IFN-I inducible miR-3614-5p targets ADAR1 isoforms and fine tunes innate immune activation.

Regulation of innate immune responses is essential for maintenance of immune homeostasis and development of an appropriate immunity against microbial infection. We show here that miR-3614-5p, product of the TRIM25 host gene, is induced by type I interferon (IFN-I) in several human non-immune and immune cell types, in particular in primary myeloid cells. Studies in HeLa cells showed that miR-3614-5p represses both p110 and p150 ADAR1 and reduces constitutive and IFN-induced A-to-I RNA editing. In line with this, activation of innate sensors and expression of IFN-ß and the pro-inflammatory IL-6 are promoted. MiR-3614-5p directly targets ADAR1 transcripts by binding to one specific site in the 3'UTR. Moreover, we could show that endogenous miR-3614-5p is associated with Ago2 and targets ADAR1 in IFN-stimulated cells. Overall, we propose that, by reducing ADAR1, IFN-I-induced miR-3614-5p contributes to lowering the activation threshold of innate sensors. Our findings provide new insights into the role of miR-3614-5p, placing it as a potential fine tuner of dsRNA metabolism, cell homeostasis and innate immunity.

Genome-Wide Gene Expression Analysis of Mtb-Infected DC Highlights the Rapamycin-Driven Modulation of Regulatory Cytokines via the mTOR/GSK-3ß Axis.

In human primary dendritic cells (DC) rapamycin-an autophagy inducer and protein synthesis inhibitor-overcomes the autophagy block induced by Mycobacterium tuberculosis (Mtb) and promotes a Th1 response via IL-12 secretion. Here, the immunostimulatory activity of rapamycin in Mtb-infected DC was further investigated by analyzing both transcriptome and translatome gene profiles. Hundreds of differentially expressed genes (DEGs) were identified by transcriptome and translatome analyses of Mtb-infected DC, and some of these genes were found further modulated by rapamycin. The majority of transcriptome-associated DEGs overlapped with those present in the translatome, suggesting that transcriptionally stimulated mRNAs are also actively translated. In silico analysis of DEGs revealed significant changes in intracellular cascades related to cytokine production, cytokine-induced signaling and immune response to pathogens. In particular, rapamycin treatment of Mtb-infected DC caused an enrichment of IFN-ß, IFN-λ and IFN-stimulated gene transcripts in the polysome-associated RNA fraction. In addition, rapamycin led to an increase of IL-12, IL-23, IL-1ß, IL-6, and TNF-α but to a reduction of IL-10. Interestingly, upon silencing or pharmacological inhibition of GSK-3ß, the rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, indicating that, in Mtb-infected DC, GSK-3ß acts as molecular switch for the regulation of the cytokine milieu. In conclusion, our study sheds light on the molecular mechanism by which autophagy induction contributes to DC activation during Mtb infection and points to rapamycin and GSK-3ß modulators as promising compounds for host-directed therapy in the control of Mtb infection.

Human Ubiquitin-Specific Peptidase 18 Is Regulated by microRNAs via the 3'Untranslated Region, A Sequence Duplicated in Long Intergenic Non-coding RNA Genes Residing in chr22q11.21.

Ubiquitin-specific peptidase 18 (USP18) acts as gatekeeper of type I interferon (IFN) responses by binding to the IFN receptor subunit IFNAR2 and preventing activation of the downstream JAK/STAT pathway. In any given cell type, the level of USP18 is a key determinant of the output of IFN-stimulated transcripts. How the baseline level of USP18 is finely tuned in different cell types remains ill defined. Here, we identified microRNAs (miRNAs) that efficiently target USP18 through binding to the 3'untranslated region (3'UTR). Among these, three miRNAs are particularly enriched in circulating monocytes which exhibit low baseline USP18. Intriguingly, the USP18 3'UTR sequence is duplicated in human and chimpanzee genomes. In humans, four USP18 3'UTR copies were previously found to be embedded in long intergenic non-coding (linc) RNA genes residing in chr22q11.21 and known as FAM247A-D. Here, we further characterized their sequence and measured their expression profile in human tissues. Importantly, we describe an additional lincRNA bearing USP18 3'UTR (here linc-UR-B1) that is expressed only in testis. RNA-seq data analyses from testicular cell subsets revealed a positive correlation between linc-UR-B1 and USP18 expression in spermatocytes and spermatids. Overall, our findings uncover a set of miRNAs and lincRNAs, which may be part of a network evolved to fine-tune baseline USP18, particularly in cell types where IFN responsiveness needs to be tightly controlled.

