Eliana M. Coccia

Viral infection and Toll-like receptor agonists induce a differential expression of type I and λ interferons in human plasmacytoid and monocyte-derived dendritic cells

In humans, the type I interferon (IFN) family consists of 13 IFN‐α subtypes, IFN‐β and IFN‐o the newly discovered IFN‐like family consists of IFN‐λ1, ‐λ2 and ‐λ3. We have investigated the expression of type I and λ IFN genes following virus infections or Toll‐like receptor (TLR) triggering in monocyte‐derived DC (MDDC) and plasmacytoid DC (pDC). We found thatall IFN‐α, ‐β, ‐o and ‐λ 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‐α, ‐β, ‐o and ‐λ subtypes in pDC, whereas TLR‐4 stimulation by LPS, or TLR‐3 stimulation bypoly I:C, induced only IFN‐β and IFN‐λ 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‐λ with IFN‐β would be of crucial importance for the maturation of DC.

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response.

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.

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

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.

IFN-αβ Released by Mycobacterium tuberculosis-Infected Human Dendritic Cells Induces the Expression of CXCL10: Selective Recruitment of NK and Activated T Cells

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-γ-inducing cytokines, IFN-αβ and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-αβ. Indeed, IFN-αβ neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4+, and CD8+ cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-αβ-induced expression of CXCL10, neutralization of IFN-αβ significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-αβ may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.

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-1β 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.

Selective expression of type I IFN genes in human dendritic cells infected with Mycobacterium tuberculosis.

Type I IFN regulates different aspects of the immune response, inducing a cell-mediated immunity. We have recently shown that the infection of dendritic cells (DC) with Mycobacterium tuberculosis (Mtb) induces IFN-α. In this work we have monitored a rapid induction of IFN-β followed by the delayed production of the IFN-α1 and/or -α13 subtypes. The Mtb infection rapidly activates the NF-κB complex and stimulates the phosphorylation of IFN regulatory factor (IRF)-3, events known to induce IFN-β expression in viral infection. In turn, the autocrine production of IFN-β induces the IFN-stimulated genes that contain binding sites for activated STATs in their promoters. Among the IFN-stimulated genes induced in DC through STAT activation are IRF-1 and IRF-7. The expression of IRF-1 appears to be dependent on the sequential activation of NF-κB and STAT-1. Once expressed, IRF-1 may further stimulate the transcription of IFN-β. Induction of IRF-7 is also regulated at the transcriptional level through the binding of phosphorylated STAT-1 and STAT-2, forming the IFN-stimulated gene factor-3 complex. In turn, the IRF-1 and IRF-7 expression appears to be required for the delayed induction of the IFN-α1/13 genes. Although correlative, our results strongly support the existence of a cascade of molecular events in Mtb-infected DC. Upon infection, constitutively expressed NF-κB and IRF-3 are activated and likely contribute to the rapid IFN-β expression. In turn, IFN-β-induced IRF-1 and IRF-7 may cooperate toward induction of IFN-α1/13 if infection persists and these factors are activated.

Characterization and Recruitment of Plasmacytoid Dendritic Cells in Synovial Fluid and Tissue of Patients with Chronic Inflammatory Arthritis

Dendritic cells (DCs) are thought to play a key role in driving the immunopathogenic response underlying chronic inflammatory arthritis. In this study, we have examined the presence and phenotype of plasmacytoid DCs (pDCs) in the synovial fluids (SF) of patients with rheumatoid arthritis (RA), psoriatic arthritis (PA), and osteoarthritis (OA) and determined the chemotactic properties of SF from these patients toward pDCs. Flow cytometry analysis showed that the percentage of pDCs, identified as a population of Lin−CD123++ cells, is 4- to 5-fold higher in RA SF and PA SF than in OA SF. The morphological and immunophenotypic characterization of pDCs isolated from PA and RA SF indicates that they are in an immature state, most likely due to inhibitory factors present in RA SF, but are still able to undergo maturation when exposed ex vivo to viral agent or unmethylated DNA. CD123+ and BDCA2+ pDCs were detected by immunohistochemistry in RA synovial tissue in which expression of the IFN-α-inducible protein MxA was also found, suggesting production of type I IFN by maturing pDCs. We also show that CXCR3 and CXCR4 are expressed by both blood-derived pDCs and pDCs isolated from RA and PA SF and that CXCL-10, CXCL-11, and CXCL-12 present in RA and PA SF stimulate chemotaxis of blood-derived pDCs. Altogether, these findings suggest that chemokine-driven recruitment of pDCs from the blood to the inflamed synovium could be important in the regulation of the immune response in chronic inflammatory arthritis.

Interferon-γ receptor 2 expression as the deciding factor in human T, B, and myeloid cell proliferation or death

Theheterodimeric interferon (IFN)‐γ receptor (IFN‐γR) is formed of two chains. Here we show that the binding chain (IFN‐γR1) was highly expressed on the membranes of T, B, and myeloid cells. Conversely, the transducing chain (IFN‐γR2) was highly expressed on the surfaces of myeloid cells, moderately expressed on B cells, and poorly expressed on the surfaces of T cells. Differential cell membrane expression of IFN‐γR2 determined the number of receptor complexes that transduced the IFN‐γ signal and resulted in a different response to IFN‐γ. After IFN‐γ stimulation, high IFN‐γR2 membrane expression induced rapid activation of signal transducer and activator of transcription‐1 (STAT‐1) and high levels of interferon regulatory factor‐1 (IRF‐1), which then triggered the apoptotic program. By contrast, low cell membrane expression resulted in slow activation of STAT‐1, lower levels of IRF‐1, and induction of proliferation. Because the forced expression of IFN‐γR2 on T cells switched their response to IFN‐γ from proliferative to apoptotic, we concluded that the surface expression of IFN‐γR2 determines whether a cell stimulated by IFN‐γ undergoes proliferation or apoptosis.

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 1and 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.

Lactobacilli and streptococci induce inflammatory chemokine production in human macrophages that stimulates Th1 cell chemotaxis

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.