Patricia Fitzgerald-Bocarsly

Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod.

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I interferon in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary interferon-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-alpha and IFN-omega from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-alpha and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-3 and IFN-alpha; resiquimod more effectively matures pDC than either IL-3 or IFN-alpha alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.

Comparative analysis of IRF and IFN-alpha expression in human plasmacytoid and monocyte-derived dendritic cells

Plasmacytoid dendritic cells (PDC) produce high levels of type I IFN upon stimulation with viruses, while monocytes and monocyte‐derived dendritic cells (MDDC) produce significantly lower levels. To find what determines the high production of type I IFN in PDC, we examined the relative levels of IRF transcription factors, some of which play critical roles in the induction of IFN. Furthermore, to determine whether the differences could result from expression of distinct IFNA subtypes, the profile of IFNA genes expressed was examined. PDC responded equally well to stimulation with HSV‐1 and Sendai virus (SV) by producing high levels of type I IFN, whereas the MDDC and monocyte response to SV were lower, and neither responded well to HSV‐1. All three populations constitutively expressed most of the IRF genes. However, real‐time RT‐PCR demonstrated increased levels of IRF‐7 transcripts in PDC compared with monocytes. As determined by intracellular flow cytometry, the PDC constitutively expressed significantly higher levels of IRF‐7 protein than the other populations while IRF‐3 levels were similar among populations. Analysis of the profile of IFNA genes expressed in virus‐stimulated PDC, monocytes and MDDC demonstrated that each population expressed IFNA1 as the major subtype but that the range of the subtypes expressed in PDC was broader, with some donor and stimulus‐dependent variability. We conclude that PDC but not MDDC are uniquely preprogrammed to respond rapidly and effectively to a range of viral pathogens with high levels of IFN‐α production due to the high levels of constitutively expressed IRF‐7.

Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history.

It has been 50 years since the initial descriptions of what are now known as plasmacytoid dendritic cells (pDC) and type I IFN. pDC, which are infrequent cells found in the peripheral blood and lymphoid organs, are the most potent producers of type I and type III IFNs in the body. pDC produce IFN-alpha in response to both DNA and RNA enveloped viruses by virtue of their ribonucleic acids signaling in the endosome through TLR9 and TLR7, respectively. This stimulation, which also occurs with DNA or RNA-containing immune complexes and synthetic TLR7 and -9 agonists, is dependent upon the transcription factor IRF-7, which is expressed at high constitutive levels in pDC. In addition to releasing as much as 3-10pg of IFN-alpha/cell, pDC are also potent modulators of the immune response. In this review, we discuss the signaling pathways in pDC, their roles in linking innate and adaptive immunity, and their roles in infectious disease and autoimmunity.

Virally stimulated plasmacytoid dendritic cells produce chemokines and induce migration of T and NK cells

The natural interferon (IFN)‐producing cell is now known to be identical to the plasmacytoid dendritic cell (PDC). These are Lin−, CD123+, CD11c−, and human leukocyte antigen‐DR+ cells that secrete large amounts of IFN‐α (1–2 IU/cell) when stimulated by enveloped viruses such as herpes simplex virus. In the current study, we have evaluated chemokine expression by virally stimulated PDC. Up‐regulation of mRNA for CCL4, CCL3, CCL5, CCL2, and CXC chemokine ligand (CXCL)10 in herpes simplex virus‐stimulated PDC was detected by RNAse protection assays. In contrast, PDC‐depleted peripheral blood mononuclear cells did not up‐regulate these mRNA species upon viral stimulation. Enzyme‐linked immunosorbent assay and/or intracellular flow cytometry confirmed production of these proteins, and studies indicated overlapping production of IFN‐α and the other cytokines/chemokines by PDC. Endocytosis plays a critical role in chemokine induction, as disruption of the pathway inhibits the response. However, transcription of viral genes is not required for chemokine induction. Autocrine IFN‐α signaling in the PDC could account for a portion of the CXCL10 and CCL2 production in virally stimulated PDC but was not responsible for the induction of the other chemokines. To evaluate the functional role of the chemokines, chemotaxis assays were performed using supernatants from virally stimulated PDC. Activated T cells and natural killer cells, but not naïve T cells, were preferentially recruited by these PDC supernatants. Migration was subsequently inhibited by addition of neutralizing antibody to CCL4 and CXCL10. We hypothesize that virally induced chemokine production plays a pivotal role in the homing of leukocytes to PDC.