Differential Responses of Human Dendritic Cells to Live or Inactivated Staphylococcus aureus: Impact on Cytokine Production and T Helper Expansion.

Understanding Staphylococcus aureus (S. aureus)-host immune system interaction is crucial to meet the tremendous medical need associated with this life-threatening bacterial infection. Given the crucial role of dendritic cells (DC) in dictating immune responses upon microbial challenge, we investigated how the bacterial viability and the conservation of structural integrity influence the response of human DC to S. aureus. To this end, human primary DC were stimulated with the methicillin-resistant S. aureus USA300 live strain, USA300 inactivated by heat (HI), ultraviolet irradiation (UVI), or paraformaldehyde treatment (PFAI) and subsequently analyzed for cell phenotype and immune-modulatory properties. Although no differences in terms of DC viability and maturation were observed when DC were stimulated with live or inactivated bacteria, the production of IL-12, IL-23, and other cytokines differed significantly. The Th1 and Th17 expansion was also more pronounced in response to live vs. inactivated S. aureus. Interestingly, cytokine production in DC treated with live and inactivated USA300 required phagocytosis, whereas blocking endosomal Toll-like receptor signaling mainly reduced the cytokine release by live and HI USA300. A further analysis of IFN-ß signaling revealed the induction of a cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING)-independent and IRF3-dependent signaling pathway(s) in UVI-stimulated DC. This study underscores the capacity of human DC to discriminate between live and inactivated S. aureus and, further, indicates that DC may represent a valuable experimental setting to test different inactivation methods with regard to the retention of S. aureus immunoregulatory properties. These and further insights may be useful for the development of novel therapeutic and prophylactic anti-S. aureus vaccine strategies.

A cell type-specific transcriptomic approach to map B cell and monocyte type I interferon-linked pathogenic signatures in Multiple Sclerosis.

Alteration in endogenous Interferon (IFN) system may profoundly impact immune cell function in autoimmune diseases. Here, we provide evidence that dysregulation in IFN-regulated genes and pathways are involved in B cell- and monocyte-driven pathogenic contribution to Multiple Sclerosis (MS) development and maintenance. In particular, by using an Interferome-based cell type-specific approach, we characterized an increased susceptibility to an IFN-linked caspase-3 dependent apoptotic cell death in both B cells and monocytes of MS patients that may arise from their chronic activation and persistent stimulation by activated T cells. Ongoing caspase-3 activation functionally impacts on MS monocyte properties influencing the STAT-3/IL-16 axis, thus, driving increased expression and massive release of the bio-active IL-16 triggering and perpetuating CD4+ T cell migration. Importantly, our analysis also identified a previously unknown multi-component defect in type I IFN-mediated signaling and response to virus pathways specific of MS B cells, impacting on induction of anti-viral responses and Epstein-barr virus infection control in patients. Taking advantage of cell type-specific transcriptomics and in-depth functional validation, this study revealed pathogenic contribution of endogenous IFN signaling and IFN-regulated cell processes to MS pathogenesis with implications on fate and functions of B cells and monocytes that may hold therapeutic potential.

Thymosins in multiple sclerosis and its experimental models: moving from basic to clinical application.

BACKGROUND: Multiple sclerosis (MS) afflicts more than 2.5 million individuals worldwide and this number is increasing over time. Within the past years, a great number of disease-modifying treatments have emerged; however, efficacious treatments and a cure for MS await discovery. Thymosins, soluble hormone-like peptides produced by the thymus gland, can mediate immune and non-immune physiological processes and have gained interest in recent years as therapeutics in inflammatory and autoimmune diseases. METHODS: Pubmed was searched with no time constraints for articles using a combination of the keywords "thymosin/s" or "thymus factor/s" AND "multiple sclerosis", mesh terms with no language restriction. RESULTS: Here, we review the state-of-the-art on the effects of thymosins on MS and its experimental models. In particular, we describe what is known in this field on the roles of thymosin-α1 (Tα1) and -ß4 (Tß4) as potential anti-inflammatory as well as neuroprotective and remyelinating molecules and their mechanisms of action. CONCLUSION: Based on the data that Tα1 and Tß4 act as anti-inflammatory molecules and as inducers of myelin repair and neuronal protection, respectively, a possible therapeutic application in MS for Tα1 and Tß4 alone or combined with other approved drugs may be envisaged. This approach is reasonable in light of the current clinical usage of Tα1 and data demonstrating the safety, tolerability and efficacy of Tß4 in clinical practice.

