Cell type specific involvement of RIG-I in antiviral responses

Toll-like receptors (TLRs) play an important role in antiviral response by recognizing viral components. Recently, a RNA helicase, RIG-I, is also suggested to recognize viral double-stranded RNA. However, how these molecules contribute to viral recognition in vivo is poorly understood. Here we would like to show that RIG-I is essential for induction of type I interferons (IFNs) after infection with RNA viruses in fibroblasts and conventional dendritic cells (DCs). RIG-I induces type I IFNs by activating IRF3 via IkappaB kinase related kinases. In contrast, plasmacytoid DC, which produce large amounts of IFN-alpha, use the TLR system rather than RIG-I for viral detection. Taken together, RIG-I and the TLR system exert antiviral responses in a cell type specific manner.     

Enhanced IL-2 in early life limits the development of TFH and protective antiviral immunity

T follicular helper cell (T_FH)–dependent antibody responses are critical for long-term immunity. Antibody responses are diminished in early life, limiting long-term protective immunity and allowing prolonged or recurrent infection, which may be important for viral lung infections that are highly prevalent in infancy. In a murine model using respiratory syncytial virus (RSV), we show that T_FH and the high-affinity antibody production they promote are vital for preventing disease on RSV reinfection. Following a secondary RSV infection, T_FH-deficient mice had significantly exacerbated disease characterized by delayed viral clearance, increased weight loss, and immunopathology. T_FH generation in early life was compromised by heightened IL-2 and STAT5 signaling in differentiating naive T cells. Neutralization of IL-2 during early-life RSV infection resulted in a T_FH-dependent increase in antibody-mediated immunity and was sufficient to limit disease severity upon reinfection. These data demonstrate the importance of T_FH in protection against recurrent RSV infection and highlight a mechanism by which this is suppressed in early life.     

Delivery of immunostimulatory RNA oligonucleotides by gelatin nanoparticles triggers an efficient antitumoral response

RNA oligonucleotides have emerged as a new class of biologicals that can silence gene expression but also stimulate immune responses through specific pattern-recognition receptors. The development of effective delivery systems remains a major challenge for the therapeutic application of the RNA oligonucleotides. In this study, we have established a novel biodegradable carrier system that is highly effective for the delivery of immunostimulatory RNA oligonucleotides. Formulation of RNA oligonucleotides with cationized gelatin nanoparticles potentiates immune activation through the Toll-like receptor 7 (TLR7) in both myeloid and plasmacytoid dendritic cells. Further, nanoparticle-delivered RNA oligonucleotides trigger production of the antitumoral cytokines IL-12 and IFN-α. Binding to gelatin nanoparticles protects RNA oligonucleotides from degradation by nucleases, facilitates their uptake by dendritic cells, and targets these nucleic acids to the endosomal compartment in which they are recognized by TLR7. In these effects, the nanoparticles are superior to the conventional transfection reagents lipofectamine, polyethylenimine, and DOTAP. In vivo, the delivery of TLR7-activating RNA oligonucleotides by gelatin nanoparticles triggers antigen-specific CD8+ T-cell and antibody responses. Indeed, immunization with RNA-loaded nanoparticles leads to an efficient antitumoral immune response in two different mouse tumor models. Thus, gelatin-based nanoparticles represent a novel delivery system for immunostimulatory RNA oligonucleotides that is both effective and nontoxic.     

Mouse and human lung fibroblasts regulate dendritic cell trafficking, airway inflammation, and fibrosis through integrin αvβ8-mediated activation of TGF-β

The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models - conditional deletion in mouse fibroblasts of the TGF-β-activating integrin αvβ8 and neutralization of αvβ8 in human COPD fibroblasts - we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvβ8-mediated activation of TGF-β by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.     