Two Discrete Promoters Regulate the Alternatively Spliced Human Interferon Regulatory Factor-5 Isoforms MULTIPLE ISOFORMS WITH DISTINCT CELL TYPE-SPECIFIC EXPRESSION, LOCALIZATION, REGULATION, AND FUNCTION

Interferon regulatory factor-5 (IRF-5) is a mediator of virus-induced immune activation and type I interferon (IFN) gene regulation. In human primary plasmacytoid dendritic cells (PDC), IRF-5 is transcribed into four distinct alternatively spliced isoforms (V1, V2, V3, and V4), whereas in human primary peripheral blood mononuclear cells two additional new isoforms (V5 and V6) were identified. The IRF-5 V1, V2, and V3 transcripts have different noncoding first exons and distinct insertion/deletion patterns in exon 6. Here we showed that V1 and V3 have distinct transcription start sites and are regulated by two discrete promoters. The V1 promoter (P-V1) is constitutively active, contains an IRF-E consensusbinding site, and is further stimulated in virus-infected cells by IRF family members. In contrast, endogenous V3 transcripts were up-regulated by type I IFNs, and the V3 promoter (P-V3) contains an IFN-stimulated responsive element-binding site that confers responsiveness to IFN through binding of the ISGF3 complex. In addition to V5 and V6, we have identified three more alternatively spliced IRF-5 isoforms (V7, V8, and V9); V5 and V6 were expressed in peripheral blood mononuclear cells from healthy donors and in immortalized B and T cell malignancies, whereas expression of V7, V8, and V9 transcripts were detected only in human cancers. The results of this study demonstrated the existence of multiple IRF-5 spliced isoforms with distinct cell type-specific expression, cellular localization, differential regulation, and dissimilar functions in virus-mediated type I IFN gene induction.

Regulation of IFN Regulatory Factor-7 and IFN-α Production by Enveloped Virus and Lipopolysaccharide in Human Plasmacytoid Dendritic Cells

Human plasmacytoid dendritic cells (PDC) are a major source of IFN-α upon exposure to enveloped viruses and TLR-7 and TLR-9 ligands. Although IFN regulatory factor-7 (IRF-7) is known to play an essential role in virus-activated transcription of IFN-α genes, the molecular mechanisms of IFN-α production in human PDC remain poorly understood. We and others have recently reported high constitutive levels of IRF-7 expression in PDC as compared with other PBMC. In this study, we demonstrate that both LPS and HSV up-regulate the expression of IRF-7 in PDC, and that this enhancement of IRF-7 is dependent on NF-κB activation. The NF-κB inhibitors MG132 and pyrrolidinedithiocarbamate efficiently inhibited the induction of IRF-7 by HSV or LPS, and also down-regulated the constitutive expression of IRF-7 in PDC and blocked the HSV-induced production of IFN-α. In addition, we found that nuclear translocation of IRF-7 occurred rapidly in response to HSV stimulation, but not in response to LPS, which is consistent with the stimulation of IFN-α production by virus and not by LPS. Although LPS by itself was not able to induce IFN-α production, it led to rapid up-regulation of TLR-4 on PDC and increased the magnitude and accelerated the kinetics of HSV-induced IFN-α production in PDC, providing a mechanism that might be operative in a scenario of mixed infection. In contrast to the current concept of IFN-α regulation established in cell lines, this study strongly supports the immediate availability of high constitutive levels of IRF-7 expression in PDC, and suggests an activation required for IRF-7 that contributes to IFN-α production in virus-stimulated PDC.

Plasmacytoid dendritic cells in HIV infection: striking a delicate balance.

pDC are the most potent IFN‐α‐producing cells in the body and serve as a vital link between innate and adaptive immunity. Deficiencies in pDC function were among the earliest observations of immune dysfunction in HIV‐1 infection. Herein, we review the status of pDC in individuals with HIV‐1 infection and the potential role of these cells in pathogenesis. We begin by reviewing the basic properties of pDC and then discuss the compromise in circulating pDC numbers and function in early and viremic HIV‐1 infection and mechanisms that might account for their depletion in HIV‐infected patients. In addition, we review the evidence that chronic production of IFN‐α, probably through the chronic activation of pDC, is central to the immune activation that is so detrimental in HIV infection. Finally, we discuss the importance of balance in pDC numbers and function and the potential value of using absolute pDC counts and function as a biomarker, along with CD4+ cell counts and VL in HIV‐1‐infected patients.