Mycobacterium tuberculosis-induced miR-155 subverts autophagy by targeting ATG3 in human dendritic cells.

Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy.

Thymosin-α1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.

BACKGROUND: B cells are key pathogenic effectors in multiple sclerosis (MS) and several therapies have been designed to restrain B cell abnormalities by directly targeting this lymphocyte population. OBJECTIVES: Moving from our data showing a Toll-like receptor (TLR)7-driven dysregulation of B cell response in relapsing-remitting multiple sclerosis (RRMS) and having found a low serum level of Thymosin-α1 (Tα1) in patients, we investigated whether the addition of this molecule to peripheral blood mononuclear cells (PBMCs) would influence the expansion of regulatory B cell subsets, known to dampen autoimmune inflammation. METHODS: Serum Tα1 level was measured by enzyme immunoassay. Cytokine expression was evaluated by Cytometric Bead Array (CBA), enzyme-linked immunosorbent assay (ELISA), and real-time reverse transcription polymerase chain reaction (RT-PCR). B cell subsets were analyzed by flow cytometry. RESULTS: Tα1 pre-treatment induces an anti-inflammatory status in TLR7-stimulated RRMS PBMC cultures, reducing the secretion of pro-inflammatory interleukin (IL)-6, IL-8, and IL-1ß while significantly increasing the regulatory IL-10 and IL-35. Indeed, Tα1 treatment enhanced expansion of CD19+CD24+CD38hi transitional-immature and CD24low/negCD38hi plasmablast-like regulatory B cell subsets, which likely inhibit both interferon (IFN)-γ and IL-17 production. CONCLUSION: Our study reveals a deficient ability of B cells from MS patients to differentiate into regulatory subsets and unveils a novel anti-inflammatory and repurposing potential for Tα1 in MS targeting B cell response.

Staphylococcus aureus Esx Factors Control Human Dendritic Cell Functions Conditioning Th1/Th17 Response.

The opportunistic pathogen Staphylococcus aureus (S. aureus) is a major cause of nosocomial- and community-acquired infections. In addition, many antibiotic-resistant strains are emerging worldwide, thus, there is an urgent unmet need to pinpoint novel therapeutic and prophylactic strategies. In the present study, we characterized the impact of infection with the pandemic methicillin-resistant USA300 S. aureus strain on human primary dendritic cells (DC), key initiators and regulators of immune responses. In particular, among staphylococcal virulence factors, the function of EsxA and EsxB, two small acidic dimeric proteins secreted by the type VII-like secretion system Ess (ESAT-6-like secretion system), was investigated in human DC setting. A comparative analysis of bacterial entry, replication rate as well as DC maturation, apoptosis, signaling pathway activation and cytokine production was performed by using wild type (wt) USA300 and three isogenic mutants carrying the deletion of esxA (ΔesxA), esxB (ΔesxB), or both genes (ΔesxAB). The S. aureus mutant lacking only the EsxA protein (ΔesxA) stimulated a stronger pro-apoptotic phenotype in infected DC as compared to wt USA300, ΔesxAB, and ΔesxB strains. When the mutant carrying the esxB deletion (ΔesxB) was analyzed, a higher production of both regulatory and pro-inflammatory mediators was found in the infected DC with respect to those challenged with the wt counterpart and the other esx mutants. In accordance with these data, supernatant derived from ΔesxB-infected DC promoted a stronger release of both IFN-γ and IL-17 from CD4+ T cells as compared with those conditioned with supernatants derived from wild type USA300-, ΔesxAB-, and ΔesxA-infected cultures. Although, the interaction of S. aureus with human DC is not yet fully understood, our data suggest that both cytokine production and apoptotic process are modulated by Esx factors, thus indicating a possible role of these proteins in the modulation of DC-mediated immunity to S. aureus.