Protection against Respiratory Syncytial Virus Infection by DNA Immunization

Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality in infants and the elderly and is a continuing challenge for vaccine development. A murine T helper cell (Th) type 2 response associates with enhanced lung pathology, which has been observed in past infant trials using formalin-inactivated RSV vaccine. In this study, we have engineered an optimized plasmid DNA vector expressing the RSV fusion (F) protein (DNA-F). DNA-F was as effective as live RSV in mice at inducing neutralizing antibody and cytotoxic T lymphocyte responses, protection against infection, and high mRNA expression of lung interferon γ after viral challenge. Furthermore, a DNA-F boost could switch a preestablished anti-RSV Th2 response towards a Th1 response. Critical elements for the optimization of the plasmid constructs included expression of a secretory form of the F protein and the presence of the rabbit β-globin intron II sequence upstream of the F-encoding sequence. In addition, anti-F systemic immune response profile could be modulated by the route of DNA-F delivery: intramuscular immunization resulted in balanced responses, whereas intradermal immunization resulted in a Th2 type of response. Thus, DNA-F immunization may provide a novel and promising RSV vaccination strategy.     

Maternal gut microbiome regulates immunity to RSV infection in offspring

Development of the immune system can be influenced by diverse extrinsic and intrinsic factors that influence the risk of disease. Severe early life respiratory syncytial virus (RSV) infection is associated with persistent immune alterations. Previously, our group had shown that adult mice orally supplemented with Lactobacillus johnsonii exhibited decreased airway immunopathology following RSV infection. Here, we demonstrate that offspring of mice supplemented with L. johnsonii exhibit reduced airway mucus and Th2 cell–mediated response to RSV infection. Maternal supplementation resulted in a consistent gut microbiome in mothers and their offspring. Importantly, supplemented maternal plasma and breastmilk, and offspring plasma, exhibited decreased inflammatory metabolites. Cross-fostering studies showed that prenatal Lactobacillus exposure led to decreased Th2 cytokines and lung inflammation following RSV infection, while postnatal Lactobacillus exposure diminished goblet cell hypertrophy and mucus production in the lung in response to airway infection. These studies demonstrate that Lactobacillus modulation of the maternal microbiome and associated metabolic reprogramming enhance airway protection against RSV in neonates.     

Notch ligand Delta-like 4 regulates disease pathogenesis during respiratory viral infections by modulating Th2 cytokines

Recent data have indicated that an important instructive class of signals regulating the immune response is Notch ligand-mediated activation. Using quantitative polymerase chain reaction, we observed that only Delta-like 4 (dll4) was up-regulated on bone marrow-derived dendritic cells after respiratory syncytial virus (RSV) infection, and that it was dependent on MyD88-mediated pathways. Using a polyclonal antibody specific for dll4, the development of RSV-induced disease was examined. Animals treated with anti-dll4 had substantially increased airway hyperresponsiveness compared with control antibody-treated animals. When the lymphocytic lung infiltrate was examined, a significant increase in total CD4+ T cells and activated (perforin+) CD8+ T cells was observed. Isolated lung CD4+ T cells demonstrated significant increases in Th2-type cytokines and a decrease in interferon gamma, demonstrating an association with increased disease pathogenesis. Parallel in vitro studies examining the integrated role of dll4 with interleukin-12 demonstrated that, together, both of these instructive signals direct the immune response toward a more competent, less pathogenic antiviral response. These data demonstrate that dll4-mediated Notch activation is one regulator of antiviral immunity.     

Organ-dependent in vivo priming of naive CD4+, but not CD8+, T cells by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) play a pivotal role as cytokine-secreting accessory cells in the antimicrobial immune defense. In contrast, the capacity of PDCs to act as antigen-presenting cells in naive T cell priming remains unclear. By studying T cell responses in mice that lack conventional DCs (cDCs), and by the use of a PDC-specific antigen-targeting strategy, we show that PDCs can initiate productive naive CD4(+) T cell responses in lymph nodes, but not in the spleen. PDC-triggered CD4(+) T cell responses differed from cDC-driven responses in that they were not associated with concomitant CD8(+) T cell priming. Our results establish PDCs as a bona fide DC subset that initiates unique CD4(+) Th cell-dominated primary immune responses.     

