneri virulence factor apyrase is released inside eukaryotic cells to hijack host cell fate.

IMPORTANCE: In this paper, we demonstrated that apyrase is released within the host cell cytoplasm during infection to target the intracellular ATP pool. By degrading intracellular ATP, apyrase contributes to prevent caspases activation, thereby inhibiting the activation of pyroptosis in infected cells. Our results show, for the first time, that apyrase is involved in the modulation of host cell survival, thereby aiding this pathogen to dampen the inflammatory response. This work adds a further piece to the puzzle of Shigella pathogenesis. Due to its increased spread worldwide, prevention and controlling strategies are urgently needed. Overall, this study highlighted apyrase as a suitable target for an anti-virulence therapy to tackle this pathogen.

SIRT5 Activation and Inorganic Phosphate Binding Reduce Cancer Cell Vitality by Modulating Autophagy/Mitophagy and ROS.

Cancer cells show increased glutamine consumption. The glutaminase (GLS) enzyme controls a limiting step in glutamine catabolism. Breast tumors, especially the triple-negative subtype, have a high expression of GLS. Our recent study demonstrated that GLS activity and ammonia production are inhibited by sirtuin 5 (SIRT5). We developed MC3138, a selective SIRT5 activator. Treatment with MC3138 mimicked the deacetylation effect mediated by SIRT5 overexpression. Moreover, GLS activity was regulated by inorganic phosphate (Pi). Considering the interconnected roles of GLS, SIRT5 and Pi in cancer growth, our hypothesis is that activation of SIRT5 and reduction in Pi could represent a valid antitumoral strategy. Treating cells with MC3138 and lanthanum acetate, a Pi chelator, decreased cell viability and clonogenicity. We also observed a modulation of MAP1LC3B and ULK1 with MC3138 and lanthanum acetate. Interestingly, inhibition of the mitophagy marker BNIP3 was observed only in the presence of MC3138. Autophagy and mitophagy modulation were accompanied by an increase in cytosolic and mitochondrial reactive oxygen species (ROS). In conclusion, our results show how SIRT5 activation and/or Pi binding can represent a valid strategy to inhibit cell proliferation by reducing glutamine metabolism and mitophagy, leading to a deleterious accumulation of ROS.

An Observational Study on Chronic Pain Biomarkers in Fibromyalgia and Osteoarthritis Patients: Which Role for Mu Opioid Receptor's Expression on NK Cells?

The evaluation of chronic pain is challenging because of the lack of specific biomarkers. We identified the Mu opioid receptor-positive (Mu+) B cell percentage of expression, named Mu-Lympho-Marker (MLM), as a candidate marker for chronic pain in fibromyalgia (FM) and osteoarthritis (OA) patients. Here, we investigate the role of MLM on natural killer (NK) cells in the same patients. Twenty-nine FM and twelve OA patients were analyzed, and twenty-three pain-free subjects were considered as the control group. Blood samples were collected to perform immunophenotyping and Western blot analysis. Biological and clinical data were statistically analyzed. The final results showed that the percentage of NK cells expressing Mu was statistically lower in FM and OA patients than in pain-free subjects, as already demonstrated for B cells. A Western blot analysis was performed in order to detect NK cells' functional status. Moreover, the correlation analysis of MLM expression with pharmacological therapy did not show any significant results. In conclusion, here, we confirm the role of MLM as a suitable marker for chronic pain and underline NK cells as a new possible immune cell type involved in the "Mu opioid receptor reserve theory".

Nicotine in Combination with SARS-CoV-2 Affects Cells Viability, Inflammatory Response and Ultrastructural Integrity.

The aims of our study are to: (i) investigate the ability of nicotine to modulate the expression level of inflammatory cytokines in A549 cells infected with SARS-CoV-2; (ii) elucidate the ultrastructural features caused by the combination nicotine+SARS-CoV-2; and (iii) demonstrate the mechanism of action. In this study, A549 cells pretreated with nicotine were either exposed to LPS or poly(I:C), or infected with SARS-CoV-2. Treated and untreated cells were analyzed for cytokine production, cytotoxicity, and ultrastructural modifications. Vero E6 cells were used as a positive reference. Cells pretreated with nicotine showed a decrease of IL6 and TNFα in A549 cells induced by LPS or poly(I:C). In contrast, cells exposed to SARS-CoV-2 showed a high increase of IL6, IL8, IL10 and TNFα, high cytopathic effects that were dose- and time-dependent, and profound ultrastructural modifications. These modifications were characterized by membrane ruptures and fragmentation, the swelling of cytosol and mitochondria, the release of cytoplasmic content in extracellular spaces (including osmiophilic granules), the fragmentation of endoplasmic reticulum, and chromatin disorganization. Nicotine increased SARS-CoV-2 cytopathic effects, elevating the levels of inflammatory cytokines, and inducing severe cellular damage, with features resembling pyroptosis and necroptosis. The protective role of nicotine in COVID-19 is definitively ruled out.

