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.

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

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

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

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

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.

Apoptotic Epitope-Specific CD8+ T Cells and Interferon Signaling Intersect in Chronic Hepatitis C Virus Infection.

CD8(+) T cells specific to caspase-cleaved antigens derived from apoptotic T cells represent a principal player in chronic immune activation. Here, we found that both apoptotic epitope-specific and hepatitis C virus (HCV)-specific CD8(+) T cells were mostly confined within the effector memory (EM) or terminally differentiated EM CD45RA(+) cell subsets expressing a dysfunctional T-helper 1-like signature program in chronic HCV infection. However, apoptotic epitope-specific CD8(+) T cells produced tumor necrosis factor α and interleukin 2 at the intrahepatic level significantly more than HCV-specific CD8(+) T cells, despite both populations expressing high levels of programmed death 1 receptor. Contextually, only apoptotic epitope-specific CD8(+) T cells correlated with both interferon-stimulated gene levels in T cells and hepatic fibrosis score. Together, these data suggest that, compared with HCV-specific CD8(+) T cells, apoptotic epitope-specific CD8(+) T cells can better sustain chronic immune activation, owing to their capacity to produce tumor necrosis factor α, and exhibit greater resistance to inhibitory signals during chronic HCV infection.

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.

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.