Interferon-producing cells fail to induce proliferation of naive T cells but can promote expansion and T helper 1 differentiation of antigen-experienced unpolarized T cells

Interferon-producing cells (IPCs) secrete high levels of type I interferon in response to certain viruses. The lack of lineage markers, the expression of major histocompatibility complex (MHC) class II and the capacity to stimulate allogeneic T cells have led these cells to be classified as a subset of dendritic cells (DCs), called plasmacytoid DCs (PDCs). However, the role of IPCs/PDCs in initiating primary immune responses remains elusive. Here we examined the antigen presenting capacity of murine IPCs in antigen specific systems. While CD8alpha+ and CD11b+ DCs induced logarithmic expansion of naive CD4 and CD8 T cells, without conferring T helper commitment at a first encounter, primary IPCs lacked the ability to stimulate naive T cells. However, when antigen-experienced, nonpolarized T cells expanded by classical DC subsets, were restimulated by IPCs, they proliferated and produced high amounts of IFN-gamma. These data indicate that IPCs can effectively stimulate preactivated or memory-type T cells and exert an immune-regulatory role. They also suggest that expansion of naive T cells and acquisition of effector function during antigen-specific T cell responses may involve different antigen-presenting cell (APC) types. Independent and coordinated control of T cell proliferation and differentiation would provide the immune system with greater flexibility in regulating immune responses.     

CD103+ pulmonary dendritic cells preferentially acquire and present apoptotic cell-associated antigen

Cells undergoing programmed cell death (apoptosis) are removed in situ by macrophages and dendritic cells (DCs) through a specialized form of phagocytosis (efferocytosis). In the lung, there are two primary DC subsets with the potential to migrate to the local lymph nodes (LNs) and initiate adaptive immune responses. In this study, we show that only CD103(+) DCs were able to acquire and transport apoptotic cells to the draining LNs and cross present apoptotic cell-associated antigen to CD8 T cells. In contrast, both the CD11b(hi) and the CD103(+) DCs were able to ingest and traffic latex beads or soluble antigen. CD103(+) DCs selectively exhibited high expression of TLR3, and ligation of this receptor led to enhanced in vivo cytotoxic T cell responses to apoptotic cell-associated antigen. The selective role for CD103(+) DCs was confirmed in Batf3(-/-) mice, which lack this DC subtype. Our findings suggest that CD103(+) DCs are the DC subset in the lung that captures and presents apoptotic cell-associated antigen under homeostatic and inflammatory conditions and raise the possibility for more focused immunological targeting to CD8 T cell responses.     

Dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta

Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-alpha/beta in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8alpha+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-alpha/beta regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8alpha+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-alpha/beta, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.     

Type I interferons protect neonates from acute inflammation through interleukin 10-producing B cells

Newborns and infants are highly susceptible to viral and bacterial infections, but the underlying mechanism remains poorly understood. We show that neonatal B cells effectively control the production of proinflammatory cytokines by both neonatal plasmacytoid and conventional dendritic cells, in an interleukin (IL) 10-dependent manner, after Toll-like receptor (TLR) 9 triggering. This antiinflammatory property of neonatal B cells may extend to other TLR agonists (Pam3CSK4, lipopolysaccharide, and R848) and viruses. In the absence of B cells or of CD5(+) B cell subsets, neonatal mice developed stronger inflammatory responses and became lethally susceptible to CpG challenge after galactosamine sensitization, whereas wild-type (WT) mice were resistant. Paradoxically, interferon (IFN)-alpha/beta enhanced the inflammatory response to CpG challenge in adult mice, whereas they helped to control neonatal acute inflammation by stimulating the secretion of IL-10 by neonatal B cells. Finally, WT neonatal B cells rescued IL-10(-/-) neonates from a lethal CpG challenge, whereas IFN-alpha/beta receptor-deficient B cells did not. Our results show that type I IFNs support a negative regulatory role of neonatal B cells on TLR-mediated inflammation, with important implications for neonatal inflammation and infection.     

Dendritic cells as arbiters of peritoneal immune responses.

