Sara E. Mertz

The Role of IFN in Respiratory Syncytial Virus Pathogenesis

Formalin-inactivated respiratory syncytial virus (RSV) vaccine preparations have been shown to cause enhanced disease in naive hosts following natural infection. In this study we demonstrate a similar pattern of enhanced disease severity following primary RSV infection of IFN-nonresponsive STAT1−/− mice. STAT1−/− mice showed markedly increased illness compared with wild-type BALB/c animals following RSV inoculation despite similar lung virus titers and rates of virus clearance. Histologically, STAT1−/− animals had eosinophilic and neutrophilic pulmonary infiltrates not present in wild-type or IFN-γ−/−-infected mice. In cytokine analyses of infected lung tissue, IFN-γ was induced in both STAT1−/− and wild-type mice, with preferential IL-4, IL-5, and IL-13 induction only in the STAT1−/− animals. Eotaxin was detected in the lungs of both wild-type and STAT1−/− mice following infection, with a 1.7-fold increase over wild-type in the STAT1−/− mice. Using a peptide epitope newly identified in the RSV fusion protein, we were able to demonstrate that wild-type memory CD4+ T cells stimulated by this peptide produce primarily IFN-γ, while STAT1−/−CD4+ cells produce primarily IL-13. These findings suggest that STAT1 activation by both type I (αβ) and type II (γ) IFNs plays an important role in establishing a protective, Th1 Ag-specific immune response to RSV infection.

Differential Type I Interferon Induction by Respiratory Syncytial Virus and Influenza A Virus In Vivo

ABSTRACT Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-α/β induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-α/β production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-α/β in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.

Lambda Interferon Is the Predominant Interferon Induced by Influenza A Virus Infection In Vivo

ABSTRACT The type I alpha/beta interferons (IFN-α/β) are known to play an important role in host defense against influenza A virus infection, but we have now discovered that the recently identified type III IFNs (IFN-λ) constitute the major response to intranasal infection with this virus. Type III IFNs were present at much higher levels than type I IFNs in the lungs of infected mice, and the enhanced susceptibility of STAT2−/− animals demonstrated that only signaling through the IFN-α/β or IFN-λ pathways was sufficient to mediate protection. This finding offers a possible explanation for the similar levels of antiviral protection found in wild-type (WT) mice and in animals lacking a functional type I IFN receptor (IFNAR−/−) but also argues that our current understanding of type III IFN induction is incomplete. While murine IFN-λ production is thought to depend on signaling through the type I IFN receptor, we demonstrate that intranasal influenza A virus infection leads to the robust type III IFN induction in the lungs of both WT and IFNAR−/− mice. This is consistent with previous studies showing that IFNAR-mediated protection is redundant for mucosal influenza virus infection and with data showing that the type III IFN receptor is expressed primarily by epithelial cells. However, the overlapping effects of these two cytokine families are limited by their differential receptor expression, with a requirement for IFN-α/β signaling in combating systemic disease.

Protection against Respiratory Syncytial Virus by a Recombinant Newcastle Disease Virus Vector

ABSTRACT Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-α/β) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-α/β receptor.

Role for Innate IFNs in Determining Respiratory Syncytial Virus Immunopathology

Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection. Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-deficient STAT1−/− mice that are incapable of generating Th1 CD4+ cells. To determine whether these effects depended only on Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the IFN-αβR (IFN-αβR−/−), the IFN-γR (IFN-γR−/−), or both receptors (IFN-αβγR−/−). Although both the IFN-γR−/− and the IFN-αβγR−/− animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains, very few CD8+ T cells could be detected in lungs of IFN-αβγR−/− animals, leaving eosinophils as the predominant leukocyte. Thus, although CD4+ Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV disease.

Chinchilla and Murine Models of Upper Respiratory Tract Infections with Respiratory Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and the elderly. While the primary infection is the most serious, reinfection of the upper airway throughout life is the rule. Although relatively little is known about either RSV infection of the upper respiratory tract or host mucosal immunity to RSV, recent literature suggests that RSV is the predominant viral pathogen predisposing to bacterial otitis media (OM). Herein, we describe mouse and chinchilla models of RSV infection of the nasopharynx and Eustachian tube. Both rodent hosts were susceptible to RSV infection of the upper airway following intranasal challenge; however, the chinchilla proved to be more permissive than the mouse. The chinchilla model will likely be extremely useful to test the role of RSV in bacterial OM and the efficacy of RSV vaccine candidates designed to provide mucosal and cytotoxic T-lymphocyte immunity. Ultimately, we hope to investigate the relative ability of these candidates to potentially protect against viral predisposal to bacterial OM.

Differences in Antibody Responses Between Trivalent Inactivated Influenza Vaccine and Live Attenuated Influenza Vaccine Correlate With the Kinetics and Magnitude of Interferon Signaling in Children

BACKGROUND Live attenuated influenza vaccine (LAIV) and trivalent inactivated influenza vaccine (TIV) are effective for prevention of influenza virus infection in children, but the mechanisms associated with protection are not well defined. METHODS We analyzed the differences in B-cell responses and transcriptional profiles in children aged 6 months to 14 years immunized with these 2 vaccines. RESULTS LAIV elicited a significant increase in naive, memory, and transitional B cells on day 30 after vaccination, whereas TIV elicited an increased number of plasmablasts on day 7. Antibody titers against the 3 vaccine strains (H1N1, H3N2, and B) were significantly higher in the TIV group and correlated with number of antibody-secreting cells. Both vaccines induced overexpression of interferon (IFN)-signaling genes but with different kinetics. TIV induced expression of IFN genes on day 1 after vaccination in all age groups, and LAIV induced expression of IFN genes on day 7 after vaccination but only in children <5 years old. IFN-related genes overexpressed in both vaccinated groups correlated with H3N2 antibody titers. CONCLUSIONS These results suggest that LAIV and TIV induced significantly different B-cell responses in vaccinated children. Early induction of IFN appears to be important for development of antibody responses.

