Inhibition of Glycolysis Impairs Retinoic Acid-Inducible Gene I-Mediated Antiviral Responses in Primary Human Dendritic Cells.

Dendritic cells (DCs) are important mediators of the induction and regulation of adaptive immune responses following microbial infection and inflammation. Sensing environmental danger signals including viruses, microbial products, or inflammatory stimuli by DCs leads to the rapid transition from a resting state to an activated mature state. DC maturation involves enhanced capturing and processing of antigens for presentation by major histocompatibility complex (MHC) class I and class II, upregulation of chemokines and their receptors, cytokines and costimulatory molecules, and migration to lymphoid tissues where they prime naive T cells. Orchestrating a cellular response to environmental threats requires a high bioenergetic cost that accompanies the metabolic reprogramming of DCs during activation. We previously demonstrated that DCs undergo a striking functional transition after stimulation of the retinoic acid-inducible gene I (RIG-I) pathway with a synthetic 5' triphosphate containing RNA (termed M8), consisting of the upregulation of interferon (IFN)-stimulated antiviral genes, increased DC phagocytosis, activation of a proinflammatory phenotype, and induction of markers associated with immunogenic cell death. In the present study, we set out to determine the metabolic changes associated with RIG-I stimulation by M8. The rate of glycolysis in primary human DCs was increased in response to RIG-I activation, and glycolytic reprogramming was an essential requirement for DC activation. Pharmacological inhibition of glycolysis in monocyte-derived dendritic cells (MoDCs) impaired type I IFN induction and signaling by disrupting the TBK1-IRF3-STAT1 axis, thereby countering the antiviral activity induced by M8. Functionally, the impaired IFN response resulted in enhanced viral replication of dengue, coronavirus 229E, and Coxsackie B5.

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection.

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent but Neuropilin-1-dependent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients.

In Vitro Characterization of the Innate Immune Pathways Engaged by Live and Inactivated Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) infection can lead to inflammation of the central nervous system. The disease can be effectively prevented by whole inactivated virus vaccines. Here, we investigated the innate immune profile induced in vitro by the antigen component of the vaccines, inactivated TBEV (I-TBEV), to gain insights into the mechanism of action of the TBE vaccine as compared to the live virus. To this end, we exposed human peripheral blood mononuclear cells (PBMCs) to inactivated and live TBEV and assessed cellular responses by RNA sequencing. Both inactivated and live TBEV significantly induced an interferon-dominated gene signature and an increased RIG-I-like receptor (RLR) expression. Using pathway-specific inhibitors, we assessed the involvement of pattern recognition receptors in the sensing of inactivated or live TBEV. Only RLR pathway inhibition significantly suppressed the downstream cascade induced by I-TBEV, while responses to the replicating virus were impacted by the inhibition of RIG-I-like, as well as Toll-like, receptors. Our results show that inactivated and live TBEV predominantly engaged an interferon response in our in vitro PBMC platform, and indicate RLRs as the main pattern recognition receptors involved in I-TBEV sensing.

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 plasmacytoid dendritic cells at the crossroad of type I interferon-regulated B cell differentiation and antiviral response to tick-borne encephalitis virus.

The Tick-borne encephalitis virus (TBEV) causes different disease symptoms varying from asymptomatic infection to severe encephalitis and meningitis suggesting a crucial role of the human host immune system in determining the fate of the infection. There is a need to understand the mechanisms underpinning TBEV-host interactions leading to protective immunity. To this aim, we studied the response of human peripheral blood mononuclear cells (PBMC) to the whole formaldehyde inactivated TBEV (I-TBEV), the drug substance of Encepur, one of the five commercially available vaccine. Immunophenotyping, transcriptome and cytokine profiling of PBMC revealed that I-TBEV generates differentiation of a sub-population of plasmacytoid dendritic cells (pDC) that is specialized in type I interferon (IFN) production. In contrast, likely due to the presence of aluminum hydroxide, Encepur vaccine was a poor pDC stimulus. We demonstrated I-TBEV-induced type I IFN together with Interleukin 6 and BAFF to be critical for B cell differentiation to plasmablasts as measured by immunophenotyping and immunoglobulin production. Robust type I IFN secretion was induced by pDC with the concerted action of both viral E glycoprotein and RNA mirroring previous data on dual stimulation of pDC by both S. aureus and influenza virus protein and nucleic acid that leads to a type I IFN-mediated sustained immune response. E glycoprotein neutralization or high temperature denaturation and inhibition of Toll-like receptor 7 signalling confirmed the importance of preserving the functional integrity of these key viral molecules during the inactivation procedure and manufacturing process to produce a vaccine able to stimulate strong immune responses.

