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Highlights • Thymosins are thymic hormone-like peptides that can mediate immune and non-immune physiological processes. • Thymosin-α1 (Tα1) displays powerful pleiotropic activities by promoting anti-inflammatory and regulatory milieu in different settings. • Thymosin-β4 (Tβ4) provides neuroprotection, immunosuppression, and neurorestoration in the central nervous system. • This review describes what is known on the effects and the potential mechanisms of action of treatment with Tβ4 or Tα1 in Multiple Sclerosis and its experimental models., 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 -β4 (Tβ4) as potential anti-inflammatory as well as neuroprotective and remyelinating molecules and their mechanisms of action. Conclusion: Based on the data that Tα1 and Tβ4 act as anti-inflammatory molecules and as inducers of myelin repair and neuronal protection, respectively, a possible therapeutic application in MS for Tα1 and Tβ4 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 Tβ4 in clinical practice.

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., Author summary Though vaccination is generally considered effective in reducing tick-borne encephalitis (TBE) incidence, several studies have shown that the antibody response to TBEV vaccination declines with age resulting in more frequent TBE cases among 50+ year-old vaccinees. These observations together with the lack of a specific antiviral drug impose to pinpoint novel host- and pathogen-directed therapies and to improve the control of vaccine efficacy. Thus, we interrogated in vitro human PBMC, whose response to TBEV may provide a picture closer to what occurs in vivo in humans after vaccination or natural infection compared to animal models. The role of E glycoprotein and viral RNA in promoting antiviral and B cell-mediated responses was investigated. Thus, these key viral molecules should be considered, in future, for novel subunit vaccine formulations than the current whole inactivated TBEV-based vaccines, which require laborious manipulation in biosafety level-3 laboratory and animal testing for manufacturing and batch release.

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-β, 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-1β, IL-6, and TNF-α but to a reduction of IL-10. Interestingly, upon silencing or pharmacological inhibition of GSK-3β, the rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, indicating that, in Mtb-infected DC, GSK-3β 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-3β modulators as promising compounds for host-directed therapy in the control of Mtb infection.

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: 1) the Rabbit Pyrogen Test (RPT), 2) the Bacterial Endotoxin Test, 3) the Recombinant Factor C test and the Monocyte Activation Test (MAT). Here, we explored the possibility to replace 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 cellular 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 set-up of a very sensitive assay (limit of detection, LOD = 0.04 EU/ml; Sensitivity = 0.1 EU/ml). Both quantitative Method A and semi-quantitative Method B were used for data analysis. Our studies revealed that for vaccine without intrinsic pyrogenicity, as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD in order 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 MAT chapter 2.6.30 and propose a re-evaluation of the validity criteria of the 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.

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-β 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.

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.

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.

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.

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., Author summary Mycobacterium tuberculosis (Mtb) is one of the most successful pathogens in human history and remains the second leading cause of death from an infectious agent worldwide. The major reason of Mtb success relies on its ability to evade host immunity. Autophagy, a cellular mechanism involved in intracellular pathogen elimination, is one of the pathways hijacked by Mtb to elude the control of dendritic cells (DC), major cellular effectors of immune response. Recently, it has become clear that Mtb infection not only alters cellular gene expression, but also controls the level of small RNA molecules, namely microRNAs (miRNAs), which function as negative regulators of mRNA translation into protein. In the present study, we observed that the infection of human DC with Mtb leads to a strong induction of host miR-155, a critical regulator of host immune response. By mean of miR-155 induction, Mtb reduces Atg3 protein content, a crucial enzyme needed for the initial phase of the autophagic process. Interestingly, miR-155 silencing during Mtb infection restores Atg3 level and rescues autophagy. These findings contribute to better elucidate Mtb-triggered escape mechanisms and highlight the importance to develop host-directed therapies to combat tuberculosis based on autophagy boosting.

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.

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.

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.

