Impaired Mitochondrial Morphology and Functionality in Lonp1wt/- Mice

LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations ofLONP1have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in whichLonp1was ablated. The homozygousLonp(-/-)mouse was not vital, while the heterozygousLonp1(wt/-)showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) fromLonp1(wt/-)mice showed a reduced expression ofLonp1andTfam, whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of theLonp1gene leads to impairment of mitochondrial ultrastructure and functions in vivo

Evidence for mitochondrial Lonp1 expression in the nucleus

The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress

Prognostic immune markers identifying patients with severe COVID-19 who respond to tocilizumab

Introduction: A growing number of evidences suggest that the combination of hyperinflammation, dysregulated T and B cell response and cytokine storm play a major role in the immunopathogenesis of severe COVID-19. IL-6 is one of the main pro-inflammatory cytokines and its levels are increased during SARS-CoV-2 infection. Several observational and randomized studies demonstrated that tocilizumab, an IL-6R blocker, improves survival in critically ill patients both in infectious disease and intensive care units. However, despite transforming the treatment options for COVID-19, IL-6R inhibition is still ineffective in a fraction of patients. Methods: In the present study, we investigated the impact of two doses of tocilizumab in patients with severe COVID-19 who responded or not to the treatment by analyzing a panel of cytokines, chemokines and other soluble factors, along with the composition of peripheral immune cells, paying a particular attention to T and B lymphocytes. Results: We observed that, in comparison with non-responders, those who responded to tocilizumab had different levels of several cytokines and different T and B cells proportions before starting therapy. Moreover, in these patients, tocilizumab was further able to modify the landscape of the aforementioned soluble molecules and cellular markers. Conclusions: We found that tocilizumab has pleiotropic effects and that clinical response to this drug remain heterogenous. Our data suggest that it is possible to identify patients who will respond to treatment and that the administration of tocilizumab is able to restore the immune balance through the re-establishment of different cell populations affected by SARS-COV-2 infection, highlighting the importance of temporal examination of the pathological features from the diagnosis

Detailed characterization of SARS-CoV-2-specific T and B cells after infection or heterologous vaccination

: The formation of a robust long-term antigen (Ag)-specific memory, both humoral and cell-mediated, is created following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination. Here, by using polychromatic flow cytometry and complex data analyses, we deeply investigated the magnitude, phenotype, and functionality of SARS-CoV-2-specific immune memory in two groups of healthy subjects after heterologous vaccination compared to a group of subjects who recovered from SARS-CoV-2 infection. We find that coronavirus disease 2019 (COVID-19) recovered patients show different long-term immunological profiles compared to those of donors who had been vaccinated with three doses. Vaccinated individuals display a skewed T helper (Th)1 Ag-specific T cell polarization and a higher percentage of Ag-specific and activated memory B cells expressing immunoglobulin (Ig)G compared to those of patients who recovered from severe COVID-19. Different polyfunctional properties characterize the two groups: recovered individuals show higher percentages of CD4+ T cells producing one or two cytokines simultaneously, while the vaccinated are distinguished by highly polyfunctional populations able to release four molecules, namely, CD107a, interferon (IFN)-γ, tumor necrosis factor (TNF), and interleukin (IL)-2. These data suggest that functional and phenotypic properties of SARS-CoV-2 adaptive immunity differ in recovered COVID-19 individuals and vaccinated ones

Human astrocytes and microglia show augmented ingestion of synapses in Alzheimer's disease via MFG-E8

Synapse loss correlates with cognitive decline in Alzheimer's disease (AD). Data from mouse models suggests microglia are important for synapse degeneration, but direct human evidence for any glial involvement in synapse removal in human AD remains to be established. Here we observe astrocytes and microglia from human brains contain greater amounts of synaptic protein in AD compared with non-disease controls, and that proximity to amyloid-β plaques and the APOE4 risk gene exacerbate this effect. In culture, mouse and human astrocytes and primary mouse and human microglia phagocytose AD patient-derived synapses more than synapses from controls. Inhibiting interactions of MFG-E8 rescues the elevated engulfment of AD synapses by astrocytes and microglia without affecting control synapse uptake. Thus, AD promotes increased synapse ingestion by human glial cells at least in part via an MFG-E8 opsonophagocytic mechanism with potential for targeted therapeutic manipulation.</p

Influenza vaccination for immunocompromised patients: systematic review and meta-analysis from a public health policy perspective.