Altered Tregs Differentiation and Impaired Autophagy Correlate to Atherosclerotic Disease.

Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and mortality in developed countries. Formerly, atherosclerosis was considered as a mere passive accumulation of lipids in blood vessels. However, it is now clear that atherosclerosis is a complex and multifactorial disease, in which the involvement of immune cells and inflammation play a key role. A variety of studies have shown that autophagy-a cellular catalytic mechanism able to remove injured cytoplasmic components in response to cellular stress-may be proatherogenic. So far, in this context, its role has been investigated in smooth muscle cells, macrophages, and endothelial cells, while the function of this catabolic protective process in lymphocyte functionality has been overlooked. The few studies carried out so far, however, suggested that autophagy modulation in lymphocyte subsets may be functionally related to plaque formation and development. Therefore, in this research, we aimed at better clarifying the role of lymphocyte subsets, mainly regulatory T cells (Tregs), in human atherosclerotic plaques and in animal models of atherosclerosis investigating the contribution of autophagy on immune cell homeostasis. Here, we investigate basal autophagy in a mouse model of atherosclerosis, apolipoprotein E (ApoE)-knockout (KO) mice, and we analyze the role of autophagy in driving Tregs polarization. We observed defective maturation of Tregs from ApoE-KO mice in response to tumor growth factor-ß (TGFß). TGFß is a well-known autophagy inducer, and Tregs maturation defects in ApoE-KO mice seem to be related to autophagy impairment. In this work, we propose that autophagy underlies Tregs maturation, advocating that the study of this process in atherosclerosis may open new therapeutic strategies.

Identification of MOR-Positive B Cell as Possible Innovative Biomarker (Mu Lympho-Marker) for Chronic Pain Diagnosis in Patients with Fibromyalgia and Osteoarthritis Diseases.

Fibromyalgia (FM) diagnosis follows the American College of Rheumatology (ACR) criteria, based on clinical evaluation and written questionnaires without any objective diagnostic tool. The lack of specific biomarkers is a tragic aspect for FM and chronic pain diseases in general. Interestingly, the endogenous opioid system is close to the immune one because of the expression of opioid receptors on lymphocytes membrane. Here we analyzed the role of the Mu opioid receptor on B lymphocytes as a specific biomarker for FM and osteoarthritis (OA) patients. We enrolled three groups of females: FM patients, OA patients (chronic pain control group) and healthy subjects (pain-free negative control group). We collected blood samples to apply immunophenotyping analysis. Written tests were administrated for psychological analysis. Data were statistically analyzed. Final results showed that the percentage of Mu-positive B cells were statistically lower in FM and OA patients than in pain-free subjects. A low expression of Mu-positive B cell was not associated with the psychological characteristics investigated. In conclusion, here we propose the percentage of Mu-positive B cells as a biological marker for an objective diagnosis of chronic pain suffering patients, also contributing to the legitimacy of FM as a truly painful disease.

Biomarkers of DNA damage in COPD patients undergoing pulmonary rehabilitation: Integrating clinical parameters with genomic profiling.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by severe respiratory symptoms. COPD shows several hallmarks of aging, and an increased oxidative stress, which is responsible for different clinical and molecular COPD features, including an increased frequency of DNA damage. The current pharmacological treatment options for COPD are mostly symptomatic, and generally do not influence disease progression and survival. In this framework, pulmonary rehabilitation is the most effective therapeutic strategy to improve physical performance, reducing hospital readmissions and mortality. Response to rehabilitation may greatly differ among patients calling for a personalized treatment. In this paper we will investigate in a group of COPD patients those variables that may predict the response to a program of pulmonary rehabilitation, integrating clinical parameters with cellular and molecular measurements, offering the potential for more effective and individualized treatment options. A group of 89 consecutive COPD patients admitted to a 3-weeks Pulmonary Rehabilitation (PR) program were evaluated for clinical and biological parameters at baseline and after completion of PR. DNA fragmentation in cryopreserved lymphocytes was compared by visual scoring and using the Comet Assay IV analysis system. The comparison of DNA damage before and after PR showed a highly significant increase from 19.6 ± 7.3 at admission to 21.8 ± 7.2 after three weeks of treatment, with a significant increase of 2.46 points (p < 0.001). Higher levels of DNA damage were observed in the group of non- responders and in those patients receiving oxygen therapy. The overall variation of %TI during treatment significantly correlated with the level of pCO2 at admission and negatively with the level of IL-6 at admission. Measuring the frequency of DNA damage in COPD patients undergoing pulmonary rehabilitation may provide a meaningful biological marker of response and should be considered as additional diagnostic and prognostic criterion for personalized rehabilitation programs.