Although data show the importance of type I interferons (IFNs) in the regulation of the innate and adaptive immunity elicited in response to viral, bacterial and parasitic infections, the functional activities of these cytokines during fungal infections are poorly understood. We examined here the impact of IFN-β on the response of human monocyte-derived dendritic cells (DCs) infected in vitro with Aspergillus fumigatus. Having found that A. fumigatus-infected DCs do not express IFN-β, we evaluated the effect of the exogenous addition of IFN-β on the maturation of human DCs induced by the infection with A. fumigatus conidia. Although the phagocytosis of the fungus was not affected by IFN-β treatment, the expression of CD86 and CD83 induced upon A. fumigatus challenge was enhanced in IFN-β-conditioned DCs, which also showed an increased expression of IL-27 and IL-12p70, members of IL-12 family. Through these modifications, IFN-β improved the capacity of DCs to promote an anti-Aspergillus T helper type 1 response, as evaluated by mixed leucocyte reaction, which plays a crucial role in the control of invasive aspergillosis. Our results identified a novel effect of IFN-β on anti-Aspergillus immune responses which, in turn, might open new perspectives on the use of IFN-β in immunotherapy for fungal infections aimed at enhancing the immunological functions of DCs.

Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen-presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon-α and granulocyte-macrophage colony-stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat-inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb-stimulated cells (SN Mtb) remained CD14(+) and poorly differentiated into CD1a(+) cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin-1β, interleukin-6, interleukin-10 and tumor necrosis factor-α upon Mtb stimulation. However, only interleukin-10 neutralization through the addition of soluble interleukin-10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin-10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen-activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin-10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non-infected monocytes into DCs through the release of interleukin-10. These results shed light on new aspects of the host-pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence.

Type I IFN are cytokines which play a central role in host resistance to viral or microbial infections and are important components linking innate and adaptive immunity. We and others have previously demonstrated that the production of IFN-beta by DC following bacterial infections or TLR triggering influences, in an autocrine manner, their maturation. In this study, we investigated whether IFN-beta release modulates the phenotype of the immature DC and their response to a subsequent TLR stimulation. The induction of CD86, HLA-DR, CD38 and B7H1 and the absence of CCR7 and CD83 expression upon IFN-beta treatment suggest that IFN-beta-primed DC remain at the site of infection acquiring an activated phenotype. These results prompted us to investigate the response of IFN-beta-primed DC to TLR stimulation. While IFN-beta pretreatment increases slightly the expression of maturation markers in TLR2- or TLR4-stimulated DC, it is able to modulate selectively the secretion of inflammatory and immuno-regulating cytokines. Interestingly, IL-27p28 subunit was induced by IFN-beta alone or during LPS-induced maturation of DC in a type I IFN-dependent manner through IFN regulatory factor-1 (IRF-1) activation. Taken together, our results shed light on the capacity of IFN-beta to finely tune DC response to invading pathogens.

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.DC maturation and production of pro-inflammatory cytokines were analyzed.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.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.

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.

Plasmacytoid dendritic cells (pDCs) display altered immune-phenotype in multiple sclerosis (MS) patients and are found actively recruited in postmortem MS brain lesions, implying that their immune regulation may represent an important aspect of MS pathogenesis. Because of the reported Toll-like receptor 7 (TLR7) implication in autoimmunity, in this study we characterized how IFN-β therapy impacts on pDC activation to TLR7 triggering in MS patients, aspect only poorly investigated so far. In vivo IFN-β administration regulates pDC functions in TLR7-treated peripheral blood mononuclear cell (PBMC) cultures differently from what is observed in isolated cells, suggesting that IFN-β may activate inhibitory mechanisms in MS peripheral blood involved in turning off pDC response to dampen the ongoing inflammation. Indeed, IL-10, a key regulatory cytokine found increased upon TLR7 stimulation in in vivo IFN-β-exposed PBMCs, directly reduced pDC-mediated IFN-α production. IFN-β therapy also shaped T-cell responses by decreasing TLR7-induced pDC maturation and inducing T-cell inhibitory molecules. Accordingly, raised pDC-induced IL-27 and decreased IL-23 expression, together with high IL-10 level, contribute to inhibit Th17 cell differentiation. Our study uncovered a role for IFN-β in the regulation of TLR7-mediated pDC responses in MS toward an anti-inflammatory phenotype opening new opportunities to better understand mechanisms of action of this drug in controlling MS immunopathogenesis.

