Lara E. Kallal

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 γ, demonstrating an association with increased disease pathogenesis. Parellel 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.

A novel inactivated intranasal respiratory syncytial virus vaccine promotes viral clearance without TH2 associated Vaccine-Enhanced disease

Background Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960s led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations. Principal Findings In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines. Conclusions These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.

Type 1 Interferon Induction of Natural Killer Cell Gamma Interferon Production for Defense during Lymphocytic Choriomeningitis Virus Infection

ABSTRACT Natural killer (NK) cells are equipped to innately produce the cytokine gamma interferon (IFN-γ) in part because they basally express high levels of the signal transducer and activator of transcription 4 (STAT4). Type 1 interferons (IFNs) have the potential to activate STAT4 and promote IFN-γ expression, but concurrent induction of elevated STAT1 negatively regulates access to the pathway. As a consequence, it has been difficult to detect type 1 IFN stimulation of NK cell IFN-γ during viral infections in the presence of STAT1 and to understand the evolutionary advantage for maintaining the pathway. The studies reported here evaluated NK cell responses following infections with lymphocytic choriomeningitis virus (LCMV) in the compartment handling the earliest events after infection, the peritoneal cavity. The production of type 1 IFNs, both IFN-α and IFN-β, was shown to be early and of short duration, peaking at 30 h after challenge. NK cell IFN-γ expression was detected with overlapping kinetics and required activating signals delivered through type 1 IFN receptors and STAT4. It took place under conditions of high STAT4 levels but preceded elevated STAT1 expression in NK cells. The IFN-γ response reduced viral burdens. Interestingly, increases in STAT1 were delayed in NK cells compared to other peritoneal exudate cell (PEC) populations. Taken together, the studies demonstrate a novel mechanism for stimulating IFN-γ production and elucidate a biological role for type 1 IFN access to STAT4 in NK cells. IMPORTANCE Pathways regulating the complex and sometimes paradoxical effects of cytokines are poorly understood. Accumulating evidence indicates that the biological consequences of type 1 interferon (IFN) exposure are shaped by modifying the concentrations of particular STATs to change access to the different signaling molecules. The results of the experiments presented conclusively demonstrate that NK cell IFN-γ can be induced through type 1 IFN and STAT4 at the first site of infection during a period with high STAT4 but prior to induction of elevated STAT1 in the cells. The response mediates a role in viral defense. Thus, a very early pathway to and source of IFN-γ in evolving immune responses to infections are identified by this work. The information obtained helps resolve long-standing controversies and advances the understanding of mechanisms regulating key type 1 IFN functions, in different cells and compartments and at different times of infection, for accessing biologically important functions. Pathways regulating the complex and sometimes paradoxical effects of cytokines are poorly understood. Accumulating evidence indicates that the biological consequences of type 1 interferon (IFN) exposure are shaped by modifying the concentrations of particular STATs to change access to the different signaling molecules. The results of the experiments presented conclusively demonstrate that NK cell IFN-γ can be induced through type 1 IFN and STAT4 at the first site of infection during a period with high STAT4 but prior to induction of elevated STAT1 in the cells. The response mediates a role in viral defense. Thus, a very early pathway to and source of IFN-γ in evolving immune responses to infections are identified by this work. The information obtained helps resolve long-standing controversies and advances the understanding of mechanisms regulating key type 1 IFN functions, in different cells and compartments and at different times of infection, for accessing biologically important functions.