In vitro assessment of tick-borne encephalitis vaccine: Suitable human cell platforms and potential biomarkers.

Tick-borne encephalitis (TBE) virus causes a severe disease that can lead to permanent neurological complications. The whole inactivated TBE vaccine is highly effective, as proven by high seroconversion rates and near eradication of the disease in countries where vaccination programs have been implemented. TBE vaccine potency testing currently requires the use of in vivo methods that present issues of reproducibility as well as animal discomfort. As an alternative, public and private entities are currently exploring a batch-to-batch consistency approach that would demonstrate conformity of a newly produced vaccine batch with a batch of proven in vivo efficacy with respect to a range of measurable in vitro quality parameters. To identify a suitable cellular platform to be used in a panel of in vitro batch-to-batch assessments for the TBE vaccine, we exposed human cell-based systems, both of primary origin and cell line-derived, to vaccine formulations of high and low quality. Following stimulation, cell responses were evaluated by assessing the expression of selected genes by RT-qPCR. Our findings show that the expression of interferon-stimulated genes differed after treatment with non-adjuvanted vaccine batches of different quality in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived dendritic cells, but not in monocyte-free PBMC suspensions nor in cell line-derived immune cells. These results indicate suitable platforms and potential biomarkers for a cell-based assay that, together with other immu­nochemical analyses, could serve for batch-to-batch assessment of the TBE vaccine, reducing, and eventually replacing, in vivo methods for potency testing.

ISG15 protects human Tregs from interferon alpha-induced contraction in a cell-intrinsic fashion.

OBJECTIVES: Type I interferons (IFNs) inhibit regulatory T-cell (Treg) expansion and activation, making them beneficial in antiviral responses, but detrimental in autoimmune diseases. Herein, we investigate the role of ISG15 in human Tregs in the context of refractoriness to type I IFN stimulation. METHODS: ISG15 expression and Treg dynamics were analysed in vitro and ex vivo from patients with chronic hepatitis C, with lupus and ISG15 deficiency. RESULTS: ISG15 is expressed at high levels in human Tregs, renders them refractory to the IFN-STAT1 signal, and protects them from IFN-driven contraction. In vitro, Tregs from healthy controls upregulate ISG15 upon activation to higher levels than conventional CD4 T cells, and ISG15-silenced Tregs are more susceptible to IFNα-induced contraction. In human ISG15 deficiency, patient Tregs display an elevated IFN signature relative to Tregs from healthy control. In vivo, in patients with chronic hepatitis C, 2 days after starting pegIFN/ribavirin therapy, a stronger ISG15 inducibility correlates with a milder Treg depletion. Ex vivo, in systemic lupus erythematosus patients, higher levels of ISG15 are associated to reduced STAT1 phosphorylation in response to IFNα, and also to increased frequencies of Tregs, characterising active disease. CONCLUSION: Our results reveal a Treg-intrinsic role of ISG15 in dictating their refractoriness to the IFN signal, thus preserving the Treg population under inflammatory conditions.

Dengue Virus Targets Nrf2 for NS2B3-Mediated Degradation Leading to Enhanced Oxidative Stress and Viral Replication.

Dengue virus (DENV) is a mosquito-borne virus that infects upward of 300 million people annually and has the potential to cause fatal hemorrhagic fever and shock. While the parameters contributing to dengue immunopathogenesis remain unclear, the collapse of redox homeostasis and the damage induced by oxidative stress have been correlated with the development of inflammation and progression toward the more severe forms of disease. In the present study, we demonstrate that the accumulation of reactive oxygen species (ROS) late after DENV infection (>24 hpi) resulted from a disruption in the balance between oxidative stress and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant response. The DENV NS2B3 protease complex strategically targeted Nrf2 for degradation in a proteolysis-independent manner; NS2B3 licensed Nrf2 for lysosomal degradation. Impairment of the Nrf2 regulator by the NS2B3 complex inhibited the antioxidant gene network and contributed to the progressive increase in ROS levels, along with increased virus replication and inflammatory or apoptotic gene expression. By 24 hpi, when increased levels of ROS and antiviral proteins were observed, it appeared that the proviral effect of ROS overcame the antiviral effects of the interferon (IFN) response. Overall, these studies demonstrate that DENV infection disrupts the regulatory interplay between DENV-induced stress responses, Nrf2 antioxidant signaling, and the host antiviral immune response, thus exacerbating oxidative stress and inflammation in DENV infection.IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen that threatens 2.5 billion people in more than 100 countries annually. Dengue infection induces a spectrum of clinical symptoms, ranging from classical dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome; however, the complexities of DENV immunopathogenesis remain controversial. Previous studies have reported the importance of the transcription factor Nrf2 in the control of redox homeostasis and antiviral/inflammatory or death responses to DENV. Importantly, the production of reactive oxygen species and the subsequent stress response have been linked to the development of inflammation and progression toward the more severe forms of the disease. Here, we demonstrate that DENV uses the NS2B3 protease complex to strategically target Nrf2 for degradation, leading to a progressive increase in oxidative stress, inflammation, and cell death in infected cells. This study underlines the pivotal role of the Nrf2 regulatory network in the context of DENV infection.

