Endogenous Alpha-Synuclein is Essential for the Transfer of Pathology by Exosome-Enriched Extracellular Vesicles, Following Inoculation with Preformed Fibrils in vivo.

The main pathological hallmark of Parkinson's disease (PD) and related synucleinopathies is the presence of intracellular proteinaceous aggregates, enriched in the presynaptic protein alpha-Synuclein (α-Syn). α-Syn association with exosomes has been previously documented both as a physiological process of secretion and as a pathological process of disease transmission, however, critical information about the mechanisms governing this interplay is still lacking. To address this, we utilized the α-Syn preformed fibril (PFF) mouse model of PD, as a source of brain-derived exosome-enriched extracellular vesicles (ExE-EVs) and assessed their pathogenic capacity following intrastriatal injections in host wild type (WT) mouse brain. We further investigated the impact of the fibrillar α-Syn on the exosomal cargo independent of the endogenous α-Syn, by isolating ExE-EVs from PFF-injected α-Syn knockout mice. Although PFF inoculation does not alter the morphology, size distribution, and quantity of brain-derived ExE-EVs, it triggers changes in the exosomal proteome related to synaptic and mitochondrial function, as well as metabolic processes. Importantly, we showed that the presence of the endogenous α-Syn is essential for the ExE-EVs to acquire a pathogenic capacity, allowing them to mediate disease transmission by inducing phosphorylated-α-Syn pathology. Notably, misfolded α-Syn containing ExE-EVs when injected in WT mice were able to induce astrogliosis and synaptic alterations in the host brain, at very early stages of α-Syn pathology, preceding the formation of the insoluble α-Syn accumulations. Collectively, our data suggest that exosomal cargo defines their ability to spread α-Syn pathology.

Metabolic rewiring: a new master of Th17 cell plasticity and heterogeneity.

T helper type 17 (Th17) cells are characterized by inherent plasticity and heterogeneity displaying both pathogenic and tissue-protective functions. Emerging evidence has illuminated a pivotal role for metabolic reprogramming in shaping Th17 cell fate determination. Metabolic responses are regulated by a constellation of factors and environmental triggers, including cytokines, nutrients, oxygen levels, and metabolites. Dysregulation of metabolic pathways not only influences Th17 cell plasticity and effector function but also affects the outcome of Th17-linked autoimmune, inflammatory, and antitumor responses. Understanding the molecular mechanisms underpinning metabolic reprogramming can allow the enhancement of protective Th17 cell-mediated responses during infections and cancer, concomitant with the suppression of detrimental Th17 processes during autoimmune and inflammatory diseases. In the present review, we describe major metabolic pathways underlying the differentiation of Th17 cells and their crosstalk with intracellular signaling mediators, we discuss how metabolic reprogramming affects Th17 cell plasticity and functions, and, finally, we outline current advances in the exploitation of metabolic checkpoints for the development of novel therapeutic interventions for the management of tissue inflammation, autoimmune disorders, and cancer.

Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α-dependent pathways.

In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined. Here, using experimental autoimmune encephalomyelitis, an established mouse MS model, we report that therapeutic administration of activin-A ameliorates disease severity and alleviates CNS immunopathology and demyelination, associated with decreased activation of Th17 cells. In fact, activin-A signaling through activin-like kinase-4 receptor represses pathogenic transcriptional programs in Th17-polarized cells, while it enhances antiinflammatory gene modules. Whole-genome profiling and in vivo functional studies revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A-induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistically, the aryl hydrocarbon receptor, along with STAT3 and c-Maf, are recruited to promoter elements on Entpd1 and Nt5e (encoding CD39 and CD73, respectively) and other antiinflammatory genes, and control their expression in Th17 cells in response to activin-A. Notably, we show that activin-A negatively regulates the metabolic sensor, hypoxia-inducible factor-1α, and key inflammatory proteins linked to pathogenic Th17 cell states. Of translational relevance, we demonstrate that activin-A is induced in the CNS of individuals with MS and restrains human Th17 cell responses. These findings uncover activin-A as a critical controller of Th17 cell pathogenicity that can be targeted for the suppression of autoimmune CNS inflammation.

Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity.

Pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate to pyruvate during glycolysis. The PK isoform PKM2 has additional roles in regulation of gene transcription and protein phosphorylation. PKM2 has been shown to control macrophage metabolic remodeling in inflammation, but its role in T cell biology is poorly understood. Here, we report PKM2 upregulation, phosphorylation, and nuclear accumulation in murine and human CD4+ T cells following activation in vitro. Treatment of T cells with TEPP-46, an allosteric activator that induces PKM2 tetramerization and blocks its nuclear translocation, strongly reduces their activation, proliferation, and cytokine production by inhibiting essential signaling pathways and thus preventing the engagement of glycolysis. TEPP-46 limits the development of both T helper 17 (Th17) and Th1 cells in vitro and ameliorates experimental autoimmune encephalomyelitis (EAE) in vivo. Overall, our results suggest that pharmacological targeting of PKM2 may represent a valuable therapeutic approach in T cell-mediated inflammation and autoimmunity.

Development of a CRISPR/Cas9 system against ruminant animal brucellosis.

BACKGROUND: Brucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide. Not only does it affect ruminant animal populations, leading to a substantial financial burden for stockbreeders, but also poses severe public health issues. For almost four decades in southern Europe and elsewhere, eradication of the disease has been based on ambiguously effective programs, rendering massive sanitation of livestock urgent and indispensable. Gene therapy, which has been proved effective in the clinic, could possibly constitute an alternative option towards a permanent cure for brucellosis, by aiding in the deletion or inactivation of genes associated with the replication of Brucella within the host cells. RESULTS: We infected ovine macrophages with B.melitensis, to simulate the host cell/microorganism interaction in vitro, and transduced the infected cells with CRISPR/Cas9 lentiviral vectors that target Brucella's RNA polymerase subunit A (RpolA) or virulence-associated gene virB10 at a multiplicity of infection of 60. We demonstrate a significant decrease in the bacterial load per cell when infected cells are transduced with the RpolA vector and that the number of internalized brucellae per cell remains unaffected when macrophages are transduced with a conventional lentiviral vector expressing the green fluorescence protein, thus underlining the bactericidal effect of our CRISPR/Cas9 system. CONCLUSIONS: Pending in vivo verification of our findings, overall, these results may prove critical not only for the treatment of human brucellosis, but for other infectious diseases in general.

Activin-A in the regulation of immunity in health and disease.

The TGF-ß superfamily of cytokines plays pivotal roles in the regulation of immune responses protecting against or contributing to diseases, such as, allergy, autoimmunity and cancer. Activin-A, a member of the TGF-ß superfamily, was initially identified as an inducer of follicle-stimulating hormone secretion. Extensive research over the past decades illuminated fundamental roles for activin-A in essential biologic processes, including embryonic development, stem cell maintenance and differentiation, haematopoiesis, cell proliferation and tissue fibrosis. Activin-A signals through two type I and two type II receptors which, upon ligand binding, activate their kinase activity, phosphorylate the SMAD2 and 3 intracellular signaling mediators that form a complex with SMAD4, translocate to the nucleus and activate or silence gene expression. Most immune cell types, including macrophages, dendritic cells (DCs), T and B lymphocytes and natural killer cells have the capacity to produce and respond to activin-A, although not in a similar manner. In innate immune cells, including macrophages, DCs and neutrophils, activin-A exerts a broad range of pro- or anti-inflammatory functions depending on the cell maturation and activation status and the spatiotemporal context. Activin-A also controls the differentiation and effector functions of Th cell subsets, including Th9 cells, TFH cells, Tr1 Treg cells and Foxp3+ Treg cells. Moreover, activin-A affects B cell responses, enhancing mucosal IgA secretion and inhibiting pathogenic autoantibody production. Interestingly, an array of preclinical and clinical studies has highlighted crucial functions of activin-A in the initiation, propagation and resolution of human diseases, including autoimmune diseases, such as, systemic lupus erythematosus, rheumatoid arthritis and pulmonary alveolar proteinosis, in allergic disorders, including allergic asthma and atopic dermatitis, in cancer and in microbial infections. Here, we provide an overview of the biology of activin-A and its signaling pathways, summarize recent studies pertinent to the role of activin-A in the modulation of inflammation and immunity, and discuss the potential of targeting activin-A as a novel therapeutic approach for the control of inflammatory diseases.