Immunocompromised patients are vulnerable to severe or complicated influenza infection. Vaccination is widely recommended for this group. This systematic review and meta-analysis assesses influenza vaccination for immunocompromised patients in terms of preventing influenza-like illness and laboratory confirmed influenza, serological response and adverse events

IL RUOLO DELL’IMMUNITA INNATA NEL COVID-19 GRAVE

La complessa risposta immunitaria causata dall'infezione da SARS-CoV-2 può portare alla malattia chiamata COVID-19. L'immunità innata è la prima linea di difesa contro le infezioni e, come precedentemente descritto dal nostro gruppo, una sua eccessiva attivazione può causare uno stato infiammatorio persistente che contribuisce all'immunopatogenesi della forma grave di COVID-19. Pertanto, il mio progetto si è focalizzato sull'identificazione dei principali meccanismi fisiopatologici innescati dai componenti dell'immunità innata, tra cui molecole solubili e cellule come monociti (MC) e neutrofili (NP), che possono contribuire all'eterogeneità della malattia. Sono state studiate due coorti di pazienti con polmonite da COVID-19. La prima comprendeva 28 pazienti e 27 soggetti sani (HD) ed è stata studiata per caratterizzare il fenotipo, il metabolismo e le funzioni dei MC circolanti. La seconda coorte includeva 88 pazienti e 59 HD ed è stata studiata per individuare i meccanismi alla base della risposta neutrofilica generata durante l'infezione da SARS-CoV-2. I risultati ottenuti hanno dimostrato che i MC nei pazienti COVID-19 gravi rispetto ai HD, sono caratterizzati da una profonda alterazione metabolica. Vi è una maggiore percentuale di MC che presentano mitocondri depolarizzati e un aumento della massa mitocondriale, insieme a profonde alterazioni ultrastrutturali. I MC disfunzionali, inoltre, esprimono bassi livelli di HLA-DR e hanno una limitata capacità di fare burst ossidativo rispetto ai MC di HD, ma mantengono la capacità di produrre citochine. Inoltre, abbiamo rilevato a livello periferico, un’importante ridistribuzione delle sottopopolazioni monocitarie, con un aumento della classe intermedia pro-infiammatoria, e una riduzione di quella non classica. Tutte le sottoclassi presentano un’overespressione dei checkpoint inibitori PD-1 e PD-L1 riconducibile al fenomeno di exhaustion. Infine, la concentrazione plasmatica di alcuni fattori coinvolti nella regolazione e migrazione dei MC è maggiore nei pazienti COVID-19, come quella di GM-CSF, che associata ad un aumento significativo di MC immaturi circolanti, suggerisce la presenza di mielopoiesi di emergenza. Analizzando la seconda coorte dello studio, abbiamo dimostrato la presenza di un’alterazione funzionale e metabolica nei granulociti circolanti dei pazienti COVID-19 gravi. La percentuale di NP immaturi aumenta e i NP degranulano di più. A livello bioenergetico, i NP dei pazienti gravi rispetto ai HD hanno una ridotta capacità respiratoria di riserva e di burst ossidativo, ma un’aumentata capacità glicolitica, che sostiene la formazione delle trappole extracellulari (NET). Inoltre, abbiamo osservato la formazione di grandi depositi intracellulari di glicogeno nei neutrofili dei pazienti rispetto ai HD e un aumento dell’espressione genica dell’enzima glicogeno fosforilasi L (PYGL). La glicogenolisi, quindi, può essere utilizzata dai neutrofili come via energetica per la formazione di NET. La quantificazione dei livelli plasmatici di citochine e chemochine nei pazienti con COVID-19 grave ha rivelato la presenza di un pattern di attivazione correlabile alla gravità della malattia. In conclusione, i dati suggeriscono che l'infezione da SARS-CoV-2 può influenzare in maniera importante l’immunità innata. I diversi profili fenotipico e metabolico di MC e NP, nella malattia, possono diventare target efficaci per lo sviluppo di nuove terapie. L’overespressione dei checkpoint inibitori sui MC rappresenta un possibile bersaglio per l'immunoterapia nella lotta contro il cancro e l'aumento dell’attività glicolitica e glicogenolitica nei NP apre la strada a nuove strategie per controllare le malattie infiammatorie.SARS-CoV-2 infection triggers a complex immune response that can cause the onset of COVID-19, and that starts by the activation of innate immunity, the first line of defense. As previously described by our group, a hyperactivation of innate immune cells leads to a strong inflammatory status, whose persistence contributes to the immunopathogenesis of severe COVID-19. Therefore, my studies aimed at the identification of pivotal pathophysiological mechanisms that are triggered by main soluble molecules and cells belonging to innate immunity, like monocytes (MC) and neutrophils (NP), and their contribution to the heterogeneity of COVID-19. Two cohorts of patients with COVID-19 pneumonia were investigated. The first comprised 28 patients and 27 healthy donors (HD) and was used to characterize metabolism, phenotype, and functions of circulating MC. The second included 88 patients and 59 HD and was studied to dissect mechanisms underlying the neutrophilic response during SARS-CoV-2 infection. We found that MC from severe COVID-19 patients were metabolically impaired. The percentage of MC with depolarized mitochondria (mt) was increased as well as their mt mass and mt ultrastructure was profoundly altered compared to healthy MC. In COVID-19 patients dysfunctional MC expressed low levels of HLA-DR and showed a reduced capacity to perform the oxidative burst, but they were still able to produce cytokines. A relevant redistribution of MC subsets was also detected, with an expansion of intermediate MC, which have pro-inflammatory function, and a reduction of nonclassical MC. In all subsets inhibitory checkpoints PD-1 and PD-L1 were overexpressed, probably contributing to immune exhaustion. Finally, plasma concentration of several mediators involved in MC regulation and migration was higher in COVID-19 patients such as GM-CSF, suggesting the presence of emergency myelopoiesis in severe patients. In fact, immature MC were significantly increased in peripheral blood from COVID-19 patients. Regarding NP, formal evidence of their functional status in severe COVID-19 patients was missing. We found that circulating granulocytes were altered in severe COVID-19 patients, with a higher proportion of immature NP and more degranulated NP. The analysis of NP bioenergetic profile revealed a decreased spare respiratory capacity and a defective respiratory burst in NP from COVID-19 patients versus HD. However, glycolysis and glycolytic capacity were strongly increased which sustain neutrophils extracellular traps (NET) formation. Other than glycolysis, other metabolic routes have been recently associated with NP’ differentiation and function. We discovered large intracytoplasmic deposits of glycogen in NP from COVID-19 patients compared to HD and increased levels of intracellular glycogen and mRNA levels of glycogen phosphorylase L (PYGL), which catalyses glycogenolysis. Indeed, NP can use glycogen for NET formation. The analysis of cytokine and chemokine profile in patients with severe COVID-19 revealed that they are profoundly altered delineating an activation pattern which correlates with the severity of the disease of COVID-19. In conclusion, data suggested that infection with SARS-CoV-2 can heavily affect the innate compartment of immune system. MC and NP have a remodelled phenotype and metabolism that can prove effective targets for innovative therapies. The upregulation of inhibitory checkpoints on MC represents an interesting point for immunotherapy against cancer, and increased glycolysis and glycogenolysis crucial for NET formation in NP suggest possible novel strategies to control inflammatory diseases