Aspergillus fumigatus is a saprophytic fungus responsible for approximately 90% of human cases of aspergillosis. It causes a usually fatal invasive aspergillosis (IA) in immunocompromised hosts (13). The airborne conidia of A. fumigatus present in the atmosphere are small enough (2 to 3 μm in diameter) to be continuously inhaled and to reach the human lung alveoli, where they are captured, ingested, and killed by resident phagocytes in immunocompetent individuals (20). Otherwise, in immunocompromised patients, the conidia can overcome the immune defense mechanisms, germinating into mycelia that invade the lung. The major recognized risk factors for IA are defects in phagocytic function (25), corticosteroid-induced suppression of macrophage conidiocidal activity (30, 43), and long-lasting neutropenia (15). Numerous studies have shown that innate immune mechanisms play a key role in the defense against A. fumigatus. In particular, alveolar macrophages and neutrophils are the main cells involved in the rapid killing of A. fumigatus through oxidative and nonoxidative mechanisms (33, 50). Although the importance of the innate response has been well described both in vitro and in vivo, a recent observation showed that healthy individuals or patients with clinical evidence of IA and disease regression in antifungal therapy featured positive lymphoproliferation and a high level of gamma interferon (IFN-γ) (19). Correlated to these results, studies with the murine model of IA showed that resistance to infection was associated with the production of IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12), whereas a dominant release of Th2 cytokines by interstitial lung lymphocytes was correlated with the development of disease (8, 9). The fact that the Th response may modify the outcome of IA indirectly indicates a key role for human dendritic cells (DC) in the modulation of the immune response against A. fumigatus. Indeed, at the site of primary infection, DC constitute an integral part of the innate immune system, recognizing the pathogen and secreting inflammatory cytokines, such as TNF-α, IL-6, and IL-1 (38). After interacting with the pathogen, DC mature and migrate into the lymphoid organs, where they interact with T cells, transmitting information on the type of infection encountered and inducing a T-cell response through a coordinated stimulation via T-cell receptor engagement, costimulatory molecules, and cytokine production (37). IL-12p70, type I IFN, and IL-18 are well-documented examples of Th1-promoting cytokines (12). Recently other factors, belonging to the IL-12 family, were reported to play a key role in promoting a Th1 response. In particular, IL-23 acts primarily on effector T cells, prolonging and sustaining their IFN-γ production (46) and stimulating the proliferation of memory T cells (27, 52), whereas IL-27 has a strong effect, especially on naive Th cells inducing the early production of IFN-γ (32). The interaction of human DC with A. fumigatus and its consequence for the immune defense against this pathogen have been studied previously (4, 18, 41, 44). However in the present study, we investigated the impact of A. fumigatus infection on human DC maturation, focusing on the expression of CCR7 and novel members of the IL-12 family. Interestingly, we observed that the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. In addition, analysis of inflammatory and immunoregulatory cytokines was performed in order to determine the capacity of DC to elicit an anti-Aspergillus immune response. In particular, our results with regard to the IL-12, IL-23, and IL-27 expression profiles provide new insights into the mechanisms promoting the anti-Aspergillus Th1 response.

In humans, the type I interferon (IFN) family consists of 13 IFN-alpha subtypes, IFN-beta and IFN-omicron the newly discovered IFN-like family consists of IFN-lambda1, -lambda2 and -lambda3. We have investigated the expression of type I and lambda IFN genes following virus infections or Toll-like receptor (TLR) triggering in monocyte-derived DC (MDDC) and plasmacytoid DC (pDC). We found that all IFN-alpha, -beta, -omicron and -lambda subtypes are expressed in influenza-virus-infected MDDC or pDC. Conversely, differential type I IFN gene transcription was induced in MDDC and pDC stimulated by specific TLR agonists. TLR-9 stimulation by CpG DNA induced the expression of all IFN-alpha, -beta, -omicron and -lambda subtypes in pDC, whereas TLR-4 stimulation by LPS, or TLR-3 stimulation by poly I:C, induced only IFN-beta and IFN-lambda gene expression in MDDC. The expression pattern of IFN regulatory factor (IRF)-5 and IRF-7 in MDDC and pDC was also determined. IRF-5 was constitutively expressed in the two DC subsets whereas IRF-7 was constitutive in pDC but its expression was induced along MDDC maturation. Overall, our data indicate that the coordinated expression of IFN-lambda with IFN-beta would be of crucial importance for the maturation of DC.