Interferon-α generation and immune reconstitution during antiretroviral therapy for human immunodeficiency virus infection

OBJECTIVES To quantify the effect of HIV infection and HIV-suppressive therapy on interferon-alpha (IFN-alpha) production by human blood mononuclear cells; to compare, in parallel, effects on CD4+ T-cell numbers; and to ascertain the relationship of these interferon and CD4 parameters to resistance to opportunistic infections. DESIGN Serial studies of 294 unselected patients with HIV infection during therapy, with outcomes analysis. METHODS Determination of IFN generation by blood mononuclear cells via bioassay, and T-lymphocyte subset analysis via flow cytometry; serial studies of individual patients; linear regression and chi2 contingency table analysis. RESULTS HIV burden is inversely related to interferon-alpha generation, much as it is to CD4+ T-cell counts. Both of these recover during HIV-suppressive therapy. Reconstitution of IFN-alpha generation to levels commensurate with protection against opportunistic infection occurs prior to similar restoration of CD4 counts. In the outcomes analyses, such immune reconstitution was associated with protection from recurrent or new opportunistic infection. Conversely, viral suppression without such immunologic recovery was not protective against opportunistic infection. CONCLUSIONS Rapidly responding IFN-alpha generating cells appear to participate in resistance to opportunistic intracellular infection. Recovery of IFN-alpha generation may be an early marker of immune reconstitution in AIDS.

Type III IFNs Are Produced by and Stimulate Human Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDC) are rare cells found in peripheral blood and lymphoid tissues. pDC are considered to be “professional” type I IFN-producing cells and produce 10- to 100-fold more IFN-α than other cell types in response to enveloped viruses or synthetic TLR7 and TLR9 agonists. In this study, purified pDC were found to express high levels of IFN-λ receptor mRNA, as well as cell-surface IFN-λ receptor. We have developed intracellular flow cytometry assays using Abs to IFN-λ1/3 or -λ2 to assess the expression of IFN-λ proteins by pDC. We observed that a subset of human pDC expresses only intracellular IFN-α, whereas another subset produces both IFN-α and IFN-λ after stimulation with virus or the TLR9 agonist, CpG A; the cells that coexpressed IFN-α and IFN-λ were the cells with the highest levels of IFN-α expression. Ab cross-linking of CD4 or CD303 molecules on pDC inhibited both HSV-induced IFN-λ and IFN-α production. Like the production of IFN-α, the HSV-induced IFN-λ production in pDC was mediated through TLR9 and independent of virus replication. Exogenous IFN-λ treatment of pDC resulted in increased virus-induced expression of both IFN-α and IFN-λ. In addition, both exogenous IFN-λ and -α inhibited dexamethasone-induced apoptosis of pDC. We conclude that pDC are major producers of IFN-λ1 and -λ2 in response to viral stimulation and also express functional receptors for this cytokine. Thus, IFN-λ can serve as an autocrine signal to strengthen the antiviral response of pDC by increasing IFN-α and IFN-λ production, resulting in prolonged pDC survival.

The nervous system as ectopic germinal center: CXCL13 and IgG in lyme neuroborreliosis.

Lyme neuroborreliosis (LNB) is a chronic infection in which B‐cell activation, plasma cell infiltration, and enhanced Ig production in infected tissue are prominent feature. However, little is known about how B cells and plasma cells invade and persist in target organs. To assess this issue, we developed real‐time PCR measurements of IgG and CXCL13 production. We used these RNA assays and specific enzyme‐linked immunosorbent assays for protein and demonstrated that human peripheral blood mononuclear cells (PBMCs), stimulated by Borrelia burgdorferi sonicate, produced CXCL13 and IgG. Magnetic separation of PBMC populations and flow cytometry showed that CXCL13 is produced by dendritic cells. We then measure the expression of CXCL13 and IgG in tissues and correlated the expression of these host genes with spirochetal load. We also measured expression of dbpA and BBK32, two spirochetal genes important in chronic infection. There was a strong correlation between host immune response gene expression (CXCL13 and IgG) and spirochetal load. Immunohistochemistry of infected nonhuman primates tissue confirmed that CXCL13 is expressed in the nervous system. We conclude that persistent production of CXCL13 and IgG within infected tissue, two characteristics of ectopic germinal centers, are definitive features of LNB. Ann Neurol 2005;57:813–823