Animal testing for vaccines. Implementing replacement, reduction and refinement: challenges and priorities.

Transition to in vitro alternative methods from in vivo in vaccine release testing and characterization, the implementation of the consistency approach, and a drive towards international harmonization of regulatory requirements are most pressing needs in the field of vaccines. It is critical for global vaccine community to work together to secure effective progress towards animal welfare and to ensure that vaccines of ever higher quality can reach the populations in need in the shortest possible timeframe. Advancements in the field, case studies, and experiences from Low and Middle Income Countries (LMIC) were the topics discussed by an international gathering of experts during a recent conference titled "Animal Testing for Vaccines - Implementing Replacement, Reduction and Refinement: Challenges and Priorities". This conference was organized by the International Alliance for Biological Standardization (IABS), and held in Bangkok, Thailand on December 3 and 4 2019. Participants comprised stakeholders from many parts of the world, including vaccine developers, manufacturers and regulators from Asia, Europe, North America, Australia and New Zealand. In interactive workshops and vibrant panel discussions, the attendees worked together to identify the remaining barriers to validation, acceptance and implementation of alternative methods, and how harmonization could be promoted, especially for LMICs.

Optimization of the monocyte activation test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine

Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: the rabbit pyrogen test (RPT), the bacterial endotoxin test, the recombinant factor C test, and the monocyte activation test (MAT). Here, we explored the possibility to replace the RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV). The testing was carried out using cryopreserved peripheral blood mononuclear cells as cell source. IL-6 release was selected as readout for the detection of both endotoxin and non-endotoxin contaminants. MAT applicability for pyrogen testing of the TBEV vaccine was assessed through preparatory tests and resulted in the establishment of a very sensitive assay (limit of detection (LOD) = 0.04 EU/mL; sensitivity = 0.1 EU/mL). Both quantitative Method A and semiquantitative Method B were used for data analysis. Our studies revealed that for a vaccine without intrinsic pyrogenicity, such as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD to define a stable maximum valid dilution of the product. In conclusion, we describe the challenges of MAT implementation for anti-TBEV vaccine following the current Ph. Eur. chapter 2.6.30 and propose a re-evaluation of the validity criteria of Methods A and B in order to set a semi-quantitative or limit test suitable for those products for which a reference lot comparison analysis is not applicable or favorable.

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.

Epstein-Barr virus-encoded EBNA2 alters immune checkpoint PD-L1 expression by downregulating miR-34a in B-cell lymphomas.

Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein-Barr virus (EBV)-associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III-expressing Burkitt lymphoma (BL), diffuse large B-cell lymphomas (DLBCL) or their EBNA2-transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III-expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2-transfected lymphoma cells. We identified early B-cell factor 1 (EBF1) as a repressor of miR-34a transcription. Short hairpin RNA (shRNA)-mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2-transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three-dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV-associated cancers.

Author Correction: Transcriptional dysregulation of Interferome in experimental and human Multiple Sclerosis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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.

Transcriptional dysregulation of Interferome in experimental and human Multiple Sclerosis.

Recent evidence indicates that single multiple sclerosis (MS) susceptibility genes involved in interferon (IFN) signaling display altered transcript levels in peripheral blood of untreated MS subjects, suggesting that responsiveness to endogenous IFN is dysregulated during neuroinflammation. To prove this hypothesis we exploited the systematic collection of IFN regulated genes (IRG) provided by the Interferome database and mapped Interferome changes in experimental and human MS. Indeed, central nervous system tissue and encephalitogenic CD4 T cells during experimental autoimmune encephalomyelitis were characterized by massive changes in Interferome transcription. Further, the analysis of almost 500 human blood transcriptomes showed that (i) several IRG changed expression at distinct MS stages with a core of 21 transcripts concordantly dysregulated in all MS forms compared with healthy subjects; (ii) 100 differentially expressed IRG were validated in independent case-control cohorts; and (iii) 53 out of 100 dysregulated IRG were targeted by IFN-beta treatment in vivo. Finally, ex vivo and in vitro experiments established that IFN-beta administration modulated expression of two IRG, ARRB1 and CHP1, in immune cells. Our study confirms the impairment of Interferome in experimental and human MS, and describes IRG signatures at distinct disease stages which can represent novel therapeutic targets in MS.