Macrophages have a central role in innate-immune responses to bacteria. In the present work, we show that infection of human macrophages with Gram-positive pathogenic Streptococcus pyogenes or nonpathogenic Lactobacillus rhamnosus GG enhances mRNA expression of inflammatory chemokine ligands CCL2/monocyte chemoattractant protein-1 (MCP-1), CCL3/macrophage-inflammatory protein-1α (MIP-1α), CCL5/regulated on activation, normal T expressed and secreted, CCL7/MCP-3, CCL19/MIP-3β, and CCL20/MIP-3α and CXC chemokine ligands CXCL8/interleukin (IL)-8, CXCL9/monokine induced by interferon-γ (IFN-γ), and CXCL10/IFN-inducible protein 10. Bacteria-induced CCL2, CCL7, CXCL9, and CXCL10 mRNA expression was partially dependent on ongoing protein synthesis. The expression of these chemokines and of CCL19 was dependent on bacteria-induced IFN-α/β production. CCL19 and CCL20 mRNA expression was up-regulated by IL-1β or tumor necrosis factor α (TNF-α), and in addition, IFN-α together with TNF-α further enhanced CCL19 gene expression. Synergy between IFN-α and TNF-α was also seen for CXCL9 and CXCL10 mRNA expression. Bacteria-stimulated macrophage supernatants induced the migration of T helper cell type 1 (Th1) cells, suggesting that in human macrophages, these bacteria can stimulate efficient inflammatory chemokine gene expression including those that recruit Th1 cells to the site of inflammation. Furthermore, L. rhamnosus-induced Th1 chemokine production could in part explain the proposed antiallergenic properties of this bacterium.

We previously reported that infection byMycobacterium tuberculosis, the causative agent of tuberculosis, leads to secretion of alpha/beta interferon (IFN-α/β). While IFN-α/β ordinarily stimulates formation of signal transducer and stimulator of transcription-1 (STAT-1) homodimers and IFN-stimulated gene factor-3 (ISGF-3), only ISGF-3 is found in infected human monocytes and macrophages. We have now investigated the basis for this unusual profile of transcription factor activation and its consequences for regulation of transcription, as well as the impact of infection on response to IFN-α. After infection, IFN-α stimulation of STAT-1 homodimers is inhibited in monocytes and macrophages, while stimulation of ISGF-3 increases in monocytes but tends to decline in macrophages. Effects of infection on the abundance of ISGF-3 subunits, STAT-1, STAT-2, and interferon regulatory factor 9, and on tyrosine phosphorylation of STAT-1 and STAT-2 explain the observed changes in DNA-binding activity, which correlate with increased or inhibited transcription of genes regulated by ISGF-3 and STAT-1. Infection byMycobacterium bovisBCG does not inhibit IFN-α-stimulated tyrosine phosphorylation of STAT-1, formation of homodimers, or transcription of genes regulated by STAT-1 homodimers, suggesting that inhibition of the response to IFN-α/β byM. tuberculosisis an aspect of pathogenicity. Thus, this well-known feature of infection by pathogenic viruses may also be a strategy employed by pathogenic bacteria.

Macrophage cell lines exhibit different responses to IFN-gamma depending on their maturation stage. We investigated the mechanisms underlying the differential IFN-gamma responsiveness in the less mature P388.D1 and in mature RAW264.7 cells. A reduction in the binding activity of the signal transducer and activator of transcription-1 (STAT-1) to different STAT binding elements (SBEs) was observed in P388.D1. This reduced binding activity was not due to an impaired STAT-1 activation. Studies on the expression of a negative regulator of cytokine signalling, protein-inhibiting activated STAT-1 (PIAS-1), showed that this protein was expressed constitutively at high levels in P388.D1. Forced expression of a PIAS-1 homologue, the Gu binding protein (GBP), inhibited the STAT-1-mediated gene activation in RAW264.7 cells, whereas a construct expressing the 5' portion of GBP in the antisense orientation reverts the IFN-gamma-resistant phenotype of P388.D1. Thus, our results indicate that PIAS-1 may account for the differential IFN-gamma responsiveness in macrophage cell lines at different stages of maturation.