Worldwide Epidemiology and Antibiotic Resistance of Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen, responsible for infections in the community and the healthcare setting. Although much of the attention is focused on the methicillin-resistant "variant" MRSA, the methicillin-susceptible counterpart (MSSA) remains a prime species in infections. The epidemiology of S. aureus, especially of MRSA, showed a rapid evolution in the last years. After representing a typical nosocomial multidrug-resistant pathogen, MRSA has recently emerged in the community and among farmed animals thanks to its ability to evolve and adapt to different settings. Global surveillance has shown that MRSA represents a problem in all continents and countries where studies have been carried out, determining an increase in mortality and the need to use last-resource expensive antibiotics. S. aureus can easily acquire resistance to antibiotics and MRSA is characteristically multidrug resistant. Resistance to vancomycin, the principal anti-MRSA antibiotic is rare, although isolates with decreased susceptibility are recovered in many areas. Resistance to the more recently introduced antibiotics, linezolid and daptomycin, has emerged; however, they remain substantially active against the large majority of MSSA and MRSA. Newer antistaphylococcal drugs have been developed, but since their clinical use has been very limited so far, little is known about the emergence of resistance. Molecular typing techniques have allowed to identify the major successful clones and lineages of MSSA and MRSA, including high-risk clones, and to trace their diffusion. In the face of a continuously evolving scenario, this review depicts the most common clones circulating in different geographical areas and in different settings at present. Since the evolution of S. aureus will continue, it is important to maintain the attention on the epidemiology of S. aureus in the future with a global view.

Impact of Mycobacterium tuberculosis RD1-locus on human primary dendritic cell immune functions.

Modern strategies to develop vaccines against Mycobacterium tuberculosis (Mtb) aim to improve the current Bacillus Calmette-Guerin (BCG) vaccine or to attenuate the virulence of Mtb vaccine candidates. In the present study, the impact of wild type or mutated region of difference 1 (RD1) variants on the immunogenicity of Mtb and BCG recombinants was investigated in human primary dendritic cells (DC). A comparative analysis of transcriptome, signalling pathway activation, maturation, apoptosis, cytokine production and capacity to promote Th1 responses demonstrated that DC sense quantitative and qualitative differences in the expression of RD1-encoded factors--ESAT6 and CFP10--within BCG or Mtb backgrounds. Expansion of IFN-γ producing T cells was promoted by BCG::RD1-challenged DC, as compared to their BCG-infected counterparts. Although Mtb recombinants acted as a strong Th-1 promoting stimulus, even with RD1 deletion, the attenuated Mtb strain carrying a C-terminus truncated ESAT-6 elicited a robust Th1 promoting phenotype in DC. Collectively, these studies indicate a necessary but not sufficient role for the RD1 locus in promoting DC immune-regulatory functions. Additional mycobacterial factors are likely required to endow DC with a high Th1 polarizing capacity, a desirable attribute for a successful control of Mtb infection.

Dual effect of Thymosin α 1 on human monocyte-derived dendritic cell in vitro stimulated with viral and bacterial toll-like receptor agonists.

OBJECTIVES: Thymosin α 1 (Tα1) recently gained interest as immune adjuvant for vaccines because of its ability to modulate the T-cell/dendritic cell (DC) axis and to improve antibody production. The objective of this study was to determine whether Tα1 would address in vitro the response of human primary monocyte-derived DC, crucial regulators of vaccine-induced immunity, upon exposure to different toll-like receptor (TLR) agonists or infection with viruses or bacteria. METHODS: DC maturation and production of pro-inflammatory cytokines were analyzed. RESULTS: Our data revealed a dual effect of Tα1 on DC biology upon viral or bacterial stimulation. Interestingly, Tα1 enhanced human leukocyte antigen (HLA)-I and II surface expression and secretion of IL-6, TNF-α and IL-8 when DCs were treated with viral TLR3 and TLR7/8 agonists. Similarly, in pandemic H1N1 influenza A-infected DCs, Tα1 raised the expression of maturation markers and type I and III Interferon (IFN). In contrast, following bacterial TLR2 and 4 stimulation, as well as upon Bacillus Calmette-Guerin infection, the presence of Tα1 in DC cultures drastically lowered the analyzed cellular parameters. CONCLUSION: The knowledge that Tα1 pleiotropic effect might ameliorate anti-viral immune responses and, at the same time, dampen inflammation caused by bacterial infections could lay the groundwork for a more appropriate therapeutic application of this molecule.