CCL20/CCR6 blockade enhances immunity to RSV by impairing recruitment of DC

Chemokines are important mediators of the immune response to pathogens, but can also promote chronic inflammatory states. CCR6 is a chemokine receptor found on dendritic cells and T cells, suggesting its role in both innate and adaptive immunity. We investigated the role of CCR6 in a pulmonary viral infection caused by respiratory syncytial virus (RSV), a ubiquitous virus that can cause severe pulmonary complications. We found that CCR6−/− mice had reduced pathophysiology and lower numbers of activated T cells in the lungs post-RSV challenge. Analysis of RSV-specific cytokine responses showed that CCR6−/− mice skewed preferentially toward a Th1 effector response. Correspondingly, viral clearance was rapid and early in CCR6−/− mice, suggesting an efficient immune response. Early timepoint analysis revealed that CCR6−/− mice had significantly fewer conventional DCs (cDCs) recruited into the lung, but the same number of plasmacytoid DCs. A pathogenic phenotype could be reconstituted in CCR6−/− mice by supplying cDCs into the airway, indicating that mere number of cDCs dictates the adverse response. Our data suggest that CCR6 deficiency provides an environment whereby the balance of innate immune cells mediates the efficient antiviral response to RSV.Chemokines are important mediators of the immune response to pathogens, but can also promote chronic inflammatory states. Chemokine receptor 6 (CCR6) is found on immature DC and effector/memory T cells, and binds a single ligand, CCL20, with high affinity. Here, we investigated the role of CCL20 and CCR6 in a pulmonary viral infection caused by RSV, a ubiquitous virus that can cause severe pulmonary complications. Neutralization of CCL20 during RSV infection significantly reduced lung pathology and favored a Th1 effector response. CCR6‐deficient animals recapitulated this phenotype, and additionally showed enhanced viral clearance when compared with WT mice. No differences were observed in migration of T cells to the lungs of CCR6−/− animals; however, a significant reduction was observed in numbers of conventional DC (cDC), but not plasmacytoid DC, in CCR6−/− mice. A pathogenic phenotype could be reconstituted in CCR6−/− mice by supplying cDC into the airway, indicating that mere number of cDC dictates the adverse response. Our data suggest that blockade of the CCL20/CCR6 pathway provides an environment whereby the attenuated recruitment of cDC alters the balance of innate immune cells and mediates the efficient antiviral response to RSV.

A Key Role for CC Chemokine Receptor 1 in T-Cell-Mediated Respiratory Inflammation

CC chemokine receptor 1 (CCR1) is found on a variety of cells in the immune system and has been shown to play an important role in the host response to pathogens. These studies used a murine model of virus-induced exacerbation of allergic airway disease to examine the role of CCR1 on T cells associated with immune responses taking place in the lung. Lungs of virally exacerbated allergic animals contained elevated levels of interferon-gamma and interleukin-13 and increased levels of CCR1 ligands CCL3 and CCL5. CCR1 expression on T cells was increased in virally exacerbated allergic animals over the level observed in mice sensitized to allergen or exposed to viral infection alone. Using mice deficient for CCR1, we observed decreased airway hyperreactivity and Th2 cytokine production from CD4(+) T cells when this receptor was absent. Transfer studies demonstrated that neither CD4(+) nor CD8(+) T cells from CCR1(-/-) mice migrated to the lymph node as efficiently as wild-type T cells. Intracellular cytokine staining in wild-type mice revealed that CCR1(+) CD4(+) and CD8(+) T cells are associated with interleukin-13 production. Thus, these studies identify CCR1 as a potential target for alleviating T-cell accumulation during exacerbation of asthmatic disease.

Inefficient Lymph Node Sensitization during Respiratory Viral Infection Promotes IL-17–Mediated Lung Pathology

Development of bronchus-associated lymphoid tissue has been suggested to enhance local antiviral immune responses; however, ectopic lymph node formation often corresponds to chronic inflammatory diseases. These studies investigated the role of ectopic pulmonary lymph nodes upon respiratory syncytial virus (RSV) infection using CCR7-deficient mice, which develop bronchus-associated lymphoid tissue early in life. CCR7−/− mice exhibited impaired secondary lymph node formation, enhanced effector T cell responses and pathogenic mucus production in the lung after RSV infection. IL-17 production from CD4 T cells in CCR7−/− mice was most remarkably enhanced. Wild-type animals reconstituted with CCR7−/− bone marrow recapitulated the pathogenic lung phenotype in CCR7−/− mice, whereas CCR7−/− animals reconstituted with wild-type bone marrow had normal lymph node development, diminished IL-17 production and reduced lung pathology. Mixed bone marrow chimeras revealed an alteration of immune responses only in CCR7−/− T cells, suggesting that impaired trafficking promotes local effector cell generation. Lymphotoxin-α–deficient mice infected with RSV were used to further examine locally induced immune responses and demonstrated increased mucus production and amplified cytokine responses in the lung, especially IL-17. Neutralization of IL-17 in CCR7−/− or in lymphotoxin-α–deficient animals specifically inhibited mucus hypersecretion and reduced IL-13. Thus, immune cell trafficking to secondary lymph nodes is necessary for appropriate cytokine responses to RSV as well as modulation of the local environment.

Changing partners at the dance: Variations in STAT concentrations for shaping cytokine function and immune responses to viral infections

Differential use of cellular and molecular components shapes immune responses, but understanding of how these are regulated to promote defense and health during infections is still incomplete. Examples include signaling from members of the Janus activated kinase-signal transducer and activator of transcription (JAK-STAT) cytokine family. Following receptor stimulation, individual JAK-STAT cytokines have preferences for particular key STAT molecules to lead to specific cellular responses. Certain of these cytokines, however, can conditionally activate alternative STATs as well as elicit pleiotropic and paradoxical effects. Studies examining basal and infection conditions are revealing intrinsic and induced cellular differences in various intracellular STAT concentrations to control the biological consequences of cytokine exposure. The system can be likened to changing partners at a dance based on competition and relative availability, and sets a framework for understanding the particular conditions promoting subset biological functions of cytokines as needed during evolving immune responses to infections.