Alveolar macrophages and newly recruited monocytes are targets of infection by Mycobacterium tuberculosis. Therefore, we examined the expression of interferon regulatory factor 1 (IRF-1), which plays an important role in host defense against M. tuberculosis, in undifferentiated and differentiated cells. Infection induced IRF-1 in both. IRF-1 from undifferentiated, uninfected monocytic cell lines was modified during extraction to produce specific species that were apparently smaller than intact IRF-1. After infection by M. tuberculosis or differentiation, intact IRF-1 was recovered. Subcellular fractions were assayed for the ability to modify IRF-1 or inhibit its modification. A serine protease on the cytoplasmic surface of an organelle or vesicle in the "lysosomal/mitochondrial" fraction from undifferentiated cells was responsible for the modification of IRF-1. Thus, the simplest explanation of the modification is cleavage of IRF-1 by the serine protease. Recovery of intact IRF-1 correlated with induction of a serine protease inhibitor that was able to significantly reduce the modification of IRF-1. The inhibitor was present in the cytoplasm of M. tuberculosis-infected or -differentiated cells. It is likely that induction of both IRF-1 and the serine protease inhibitor in response to infection by M. tuberculosis represent host defense mechanisms.

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.

A major challenge in tuberculosis (TB) is to improve current vaccination and therapeutic strategies and this requires a fine understanding of the mechanisms that mediate protection and pathogenesis. We need to discern how the host perceives Mycobacterium tuberculosis (Mtb) infection, what are the danger signals that activate the immune system and, in turn, how the immune response controls the life-cycle of Mtb. Cytokines, because of their nature of soluble mediators, represent key elements in mediating and tuning these complex processes. In this review, we provide an overview of recent studies on cytokines expression and function in active (mainly human) TB. Understanding of the balance between pro- and anti-inflammatory networks is crucial to refine our knowledge on the immune responses directed against Mtb.

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-α, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-γ-inducing cytokines, IL-12 and IFN-α, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-γ production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-α, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-α and IL-18 play a significant role in enhancing IFN-γ synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.

Previous work from our laboratory demonstrated that a synthetic heptapeptide (Ch7: RGSDIAG), corresponding to a conserved sequence of human immunodeficiency virus (HIV) core protein p24 (amino acids 232- 238), was able to specifically abrogate antigen-induced responses in cultures of normal human peripheral blood mononuclear cells (PBMC), probably acting at the level of monocytes. The Ch7 peptide displays sequence homology to human plasminogen. In the present report we show that a compound (6-aminoexanoic acid), known to prevent plasminogen binding to monocyte-like cells, greatly reduced the immunosuppressive capacity of Ch7. We suggest that the plasminogen receptor may represent a target structure on human monocytes for the immunosuppressive p24 sequence.

The influence of interleukin-12 (IL-12) on the induction of a specific antibody response to the T-dependent antigen sheep erythrocytes (SRBC) in cultures of human blood lymphocytes was investigated. The response, evaluated as number of antigen-induced antibody-producing cells, was greatly increased in the presence of IL-12. When a two-stage limiting dilution culture system was used, the plot of the number of seeded cells versus the logarithm of the fraction of negative cultures deviated from linearity in antigen- and IL-12-stimulated cultures. However, linearity was reached when IL-2 was added in the second stage. Under these latter conditions, since single-hit criteria were fulfilled, it was possible to estimate the frequency of SRBC-specific B cell precursors able to respond to the antigen and to show that such frequency was increased upon addition of IL-12. Thus, the enhancing effect of IL-12 may be based on an increased frequency of responding precursor cells. The results here presented demonstrate, to our knowledge for the first time, a definite role of IL-12 in the induction of a specific antibody response in human cells. Further, they stress the importance for such studies of appropriate in vitro systems. Finally, they show that the induction of primary immune responses in cultures of human peripheral blood lymphocytes mostly depends on the proper cytokine balance at different time points.