The Role of Metabolic Remodeling in Macrophage Polarization and Its Effect on Skeletal Muscle Regeneration.

Significance: Macrophages are crucial for tissue homeostasis. Based on their activation, they might display classical/M1 or alternative/M2 phenotypes. M1 macrophages produce pro-inflammatory cytokines, reactive oxygen species (ROS), and nitric oxide (NO). M2 macrophages upregulate arginase-1 and reduce NO and ROS levels; they also release anti-inflammatory cytokines, growth factors, and polyamines, thus promoting angiogenesis and tissue healing. Moreover, M1 and M2 display key metabolic differences; M1 polarization is characterized by an enhancement in glycolysis and in the pentose phosphate pathway (PPP) along with a decreased oxidative phosphorylation (OxPhos), whereas M2 are characterized by an efficient OxPhos and reduced PPP. Recent Advances: The glutamine-related metabolism has been discovered as crucial for M2 polarization. Vice versa, flux discontinuities in the Krebs cycle are considered additional M1 features; they lead to increased levels of immunoresponsive gene 1 and itaconic acid, to isocitrate dehydrogenase 1-downregulation and to succinate, citrate, and isocitrate over-expression. Critical Issues: A macrophage classification problem, particularly in vivo, originating from a gap in the knowledge of the several intermediate polarization statuses between the M1 and M2 extremes, characterizes this field. Moreover, the detailed features of metabolic reprogramming crucial for macrophage polarization are largely unknown; in particular, the role of ß-oxidation is highly controversial. Future Directions: Manipulating the metabolism to redirect macrophage polarization might be useful in various pathologies, including an efficient skeletal muscle regeneration. Unraveling the complexity pertaining to metabolic signatures that are specific for the different macrophage subsets is crucial for identifying new compounds that are able to trigger macrophage polarization and that might be used for therapeutical purposes.

Modulating the metabolism by trimetazidine enhances myoblast differentiation and promotes myogenesis in cachectic tumor-bearing c26 mice.

Trimetazidine (TMZ) is a metabolic reprogramming agent able to partially inhibit mitochondrial free fatty acid ß-oxidation while enhancing glucose oxidation. Here we have found that the metabolic shift driven by TMZ enhances the myogenic potential of skeletal muscle progenitor cells leading to MyoD, Myogenin, Desmin and the slow isoforms of troponin C and I over-expression. Moreover, similarly to exercise, TMZ stimulates the phosphorylation of the AMP-activated protein kinase (AMPK) and up-regulates the peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), both of which are known to enhance the mitochondrial biogenesis necessary for myoblast differentiation. TMZ also induces autophagy which is required during myoblast differentiation and promotes myoblast alignment which allows cell fusion and myofiber formation. Finally, we found that intraperitoneally administered TMZ (5mg/kg) is able to stimulate myogenesis in vivo both in a mice model of cancer cachexia (C26 mice) and upon cardiotoxin damage. Collectively, our work demonstrates that TMZ enhances myoblast differentiation and promotes myogenesis, which might contribute recovering stem cell blunted regenerative capacity and counteracting muscle wasting, thanks to the formation of new myofibers; TMZ is already in use in humans as an anti-anginal drug and its repositioning might impact significantly on aging and regeneration-impaired disorders, including cancer cachexia, as well as have implications in regenerative medicine.

In vitro interaction of Stenotrophomonas maltophilia with human monocyte-derived dendritic cells.