During the past few years, there has been a substantial increase in the understanding of innate immunity. Dendritic cells are emerging as key players in the orchestration of this early phase of immune responses, with a role that will translate into the subsequent type of adaptive immune response against infection. Here we provide an overview of dendritic cell differentiation and function, with particular emphasis on those features unique to the immune defense of the peritoneal cavity and in the context of peritoneal dialysis-associated immune responses. The reader is referred to the primary references included in the accompanying list for specific details in this fascinating field.     

Virus-specific CD8+ T Lymphocytes Downregulate T Helper Cell Type 2 Cytokine Secretion and Pulmonary Eosinophilia during Experimental Murine Respiratory Syncytial Virus Infection

T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8⁺ T cytolytic T cells on the differentiation and activation of functionally distinct CD4⁺ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I–restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8⁺ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8⁺ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8⁺ T cells in this process was further supported by the results in CD8⁺ T cell deficient, β₂ microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8⁺ T cell compartment. These results indicate that CD8⁺ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4⁺ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.Multiple arms of the immune system are activated in response to infection by foreign organisms. The outcomes of the infection, such as the clearance of the organisms and the generation of tissue injury, depend on these immune effector mechanisms that are mobilized against the pathogen. The role of T cells in the immune response to viral infections has been clearly established (1, 2). Mature T cells that express α/β T cell receptors can be divided into cells expressing either CD4 or CD8 surface antigen. CD8⁺ T cells recognize processed antigen in the context of MHC class I molecules, whereas CD4⁺ T cells recognize peptides in the context of MHC class II molecules (3, 4). The conventional view of CD8⁺ T cells has been primarily the killing of virally infected cells by direct cytolysis or by cytokines secreted by these T cells such as IFN-γ and TNF (1, 2). Functional subsets of CD4⁺ T cells have been described based on the cytokines produced by these cells, Th1 CD4⁺ T cells that secrete IL-2 and IFN-γ and Th2 T cells that secrete IL-4 and IL-5 (5, 6). The physiological relevance of these subsets of T cells with diverse functions have been shown in several models of viral infection including respiratory syncytial virus (RSV)¹.In the murine model of RSV infection, the roles of T cells and their products in the eradication of the virus as well as the pathogenesis of lung inflammation have been well documented (7–12). Recent studies have shown that mice sensitized to either of the two major glycoproteins of this virus developed distinct lung pathology when subsequently infected with RSV (12–15). Mice sensitized to the attachment glycoprotein (G) of RSV developed pulmonary eosinophilia that correlated with a strong induction of Th2 type response, whereas mice primed to the fusion (F) glycoprotein mounted a weak Th2 response and developed lung inflammation characterized by mononuclear cell infiltration. The underlying mechanisms that lead to these apparent distinct patterns of cytokine production and lung injury still remain unclear.The process by which CD4⁺ T cells mature and differentiate has been the subject of intense investigation (16). CD4⁺ T cells can be induced to differentiate into effector cells of either the Th1 or Th2 subsets, depending on the milieu in which these cells are activated. Cytokines such as IL-4 and IL-12 have been shown to play a crucial part in the regulation of CD4⁺ T cell differentiation (17–19). The presence of certain immune effector functions during the differentiation and expansion of CD4⁺ T cells may potentially be a key factor in determining the functional phenotypes acquired by these cells. In the murine model of experimental RSV infection, one important difference in the immune response to F and G glycoprotein of RSV is the lack of MHC class I–restricted cytolytic response to the G glycoprotein (13, 20). In the following studies, we began to investigate the impact of CD8⁺ T cells and MHC class I–restricted CTL responses on cytokine secretion and the subsequent development of pulmonary eosinophilia during experimental murine RSV infection. We have found that the induction of CD8⁺ T cell response to RSV proteins resulted in dramatically decreased levels of Th2 type cytokines and eosinophil recruitment into the lungs of RSV-infected mice previously sensitized to the RSV-G glycoprotein. The possible mechanisms underlying these observations and their potential significance are discussed.     

BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction.