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.

We studied the effects of the antiretroviral drugs 3’-azido-3’-deoxythymidine (AZT) and 2’,3’-dideoxycytidine (DDC) on the induction of a specific primary antibody response to the antigen SRC and on Pokeweed mitogen-induced polyclonal immunoglobulin (Ig) production in cultures of normal human peripheral blood lymphocytes.The results presented here show that AZT and DDC inhibited both systems in a dose-dependent way. However, inhibition of the antigen-specific antibody response occurred at drug concentrations significantly lower than those effective on the mitogen-driven polyclonal response. This was particularly striking for DDC, which was approximately 100 times more powerful, on a molar basis, on the specific antibody response than on polyclonal Ig production. Thus, in vitro systems for the induction of antigen-specific antibody responses in human lymphocytes may be important, sensitive tools for preclinical studies of the immunotoxicity of antiretroviral drugs.

International audience; Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.

Ch7 (RGSDIAG), a synthetic heptapeptide derived from a conserved region of HIV p24 (aa 232-238), was previously shown to suppress antigen-induced responses in cultures of normal human peripheral blood lymphocytes (PBL). We show in this paper that Ch7 is the shortest peptide retaining full inhibitory capacity. Further, the peptide inhibited efficiently and in a dose-dependent manner the induction of a specific antibody response to the antigens SRC (sheep red cells) and Candida albicans but did not exert any effect on the induction of immunoglobulin-secreting cells in PWM-stimulated cultures. Finally, Ch7 inhibited anti-CD3-induced lympho-proliferation but did not affect anti-CD2 activation. These results suggest that a conserved epitope of HIV p24 may be able to prevent the induction of antigen-specific antibody responses by interfering with lymphocyte activation via the T3-Ti complex, resulting in the abrogation of immune functions that are defective in HIV-infected individuals.

A method is described for the induction of a specific antibody response to Candida albicans in cultures of normal human peripheral blood lymphocytes (PBL). PBL were cultured in the presence of whole C. albicans cells and the antibody response was evaluated by the ELISPOT technique, on plastic wells coated with a purified candidal cell well mannoprotein (MP). Under the conditions described here, a specific antibody response was obtained in all of the eight donors tested. The response was antigen-dependent and antigen-specific, peaked around day 10–12 of culture and the antibodies belonged to both the IgM and the IgG isotypes. By testing the cultured cells on MP from different Candida species, the method per detection of antibodies directed against MP epitopes shared by C. albicans and C. parapsilosis.

Knowledge of the molecular events regulating the innate response toMycobacterium tuberculosis(Mtb) is critical for understanding immunological pathogenesis and protection from tuberculosis. To this aim, the regulation and the expression of regulatory and proinflammatory cytokines were investigated in human primary monocytes upon Mtb infection. We found that Mtb-infected monocytes preferentially express a proinflammatory cytokine profile, including IL-6, TNF-α, and IL-1β. Conversely, among the regulatory cytokines, Mtb elicited IL-10 and IL-23 release while no expression of IL-12p70, IL-27, and IFN-βwas observed. The analysis of the signalling pathways leading to this selective cytokine expression showed that in monocytes Mtb activates MAPK and NF-κB but is unable to stimulate IRF-3 phosphorylation, a transcription factor required for IL-12p35 and IFN-βgene expression. Thus, by inducing a specific cytokine profile, Mtb can influence the immunoregulatory properties of monocytes, which represent important target of novel vaccinal strategies against Mtb infection.

Given the variable protective efficacy provided by Mycobacterium bovis bacillus Calmette-Guérin (BCG), there is an urgent need to develop new vaccines against tuberculosis. As dendritic cells (DC) play a critical role in initiating and regulating a protective T cell response against the pathogens, the comprehension of mycobacterium-induced modulation of DC functions is critical to pinpoint new, immunological strategies. To this end, a comparative analysis of the effect induced by BCG and Mycobacterium tuberculosis (Mtb) infection on the DC immunophenotype indicated that BCG is less efficient in inducing DC maturation than Mtb. In addition, BCG-infected DC poorly expressed IFN-β and displayed a reduced production of IL-12 as compared with Mtb-stimulated cells. The impaired expression of IL-12p35 and IFN-β is likely a result of the inability of BCG to induce the activation of the IFN regulatory factor-3. Taking into account these data, we sought to investigate whether the exogenous addition of IFN-β, a cytokine that exerts important effects on the immune system, could enhance the Th1-polarizing capacity of BCG-infected DC. Interestingly, when DC infected by BCG were pretreated in vitro with IFN-β, they displayed a fully mature phenotype and released a significant amount of bioactive IL-12p70, which resulted in an enhanced Th1 response. This study demonstrates that IFN-β potentiates DC immunological functions following BCG infection, thus suggesting IFN-β as a possible candidate as vaccine adjuvant.