Stenotrophomonas maltophilia is increasingly identified as an opportunistic pathogen in immunocompromised, cancer and cystic fibrosis (CF) patients. Knowledge on innate immune responses to S. maltophilia and its potential modulation is poor. The present work investigated the ability of 12 clinical S. maltophilia strains (five from CF patients, seven from non-CF patients) and one environmental strain to survive inside human monocyte-derived dendritic cells (DCs). The effects of the bacteria on maturation of and cytokine secretion by DCs were also measured. S. maltophilia strains presented a high degree of heterogeneity in internalization and intracellular replication efficiencies as well as in the ability of S. maltophilia to interfere with normal DCs maturation. By contrast, all S. maltophilia strains were able to activate DCs, as measured by increase in the expression of surface maturation markers and proinflammatory cytokines secretion.

Purinergic signaling in atherosclerosis.

Cell surface expression of specific receptors and ecto-nucleotidases makes extracellular nucleotides such as ATP, ADP, UTP, and adenosine suitable as signaling molecules for physiological and pathological events, including tissue stress and damage. Recent data have revealed the participation of purinergic signaling in atherosclerosis, depicting a scenario in which, in addition to some exceptions reflecting dual effects of individual receptor subtypes, adenosine and most P1 receptors, as well as ecto-nucleotidases, show a protective, anti-atherosclerotic function. By contrast, P2 receptors promote atherosclerosis. In consideration of these findings, modulation of purinergic signaling would represent an innovative and valuable tool to counteract atherosclerosis. We summarize recent developments on the participation of the purinergic network in atheroma formation and evolution.

Immunoregulation through extracellular nucleotides.

Extracellular ATP (eATP), the most abundant among nucleotides, can act as a mediator during inflammatory responses by binding to plasmamembrane P2 purinergic receptors, which are widely expressed on cells of the immune system. eATP is generally considered as a classical danger signal, which stimulates immune responses in the presence of tissue damage. Converging evidence from several studies using murine models of chronic inflammation have supported this hypothesis; however, the role of eATP in the regulation of human immune function appears to be more complex. Chronic stimulation with micromolar eATP concentrations inhibits the proliferation of T and NK lymphocytes and enhances the capacity of dendritic cells to promote tolerance. The effect of eATP depends on multiple factors, such as the extent of stimulation, eATP concentration, presence/absence of other mediators in the microenvironment, and pattern of P2 receptor engagement. Small but significant differences in the pattern of P2 receptor expression in mice and humans confer the diverse capacities of ATP in regulating the immune response. Such diversity, which is often overlooked, should therefore be carefully considered when evaluating the role of eATP in human inflammatory and autoimmune diseases.

Role of the common γ chain in cell cycle progression of human malignant cell lines.

The γ-chain (γc) is a transducing element shared between several cytokine receptors whose alteration causes X-linked severe combined immunodeficiency. Recently, a direct involvement of γc in self-sufficient growth in a concentration-dependent manner was described, implying a direct relationship between the amount of the molecule and its role in cell cycle progression. In this study, we evaluate whether γc expression could interfere in cell cycle progression also in malignant hematopoietic cells. Here, we first report that in the absence of γc expression, lymphoblastoid B-cell lines (BCLs) die at a higher extent than control cells. This phenomenon is caspase-3 independent and is associated to a decreased expression of the antiapoptotic Bcl-2 family members. By contrast, increased expression of γc protein directly correlates with spontaneous cell growth in several malignant hematopoietic cell lines. We, also, find that the knockdown of γc protein through short interfering RNA is able to decrease the cell proliferation rate in these malignancies. Furthermore, an increased expression of all D-type cyclins is found in proliferating neoplastic cells. In addition, a direct correlation between the amount of γc and cyclins A2 and B1 expression is found. Hence, our data demonstrate that the amount of the γc is able to influence the transcription of genes involved in cell cycle progression, thus being directly involved in the regulatory control of cell proliferation of malignant hematopoietic cells.

Dendritic cells/natural killer cross-talk: a novel target for human immunodeficiency virus type-1 protease inhibitors.