Synthesis and Anticancer Activity of CDDO and CDDO-Me, Two Derivatives of Natural Triterpenoids

Triterpenoids are natural compounds synthesized by plants through cyclization of squalene, known for their weak anti-inflammatory activity. 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), and its C28 modified derivative, methyl-ester (CDDO-Me, also known as bardoxolone methyl), are two synthetic derivatives of oleanolic acid, synthesized more than 20 years ago, in an attempt to enhance the anti-inflammatory behavior of the natural compound. These molecules have been extensively investigated for their strong ability to exert antiproliferative, antiangiogenic, and antimetastatic activities, and to induce apoptosis and differentiation in cancer cells. Here, we discuss the chemical properties of natural triterpenoids, the pathways of synthesis and the biological effects of CDDO and its derivative CDDO-Me. At nanomolar doses, CDDO and CDDO-Me have been shown to protect cells and tissues from oxidative stress by increasing the transcriptional activity of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). At doses higher than 100 nM, CDDO and CDDO-Me are able to modulate the differentiation of a variety of cell types, both tumor cell lines or primary culture cell, while at micromolar doses these compounds exert an anticancer effect in multiple manners; by inducing extrinsic or intrinsic apoptotic pathways, or autophagic cell death, by inhibiting telomerase activity, by disrupting mitochondrial functions through Lon protease inhibition, and by blocking the deubiquitylating enzyme USP7. CDDO-Me demonstrated its efficacy as anticancer drugs in different mouse models, and versus several types of cancer. Several clinical trials have been started in humans for evaluating CDDO-Me efficacy as anticancer and anti-inflammatory drug; despite promising results, significant increase in heart failure events represented an obstacle for the clinical use of CDDO-Me

A Comprehensive Analysis of Cytokine Network in Centenarians

Cytokines have been investigated extensively in elderly people, with conflicting results. We performed a comprehensive analysis of the plasma levels of 62 cytokines and growth factors involved in the regulation of the immune system, in healthy centenarians, and middle-aged controls. We confirmed the previously observed increase in the levels of several pro-inflammatory cytokines, such as TNF-&alpha; and IL-6, and found that several other cytokines, directly or indirectly involved in inflammation (such as IFN-&alpha;, IL-23, CCL-5), were present at higher levels in centenarians. We did not observe any increase in the levels of anti-inflammatory cytokines, with the notable exception of the Th2-shifting cytokine IL-19. No relevant difference was observed in cytokines regulating T cell immunity. Several growth factors having a role in regulating immunity, such as G-CSF, GM-CSF, EGF, and VEGF, were upregulated in centenarians, too. Principal component analysis of the cytokine dataset showed that pro and anti-inflammatory cytokines were the variables that contributed the most to the variability of the data we observed