The roles of plasmacytoid dendritic cells (pDCs) and their response to interferon (IFN)-beta therapy in multiple sclerosis (MS) patients are poorly understood. We identified pDC accumulation in white matter lesions and leptomeninges of MS brains and abundant expression of the Type I IFN-induced protein MxA, mainly in perivascular CD3+ lymphocytes in lesions, indicating Type I IFN production by activated pDCs. The pDC chemoattractant chemerin was detected in intralesional cerebrovascular endothelial cells, and the chemerin receptor was expressed on infiltrating leukocytes, including pDCs. The effect of IFN-beta on pDC phenotype and function was evaluated in MS patients before and during IFN-beta treatment. Although IFN-beta did not modify the frequency and immature phenotype of circulating pDC, they showed lower expression of major histocompatibility complex Class II and blood-dendritic cell antigen 2 molecules and upregulation of CD38 and B7H1 costimulatory molecules. On exposure to CpG (a site where cytosine [C] lies next to guanine [G] in the DNA sequence [the p indicates that C and G are connected by a phosphodiester bond]) oligodeoxynucleotides in vitro, pDCs from IFN-beta-treated MS patients showed reduced expression of the pDC maturation markers CD83 and CD86 molecules; in vitro IFN-beta treatment of pDCs from healthy donors resulted in lower secretion of proinflammatory cytokines, including IFN-alpha, and a decreased ability to stimulate allogeneic T cells in response to maturative stimuli. These data indicate that IFN-beta modulates the immunologic functions of pDC, thus identifying pDCs as a novel target of IFN-beta therapy in MS patients.

TLRs interact with a growing list of pathogen-derived products and these interactions drive the activation of innate and adaptive immune responses. Dendritic cells (DC) play a key role in these events expressing a heterogeneous repertoire of TLRs. We have previously demonstrated the production of type I IFNs in DC following bacterial infections and TLR triggering. In this study, we sought to characterize the transcriptome specifically induced in human DC by IFN-β production stimulated upon LPS treatment. To this aim, by using cDNA microarrays, we compared the transcriptome of DC following LPS treatment in the absence or presence of neutralizing anti-type I IFN Abs. Interestingly, we found that the expression of TLR7 was induced during LPS-induced maturation of DC in a type I IFN-dependent manner. The induction of TLR7 in maturing DC was mainly a consequence of the transcriptional activity of IRF-1, whose binding site was located within TLR7 promoter. Moreover, we also demonstrated that “priming” of immature DC, that usually express TLR8 but not TLR7, with exogenous IFN-β induced a functionally active TLR7. In fact, treatment with the TLR7-specific ligand 3M-001 up-regulated the expression of CD83, CD86, and CD38 in IFN-β-primed DC but not in immature DC. Therefore, a robust enhancement in proinflammatory as well as regulatory cytokines was observed. These data suggest that TLR4-mediated type I IFN release activates specific transcription programs in DC amplifying the expression of pathogen sensors to correctly and combinatorially respond to a bacterial as well as viral infection.