TRIM68 negatively regulates IFN-β production by degrading TRK fused gene, a novel driver of IFN-β downstream of anti-viral detection systems.

In recent years members of the tripartite motif-containing (TRIM) family of E3 ubiquitin ligases have been shown to both positively and negatively regulate viral defence and as such are emerging as compelling targets for modulating the anti-viral immune response. In this study we identify TRIM68, a close homologue of TRIM21, as a novel regulator of Toll-like receptor (TLR)- and RIG-I-like receptor (RLR)-driven type I IFN production. Proteomic analysis of TRIM68-containing complexes identified TRK-fused gene (TFG) as a potential TRIM68 target. Overexpression of TRIM68 and TFG confirmed their ability to associate, with TLR3 stimulation appearing to enhance the interaction. TFG is a known activator of NF-κB via its ability to interact with inhibitor of NF-κB kinase subunit gamma (IKK-γ) and TRAF family member-associated NF-κB activator (TANK). Our data identifies a novel role for TFG as a positive regulator of type I IFN production and suggests that TRIM68 targets TFG for lysosomal degradation, thus turning off TFG-mediated IFN-β production. Knockdown of TRIM68 in primary human monocytes resulted in enhanced levels of type I IFN and TFG following poly(I:C) treatment. Thus TRIM68 targets TFG, a novel regulator of IFN production, and in doing so turns off and limits type I IFN production in response to anti-viral detection systems.

Respiratory syncytial virus infection modifies and accelerates pulmonary disease via DC activation and migration

In the present studies, we have established that RSV can elicit a more pathogenic environment dependent on improper DC‐associated sensitization. Our initial studies demonstrated that RSV, but not influenza, infection during an allergen exposure into the airway induced a more severe allergen response. The RSV‐induced exacerbation included an increased Th2 cytokine response and pathophysiology as monitored by AHR and mucus overproduction. DCs played a central role in the allergen‐induced responses, as instilling RSV‐infected BMDC into the airway could recapitulate a live virus challenge. With the use of CCR6−/− mice that have a primary defect in the recruitment of mDC subsets, reduced exacerbation of disease was observed when RSV was administered along with allergen. Furthermore, sensitization of mice with RSV‐infected BMDC into the airway produced a more severe immune response to a live virus challenge. Subsequently, using RSV‐infected BMDC from CCR7−/− mice (that do not migrate efficiently to LNs) to sensitize the exacerbated response demonstrated that the response was dependent on DC migration to the LN. Finally, the ability of RSV‐infected DCs to elicit an exacerbated, allergen‐induced pathogenic response could be maintained for as long as 3 weeks, suggesting that RSV‐infected DCs themselves created an altered immune environment that impacts off‐target mucosal responses that could have prolonged effects.

CCR7 impairs hematopoiesis after hematopoietic stem cell transplantation increasing susceptibility to invasive aspergillosis

Hematopoietic stem cell transplantation (HSCT) is limited by patient susceptibility to opportunistic infections. One of the most devastating infections after HSCT is invasive aspergillosis (IA), a life-threatening disease caused by Aspergillus fumigatus. Transplantation of hematopoietic stem cells (HSCs) and myeloid progenitor cells (MPCs) has been shown to mediate protection against IA, but little is known about the factors that regulate HSC and MPC cell expansion after transplantation. Herein, we investigated the role of CCR7 in a murine model of IA after combined HSC and MPC transplantation into lethally irradiated wild-type (WT) mice. Nonirradiated CCR7(-/-) mice had expanded populations of HSCs in the bone marrow and spleen, compared with WT mice. Irradiated WT mice reconstituted with CCR7(-/-) HSCs and MPCs had increased survival, decreased fungal burden, and enhanced myeloid leukocyte numbers during IA, compared with WT controls. In addition, WT mice reconstituted with WT HSCs and MPCs and treated with anti-CCR7 exhibited accelerated myeloid cell expansion similar to that observed in CCR7(-/-)→WT chimeras. Thus, removal of the inhibitory effects of CCR7 through genetic alteration or ligand immunoneutralization enhanced myeloid reconstitution, thereby accelerating fungal clearance in a murine model of IA.