Plasmacytoid dendritic cells are present in lymphoid and nonlymphoid tissue and contribute substantially to both innate and adaptive immunity. Recently, we have described several monoclonal antibodies that recognize a plasmacytoid dendritic cell-specific antigen, which we have termed BDCA-2. Molecular cloning of BDCA-2 revealed that BDCA-2 is a novel type II C-type lectin, which shows 50.7% sequence identity at the amino acid level to its putative murine ortholog, the murine dendritic cell-associated C-type lectin 2. Anti-BDCA-2 monoclonal antibodies are rapidly internalized and efficiently presented to T cells, indicating that BDCA-2 could play a role in ligand internalization and presentation. Furthermore, ligation of BDCA-2 potently suppresses induction of interferon alpha/beta production in plasmacytoid dendritic cells, presumably by a mechanism dependent on calcium mobilization and protein-tyrosine phosphorylation by src-family protein-tyrosine kinases. Inasmuch as production of interferon alpha/beta by plasmacytoid dendritic cells is considered to be a major pathophysiological factor in systemic lupus erythematosus, triggering of BDCA-2 should be evaluated as therapeutic strategy for blocking production of interferon alpha/beta in systemic lupus erythematosus patients.     

The development of respiratory syncytial virus-specific immune complexes in nasopharyngeal secretions following natural infection

Sequential samples of nasopharyngeal secretions (NPS) from 61 infants and children with respiratory syncytial virus (RSV) infection were examined for the presence of RSV-specific immune complexes in order to determine their possible role in recovery from or pathogenesis of RSV infection. Immune complexes in NPS were identified by the Raji cell assay using indirect immunofluorescence and RSV antigen in these complexes was also detected by indirect immunofluorescence. RSV-specific immune complexes were detected in 56% of subjects tested, being present as early as 3-6 days after the onset of illness and persisting for up to 36 days after the onset of illness. The appearance of immune complexes was temporally associated with the disappearance of RSV antigen from airway epithelial cells, suggesting a role for immune complexes in eradication of infection. RSV-specific immune complexes were identified with approximately equal frequency in patients with all forms of illness due to RSV, an observation which tends to rule out a role for immune complexes in the pathogenesis of RSV disease.     

Plasmacytoid dendritic cells lead the charge against tumors

Imiquimod is a TLR agonist that is used as an antitumor agent, mainly against skin tumors. Its clinical benefits are well described in several studies; however, the mechanisms behind its antitumor effects are not completely understood. In this issue of the JCI, Drobits and colleagues demonstrate that topical application of imiquimod suppresses cutaneous melanoma by TLR7-dependent recruitment and transformation of plasmacytoid dendritic cells into killer cells; this occurs independently of conventional adaptive immune mechanisms.     

Differential antigen presentation regulates the changing patterns of CD8+ T cell immunodominance in primary and secondary influenza virus infections

The specificity of CD8+ T cell responses can vary dramatically between primary and secondary infections. For example, NP366-374/Db- and PA224-233/Db-specific CD8+ T cells respond in approximately equal numbers to a primary influenza virus infection in C57BL/6 mice, whereas NP366-374/Db-specific CD8+ T cells dominate the secondary response. To investigate the mechanisms underlying this changing pattern of immunodominance, we analyzed the role of antigen presentation in regulating the specificity of the T cell response. The data show that both dendritic and nondendritic cells are able to present the NP366-374/Db epitope, whereas only dendritic cells effectively present the PA224-233/Db epitope after influenza virus infection, both in vitro and in vivo. This difference in epitope expression favored the activation and expansion of NP366-374/Db-specific CD8+ memory T cells during secondary infection. The data also show that the immune response to influenza virus infection may involve T cells specific for epitopes, such as PA224-233/Db, that are poorly expressed at the site of infection. In this regard, vaccination with the PA224-233 peptide actually had a detrimental effect on the clearance of a subsequent influenza virus infection. Thus, differential antigen presentation impacts both the specificity of the T cell response and the efficacy of peptide-based vaccination strategies.