The transcription factor STAT-4 plays a pivotal role in the IL-12-mediated development of naive CD4+ T cells into the Th1 phenotype. Initially thought to be restricted to the lymphoid lineage, STAT-4 was subsequently shown to be expressed in the myeloid compartment, mainly in activated monocytes, macrophages, and dendritic cells (DC). Here, we have studied STAT-4 in human monocyte-derived DC, and we demonstrated that its expression can be induced by multiple stimuli, such as the ligands for TLR-4, TLR-2, and TLR-3, different pathogens, CD40 ligand, and the proinflammatory cytokines TNF-α and IL-1β. It is interesting that we found that STAT-4 is tyrosine-phosphorylated in response to type I IFN but not IL-12 in human mature DC. Cloning and functional analysis of the STAT-4 promoter showed that a NF-κB binding site, localized at –969/–959 bp upstream of the transcriptional start site, is involved in the regulation of this gene in primary human DC. EMSAs using a probe containing this NF-κB binding sequence and chromatin immunoprecipitation indicated that p65/p50 and p50/p50 dimers were the main NF-κB/Rel proteins involved in STAT-4 gene expression in maturing DC. The mutation of this κB site or the overexpression of the repressor IκBα exerted an inhibitory effect on a STAT-4 promoter-driven reporter as well as on STAT-4 expression. Altogether, these results indicate that STAT-4 can be finely tuned along with DC maturation through NF-κB activation and that its induction may be involved in preparing the DC to be receptive to the cytokine environment present in lymphoid organs.

Background Calcium channel blockers are widely used in transplantation. Their immunosuppressive activity is well known and has been demonstrated both in vitro and in vivo. Nevertheless, their effect on cytokine production has never been reported. Methods One-way mixed lymphocyte cultures (MLCs) have been obtained from healthy human subjects. Cytokine production has been assessed by three different methods: by enzyme-linked immunosorbent assay on supernatants of MLC, by enzyme-linked immunospot method on MLC cells for measuring cytokine-producing cells, and by the reverse transcription polymerase chain reaction technique on MLC cells for measuring cytokine mRNAs. Results An interesting effect on proinflammatory monokines was observed: in this study, we demonstrate that the calcium antagonist diltiazem enhances interleukin-1beta and slightly reduces interleukin-6 production in MLC, but it has no effect on tumor necrosis factor-alpha levels. Conclusion For the first time, a modulation of monokine production by diltiazem can be demonstrated. This evidence suggests that calcium antagonist drugs may exert effects on monocytes and possibly on other antigen-presenting cells.

Given the role played by chemokines in the selective homing of immune cells, we sought to characterize the profile of chemokines produced by human dendritic cells (DC) following in vitro Aspergillus fumigatus infection and their ability to recruit cells involved in the antifungal defense. At the onset of A. fumigatus infection, DC released elevated amounts of CXCL8 that promote the migration of polymorphonuclear cells (PMN). Moreover, soluble factors released from A. fumigatus-infected DC increased also the surface expression of two activation markers, CD11b and CD18, on PMN. A. fumigatus infection resulted also in CCL3, CCL4, CXCL10 and CCL20 productions that induce the migration of effector memory Th1 cells. Moreover, the late expression of CCL19 suggests that A. fumigatus-infected DC could be implicated in the migration of CCR7+ naive T cells and mature DC in lymph nodes. Together these results suggested the involvement of human DC in the regulation of innate and adaptive immunity against A. fumigatus through the recruitment of cells active in the fungal destruction.

In human monocyte-derived dendritic cells (DC), infection with Mycobacterium tuberculosis and viruses or stimulation with Toll-like receptor type 3 and 4 agonists causes the release of type I interferon (IFN). Here, we describe that the IFN-β released upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) is responsible for a rapid and sustained signal transducer and activator of transcription 1 and 2 activation and expression of IFN-stimulated genes, such as the transcription factor IFN regulatory factor 7 and the chemokine CXC chemokine ligand 10. The autocrine production of IFN-β from LPS and poly I:C-matured DC (mDC) induced a temporary saturation of the response to type I IFN and a marked decline in the level of the two IFN receptor (IFNAR) subunits. It is interesting that we found that upon clearing of the released cytokines, LPS-stimulated DC reacquired full responsiveness to IFN-β but only partial responsiveness to IFN-α, and their maturation process was unaffected. Monitoring of surface and total levels of the receptor subunits showed that maximal expression of IFNAR2 resumed within 24 h of clearing, and IFNAR1 expression remained low. Thus, mDC can modulate their sensitivity to two IFN subtypes through a differential regulation of the IFNAR subunits.