Novel evidence of Thymosin α1 immunomodulatory properties in SARS-CoV-2 infection: Effect on innate inflammatory response in a peripheral blood mononuclear cell-based in vitro model.

The peculiar property of Thymosin alpha 1 (Tα1) to act as master regulator of immune homeostasis has been successfully defined in different physiological and pathological contexts ranging from cancer to infection. Interestingly, recent papers also demonstrated its mitigating effect on the "cytokine storm" as well as on the T-cell exhaustion/activation in SARS-CoV-2 infected individuals. Nevertheless, in spite of the increasing knowledge on Tα1-induced effects on T cell response confirming the distinctive features of this multifaceted peptide, little is known on its effects on innate immunity during SARS-CoV-2 infection. Here, we interrogated peripheral blood mononuclear cell (PBMC) cultures stimulated with SARS-CoV-2 to disclose Tα1 properties on the main cell players of early response to infection, namely monocytes and myeloid dendritic cells (mDC). Moving from ex vivo data showing an enhancement in the frequency of inflammatory monocytes and activated mDC in COVID-19 patients, a PBMC-based experimental setting reproduced in vitro a similar profile with an increased percentage of CD16+ inflammatory monocytes and mDC expressing CD86 and HLA-DR activation markers in response to SARS-CoV-2 stimulation. Interestingly, the treatment of SARS-CoV-2-stimulated PBMC with Tα1 dampened the inflammatory/activation status of both monocytes and mDC by reducing the release of pro-inflammatory mediators, including TNF-α, IL-6 and IL-8, while promoting the production of the anti-inflammatory cytokine IL-10. This study further clarifies the working hypothesis on Tα1 mitigating action on COVID-19 inflammatory condition. Moreover, these evidence shed light on inflammatory pathways and cell types involved in acute SARS-CoV-2 infection and likely targetable by newly immune-regulating therapeutic approaches.

Analysis of coding and non-coding transcriptome of peripheral B cells reveals an altered interferon response factor (IRF)-1 pathway in multiple sclerosis patients.

Several evidences emphasize B-cell pathogenic roles in multiple sclerosis (MS). We performed transcriptome analyses on peripheral B cells from therapy-free patients and age/sex-matched controls. Down-regulation of two transcripts (interferon response factor 1-IRF1, and C-X-C motif chemokine 10-CXCL10), belonging to the same pathway, was validated by RT-PCR in 26 patients and 21 controls. IRF1 and CXCL10 transcripts share potential seeding sequences for hsa-miR-424, that resulted up-regulated in MS patients. We confirmed this interaction and its functional effect by transfection experiments. Consistent findings indicate down-regulation of IRF1/CXCL10 axis, that may plausibly contribute to a pro-survival status of B cells in MS.

Fatty acid metabolism complements glycolysis in the selective regulatory T cell expansion during tumor growth.

The tumor microenvironment restrains conventional T cell (Tconv) activation while facilitating the expansion of Tregs. Here we showed that Tregs' advantage in the tumor milieu relies on supplemental energetic routes involving lipid metabolism. In murine models, tumor-infiltrating Tregs displayed intracellular lipid accumulation, which was attributable to an increased rate of fatty acid (FA) synthesis. Since the relative advantage in glucose uptake may fuel FA synthesis in intratumoral Tregs, we demonstrated that both glycolytic and oxidative metabolism contribute to Tregs' expansion. We corroborated our data in human tumors showing that Tregs displayed a gene signature oriented toward glycolysis and lipid synthesis. Our data support a model in which signals from the tumor microenvironment induce a circuitry of glycolysis, FA synthesis, and oxidation that confers a preferential proliferative advantage to Tregs, whose targeting might represent a strategy for cancer treatment.

Interferon-ß 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-ß (IFN-ß) 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-ß-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-ß 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-ß 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.

Pro- and anti-inflammatory cytokines in tuberculosis: a two-edged sword in TB pathogenesis.

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.

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.

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.

Bystander inhibition of dendritic cell differentiation by Mycobacterium tuberculosis-induced IL-10.

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-1ß, 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.

Epstein-Barr virus latent infection and BAFF expression in B cells in the multiple sclerosis brain: implications for viral persistence and intrathecal B-cell activation.

A cardinal feature of multiple sclerosis (MS) is the persistent intrathecal synthesis of antibodies. Our previous finding that a large fraction of B cells infiltrating the MS brain are infected with Epstein-Barr virus (EBV) raises the possibility that this virus, because of its ability to establish a latent infection in B cells and interfere with their differentiation, contributes to B-cell dysregulation in MS. The aim of this study was to gain further insight into EBV latency programs and their relationship to B-cell activation in the MS brain. Immunohistochemical analysis of postmortem MS brain samples harboring large EBV deposits revealed that most B cells in white matter lesions, meninges, and ectopic B-cell follicles are CD27+ antigen-experienced cells and coexpress latent membrane protein 1 and latent membrane protein 2A, 2 EBV-encoded proteins that provide survival and maturation signals to B cells. By combining laser-capture microdissection with preamplification reverse transcription-polymerase chain reaction techniques, EBV latency transcripts (latent membrane protein 2A, EBV nuclear antigen 1) were detected in all MS brain samples analyzed. We also found that B cell-activating factor of the tumor necrosis factor family is expressed in EBV-infected B cells in acute MS lesions and ectopic B-cell follicles. These findings support a role for EBV infection in B-cell activation in the MS brain and suggest that B cell-activating factor of the tumor necrosis factor family produced by EBV-infected B cells may contribute to this process resulting in viral persistence and, possibly, disruption of B-cell tolerance.

IFN regulation and functions in myeloid dendritic cells.

A central issue in dendritic cells (DC) biology is to understand how type I IFNs modulate the immuno-regulatory properties of DC. In this review I will address this issue in light of the recent experimental evidence on the expression and function of these cytokines in myeloid DC. This knowledge may have important therapeutic implications in infectious and neoplastic diseases and open new perspectives in the use of IFNs as vaccine adjuvants and in the development of DC-based vaccines.

Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions.

As a result of their close association with the blood-brain barrier, astrocytes play an important role in regulating the homing of different leukocyte subsets to the inflamed central nervous system (CNS). In this study, we investigated whether human astrocytes produce chemokines that promote the migration of myeloid dendritic cells (DCs). By reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, we show that cultured human astrocytes stimulated with interleukin-1beta and tumor necrosis factor produce CCL2, CCL3, CCL4, CCL5, CCL20, and CXCL12 that act on immature DCs, but not CCL19 and CCL21, 2 chemokines specific for mature DCs. Compared with controls, supernatants of cytokine-stimulated astrocytes are more effective in promoting the migration of immature monocyte-derived DCs (iMDDCs). Desensitization of CXCR4 (receptor for CXCL12), CCR1-3-5 (shared receptors for CCL3-4-5), and CCR6 (receptor for CCL20) on iMDDC reduces cell migration toward astrocyte supernatants, indicating that astrocytes release biologically relevant amounts of iMDDC-attracting chemokines. By immunohistochemistry, we show that CXCL12 and, to a lesser extent, CCL20 are expressed by reactive astrocytes in multiple sclerosis lesions. These data lend support to the idea that astrocyte-derived chemokines may contribute to immature DC recruitment to the inflamed CNS.