Type I Interferon Inhibition and Dendritic Cell Activation during Gammaherpesvirus Respiratory Infection

ABSTRACT The respiratory tract is a major mucosal site for microorganism entry into the body, and type I interferon (IFN) and dendritic cells constitute a first line of defense against viral infections. We have analyzed the interaction between a model DNA virus, plasmacytoid dendritic cells, and type I IFN during lung infection of mice. Our data show that murine gammaherpesvirus 68 (γHV68) inhibits type I IFN secretion by dendritic cells and that plasmacytoid dendritic cells are necessary for conventional dendritic cell maturation in response to γHV68. Following γHV68 intranasal inoculation, the local and systemic IFN-α/β response is below detectable levels, and plasmacytoid dendritic cells are activated and recruited into the lung with a tissue distribution that differs from that of conventional dendritic cells. Our results suggest that plasmacytoid dendritic cells and type I IFN have important but independent roles during the early response to a respiratory γHV68 infection. γHV68 infection inhibits type I IFN production by dendritic cells and is a poor inducer of IFN-α/β in vivo, which may serve as an immune evasion strategy.

The psychoactive substance of cannabis Δ9-tetrahydrocannabinol (THC) negatively regulates CFTR in airway cells

BACKGROUND Marijuana consumption is on the rise in the US but the health benefits of cannabis smoking are controversial and the impact of cannabis components on lung homeostasis is not well-understood. Lung function requires a fine regulation of the ion channel CFTR, which is responsible for fluid homeostasis and mucocilliary clearance. The goal of this study was to assess the effect that exposure to Δ9-tetrahydrocannabinol (THC), the psychoactive substance present in marijuana, has on CFTR expression and function. METHODS Cultures of human bronchial epithelial cell line 16HBE14o- and primary human airway epithelial cells were exposed to THC. The expression of CFTR protein was determined by immunoblotting and CFTR function was measured using Ussing chambers. We also used specific pharmacological inhibitors of EGFR and ERK to determine the role of this pathway in THC-induced regulation of CFTR. RESULTS THC decreased CFTR protein expression in primary human bronchial epithelial cells. This decrease was associated with reduced CFTR-mediated short-circuit currents. THC also induced activation of the ERK MAPK pathway via activation of EGFR. Inhibition of EGFR or MEK/ERK prevented THC-induced down regulation of CFTR protein expression. CONCLUSIONS AND GENERAL SIGNIFICANCE THC negatively regulates CFTR and this is mediated through the EGFR/ERK axis. This study provides the first evidence that THC present in marijuana reduces the expression and function of CFTR in airway epithelial cells.

A Systems Analysis Approach to Define the Protective Host Immune Responses of Children with Mild Respiratory Syncytial Virus Infection

Abstract Background Almost all children are infected with RSV by 2 yrs of age, yet <3% are hospitalized. The immune response of children with mild RSV infection who do not develop severe disease is not fully understood. Characterization of such response may have implications for vaccine development. We sought to define the transcriptomic and cellular immune profiles associated with mild RSV disease in children. Methods We enrolled 190 previously healthy children <2 yrs of age: 125 with RSV infection treated as outpatients (OP; mild disease; n = 41) or inpatients (IP; severe disease; n = 84), and age-matched healthy controls (HC; n = 65). Nasopharyngeal RSV loads, blood RNA transcriptome and WBC immunophenotyping were analyzed according to disease severity. Results OP were older (7.6 m) than IP (2.6 m; P < 0.01). Median duration of symptoms at enrollment in both groups was 4 days, yet RSV loads were higher in OP vs. IP (5.5 log10 and 4.9 log10 copies/mL respectively; P = 0.05). To control for differences in age, transcriptional signatures were derived in an age-matched discovery cohort (OP n = 24; IP n = 24; HC n = 24) and subsequently validated in an independent validation cohort (n = 55). Compared with IP, the OP signature was characterized by greater activation of interferon (IFN) and plasma cell genes, less suppression of T-cell, and cytotoxic/NK cell genes and less activation of inflammation and neutrophil genes (P < 0.01; Fig 1). Overall, plasma cell genes positively correlated with number of plasmablasts, plasma cells, and T follicular helper cells (r=0.6; P < .01), while IFN gene expression inversely correlated with T-cells, DC and B cells numbers (r=-0.4 P < .05). Neutrophil numbers correlated with neutrophil (r=0.5; P < =.02) and inflammation gene expression (r=0.8; P < 0.001). Last, monocyte numbers were higher in IP and OP vs. HC (P < 0.01) however; HLA-DR low monocytes were increased only in IP. Conclusion Children with mild disease demonstrated a distinct cellular and transcriptional immune response to RSV. Despite that RSV loads were higher in these children, IFN expression and monocyte activation were increased and T and NK cell genes less suppressed, suggesting that robust immune responses are associated with improved clinical outcomes in children with RSV infection. Disclosures O. Ramilo, Abbvie: Board Member, Consulting fee; Regeneron: Board Member, Consulting fee; Janssen: Board Member and Investigator, Consulting fee and Research grant; NIH: Grant Investigator, Research grant; A. Mejias, Janssen: Investigator and Scientific Advisor, Consulting fee and Research support; Abbvie: Consultant and Scientific Advisor, Speaker honorarium; Novartis: CME lecture, Speaker honorarium; NIH: Investigator, Research grant