Fabiana Rizzo

Epstein–Barr virus persistence and infection of autoreactive plasma cells in synovial lymphoid structures in rheumatoid arthritis

Objectives Rheumatoid arthritis (RA) is associated with an increased Epstein–Barr virus (EBV) blood DNA load, a robust immune response to EBV and cross-reactive circulating antibodies to viral and self-antigens. However, the role of EBV in RA pathogenesis remains elusive. Here, we investigated the relationship between synovial EBV infection, ectopic lymphoid structures (ELS) and immunity to citrullinated self and EBV proteins. Methods Latent and lytic EBV infection was investigated in 43 RA synovial tissues characterised for presence/absence of ELS and in 11 control osteoarthritis synovia using RT-PCR, in situ hybridisation and immunohistochemistry. Synovial production of anti-citrullinated protein (ACPA) and anti-citrullinated EBV peptide (VCP1/VCP2) antibodies was investigated in situ and in vivo in the severe combined immunodeficiency (SCID)/RA chimeric model. Results EBV dysregulation was observed exclusively in ELS+ RA but not osteoarthritis (OA) synovia, as revealed by presence of EBV latent (LMP2A, EBV-encoded small RNA (EBER)) transcripts, EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, a large proportion of ACPA-producing plasma cells surrounding synovial germinal centres were infected with EBV. Furthermore, ELS-containing RA synovia transplanted into SCID mice supported production of ACPA and anti-VCP1/VCP2 antibodies. Analysis of CD4+ and CD8+ T-cell localisation and granzyme B expression suggests that EBV persistence in ELS-containing synovia may be favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. Conclusions We demonstrated active EBV infection within ELS in the RA synovium in association with local differentiation of ACPA-reactive B cells.

EBV stimulates TLR- and autophagy-dependent pathways and impairs maturation in plasmacytoid dendritic cells: implications for viral immune escape.

Plasmacytoid DCs (pDCs) are crucial mediators in the establishment of immunity against most viruses, given their extraordinary capacity to produce a massive quantity of type I IFN. In this study we investigate the response of pDCs to infection with EBV, a γ‐herpes virus that persists with an asymptomatic infection in immunocompetent hosts, although in certain conditions it can promote development of cancers or autoimmune diseases. We show that high amounts of type I IFNs were released from isolated pDCs after exposure to EBV by a mechanism requiring TLRs and a functional autophagic machinery. We next demonstrate that EBV can infect pDCs via viral binding to MHC class II molecule HLA‐DR and that pDCs express EBV‐induced latency genes. Furthermore, we observe that EBV is able to induce activation but not maturation of pDCs, which correlates with an impaired TNF‐α release. Accordingly, EBV‐infected pDCs are unable to mount a full T‐cell response, suggesting that impaired pDC maturation, combined with a concomitant EBV‐mediated upregulation of the T‐cell inhibitory molecules B7‐H1 and ICOS‐L, could represent an immune‐evasion strategy promoted by the virus. These mechanisms might lead to persistence in immunocompetent hosts or to dysregulated immune responses linked to EBV‐associated diseases.

Interferon‐β therapy specifically reduces pathogenic memory B cells in multiple sclerosis patients by inducing a FAS‐mediated apoptosis

Growing evidences put B lymphocytes on a central stage in multiple sclerosis (MS) immunopathology. While investigating the effects of interferon‐β (IFN‐β) therapy, one of the most used first‐line disease‐modifying drugs for the treatment of relapsing‐remitting MS, in circulating B‐cell sub‐populations, we found a specific and marked decrease of CD27+ memory B cells. Interestingly, memory B cells are considered a population with a great disease‐driving relevance in MS and resulted to be also target of B‐cell depleting therapies. In addition, Epstein–Barr virus (EBV), associated with MS etiopathogenesis, harbors in this cell type and an IFN‐β‐induced reduction of the memory B‐cell compartment, in turn, resulted in a decreased expression of the EBV gene latent membrane protein 2A in treated patients. We found that in vivo IFN‐β therapy specifically and highly induced apoptosis in memory B cells, in accordance with a strong increase of the apoptotic markers Annexin‐V and active caspase‐3, via a mechanism requiring the FAS‐receptor/TACI (transmembrane activator and CAML interactor) signaling. Thus, efficacy of IFN‐β therapy in MS may rely not only on its recognized anti‐inflammatory activities but also on the specific depletion of memory B cells, considered to be a pathogenic cell subset, reducing their inflammatory impact in target organs.

IFN-β and multiple sclerosis: Cross-talking of immune cells and integration of immunoregulatory networks

Multiple sclerosis (MS) is characterized by autoimmune inflammation affecting the central nervous system and subsequent neurodegeneration. Historically, damage was thought to be mediated exclusively by auto-antigen-activated pro-inflammatory T cells. However, more recently, we are gaining increasing knowledge on the pathogenic role played in MS by B cells, dendritic cells and monocytes. IFN-β therapy was one the first approved therapy for MS for its ability to reduce relapse rate and MRI lesion activity and to significantly decrease risk of disability progression. IFN-β-mediated mechanisms of action, even if not completely understood, mainly rely on its multifaceted pleiotropic effects resulting in sustained anti-inflammatory properties directed toward almost every immune cell type. Here, we will discuss in detail literature data characterizing the pathogenic activity of the different immune cell subsets involved in MS pathogenesis and how IFN-β therapy regulates their function by modulating bystander responses. We believe that the effectiveness of this drug in MS treatment, even if in use for a long time, can unveil new insights on this disease and still teach a lesson to researchers in the MS field.

IFN‐β therapy modulates B‐cell and monocyte crosstalk via TLR7 in multiple sclerosis patients

The implication of B lymphocytes in the immunopathology of multiple sclerosis (MS) is increasingly recognized. Here we investigated the response of B cells to IFN‐β, a first‐line therapy for relapsing‐remitting MS patients, upon stimulation with TLR. IFN‐β restored the frequency of TLR7‐induced IgM and IgG‐secreting cells in MS patients to the levels found in healthy donors, showing a specific deficiency in the TLR7 pathway. However, no difference was observed in the TLR9 response. Furthermore, in MS‐derived PBMCs, TLR7‐mediated production of IL‐6 and the ex vivo expression of B‐cell‐activating factor of the TNF family, two crucial cytokines for B‐cell differentiation and survival, were induced by IFN‐β. Depletion of monocytes, which are key producers of both IL‐6 and B‐cell‐activating factor of the TNF family, showed that TLR7‐mediated B‐cell differentiation into Ig‐secreting cells is strongly dependent on the cross‐talk between B cells and monocytes. Accordingly, impaired expression of TLR7 mRNA was observed in PBMCs and monocytes isolated from MS‐affected individuals as compared with those from healthy donors, which was rescued by IFN‐β therapy. Collectively, our data unveil a novel TLR7‐regulated mechanism in in vivo IFN‐β‐stimulated whole leukocytes that could be exploited to define new TLR7‐based strategies for the treatment of MS.

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.

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.

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-1β 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.