BACKGROUND: HIV-1 Protease Inhibitors, namely PIs, originally designed to inhibit HIV-1 aspartic protease, can modulate the immune response by mechanisms largely unknown, and independent from their activity on viral replication. Here, we analyzed the ability of PIs to interfere with differentiation program of monocytes toward dendritic cell (DCs) lineage, a key process in the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Monocytes from healthy donors were isolated and induced to differentiate in vitro in the presence or absence of saquinavir, ritonavir, nelfinavir, indinavir or amprenavir (sqv, rtv, nlfv, idv, apv, respectively). These drugs demonstrated a differential ability to sustain the generation of immature DCs (iDCs) with an altered phenotype, including low levels of CD1a, CD86, CD36 and CD209. DCs generated in the presence of rtv also failed to acquire the typical phenotype of mature DCs (mDCs), and secreted lower amounts of IL-12 and IL-15. Accordingly, these aberrant mDCs failed to support activation of autologous Natural Killer (NK) cells, and resulted highly susceptible to NK cell-mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: Our findings uncover novel functional properties of PIs within the DC-NK cell cross-talk, unveiling the heterogeneous ability of members of this class drugs to drive the generation of atypical monocyte-derived DCs (MDDCs) showing an aberrant phenotype, a failure to respond appropriately to bacterial endotoxin, a weak ability to prime autologous NK cells, and a high susceptibility to NK cell killing. These unexpected properties might contribute to limit inflammation and viral spreading in HIV-1 infected patients under PIs treatment, and open novel therapeutical perspectives for this class drugs as immunomodulators in autoimmunity and cancer.

The cellular amount of the common gamma-chain influences spontaneous or induced cell proliferation.

Mutations of the IL2RG encoding the common gamma-chain (gamma(c)) lead to the X-linked SCID disease. Gene correction through ex vivo retroviral transduction restored the immunological impairment in the most of treated patients, although lymphoproliferative events occurred in five of them. Even though in two cases it was clearly documented an insertional mutagenesis in LMO2, it is conceivable that gamma(c) could have a role per se in malignant lymphoproliferation. The gamma(c) is a shared cytokine receptor subunit, involved also in growth hormone (GH) receptor signaling. Through short interfering RNA or using X-linked SCID B lymphoblastoid cell lines lacking gamma(c), we demonstrate that self-sufficient growth was strongly dependent on gamma(c) expression. Furthermore, a correlation between gamma(c) amount and the extent of constitutive activation of JAK3 was found. The reduction of gamma(c) protein expression also reduced GH-induced proliferation and STAT5 nuclear translocation in B lymphoblastoid cell lines. Hence, our data demonstrate that gamma(c) plays a remarkable role in either spontaneous or GH-induced cell cycle progression depending on the amount of protein expression, suggesting a potential role as enhancing cofactor in lymphoproliferation.

Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells.

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.

Thymoma followed by paroxysmal nocturnal hemoglobinuria: a unique clinical association in the context of multiorgan autoimmunity with a potential role for CD8+ T lymphocytes.

Seven years after a surgically resected thymoma, a female patient affected by myasthenia gravis and Good's syndrome presented with paroxysmal nocturnal hemoglobinuria (PNH). Co-culture experiments and spectratyping analyses indicated that CD8+ lymphocytes were involved in damaging hematopoietic precursors. While PNH clones have been identified in various hematological disorders, the sequential association of thymoma and PNH has been unreported so far.

Fatal immunodeficiency in a patient with thymoma and Good's syndrome.

Thymoma is a rare tumor characterized by an association with autoimmune diseases. Immunological abnormalities are increasingly being recognized in thymoma patients and are the cause of a peculiar susceptibility to infections. The authors present the clinical history of a thymoma patient affected by fatal immunodeficiency.

Cutaneous manifestations as presenting sign of autoimmune lymphoproliferative syndrome in childhood.

Autoimmune lymphoproliferative syndrome is a disorder due to a defect of lymphocyte apoptosis, whose clinical manifestations consist of hyperplasia of lymphoid tissues and autoimmune diseases. We report on a 26-month-old child who presented with frequent eruptions of weals and angioedema without any apparent triggering factor, who subsequently developed an erythematopapular rash with a histological pattern of a lymphoplasmacellular infiltrate. Familial anamnesis revealed a history of lymphoadenomegaly and massive spleen and liver enlargement in her sister. Functional and molecular analysis led to a diagnosis of type 1a autoimmune lymphoproliferative syndrome. Immunophenotyping of the cutaneous lesion revealed the presence of an inflammatory infiltrate with a considerably high number of Langerhans cells. Cutaneous features such as urticaria, angioedema and vasculitis in children with a personal and familial history of hyperplasia of lymphoid tissues may be a presenting sign of a systemic disease, such as autoimmune lymphoproliferative syndrome.