INTRODUCTION
Most cases of infantile viral bronchiolitis are caused by respiratory syncytial virus (RSV) infection (
1). Infants previously hospitalized with RSV disease are highly likely to experience recurrent wheeze (
2,
3), and delaying RSV infection (with the monoclonal antibody palivizumab) reduces the prevalence of wheezing during the first year of life (
4). The mechanisms responsible for postbronchiolitic wheeze are not fully defined, but it is possible that severe viral lung infections during infancy cause long-term changes in the mucosal immune responses in the lung.
To investigate the long-term effects of viral lung infection in early life, we developed a mouse model of neonatal RSV infection followed by adult reinfection (
5). Both CD4 and CD8 T cells play a role in this phenomenon, as does the major histocompatibility complex (MHC) genotype (
6,
7), but these factors may not fully explain the delayed effects of neonatal sensitization, since T cell depletion during secondary reinfection does not completely abrogate disease. The presence of weight loss as early as day 2 after infection (
6) suggests the possibility that innate responses also play a role.
When neonatal mice are infected with a recombinant RSV expressing interleukin-4 (IL-4), weight loss is not exacerbated during adult reinfection, despite greatly enhanced Th2 immunity (
8). In addition, while neonatal infection with RSV expressing gamma interferon (IFN-γ) is protective against disease during subsequent reinfection, it does not enhance Th1 immunity; rather, the clearest correlate of protection is reduced natural killer (NK) cell recruitment during secondary reinfection. Since strong NK cell responses can cause severe pathology during adult RSV infection (
9,
10), we wished to determine whether the innate response plays a role in orchestrating or enhancing the pathology seen during secondary reinfection in mice neonatally infected with RSV.
In the present studies, we found that RSV infection during infancy (but not adulthood) led to a rapid increase of highly activated NK cells in the airways and lungs. Removal of NK cells or the alveolar macrophages necessary for their recruitment resulted in a reduction in subsequent disease. Thus, cells of the innate immune system play an important role in the long-term effects of neonatal RSV infection.
MATERIALS AND METHODS
Virus stocks and mouse infection.
RSV strain A2 was grown in HEp-2 cells, and the viral titer was determined by plaque assay. Time-mated pregnant BALB/c mice (Harlan, Motspur Park, United Kingdom) were purchased at <14 days of gestation, and pups were weaned at age 3 weeks. Mice were infected intranasally (i.n.) with 4 × 104 focus-forming units (FFU)/g body weight virus at 4 days (neonatal dose was ∼105 focus-forming units [FFU]) while they were under isoflurane anesthesia. Secondary RSV challenge was given i.n. at 106 FFU in 100 μl at 8 weeks after priming. Weight was measured daily to monitor disease severity. All work was approved and licensed by the United Kingdom Home Office.
Cell depletion.
To deplete NK cells, 100 μl of rabbit anti-mouse asialo-GM1 polyclonal antibodies (Wako Chemicals, Japan) or control antibodies was administered intravenously (i.v.) on days −1 and +2 of infection; to deplete basophils, anti-FcεR1 (MAR1) was administered intraperitoneally on days −1 and +2 of secondary reinfection. NK cell depletion was performed either during secondary reinfection or primary infection or between the primary and secondary infections. For macrophage depletion, mice were treated prior to secondary reinfection with 100 μl of a clodronate-containing liposome (CL) suspension (a gift from Nico van Rooijen, Boehringer GmbH, Germany) or the control treatment i.n. while the they were under light anesthesia with isoflurane.
Quantification of viral RNA.
RNA was extracted from the lung using the RNA stat-60 reagent (AMS Biotech Ltd.), and cDNA was generated with random hexamers using Omniscript reverse transcriptase (Qiagen). Real-time PCR was carried out with a sequence in the RSV L gene using 900 nM forward primer (5′-GAACTCAGTGTAGGTAGAATGTTTGCA-3′), 300 nM reverse primer (5′-TTCAGCTATCATTTTCTCTGCCAAT-3′), and 100 nM probe (5′-FAM-TTTGAACCTGTCTGAACAT-TAMRA-3′, where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine) on an ABI Prism 7000 sequence detection system. This detects viral genomic RNA, viral anti-genomic RNA, and intracellular RSV L mRNA, referred to here as RSV L RNA gene. The L-gene copy number was quantified relative to that for a standard plasmid (
5).
Cell recovery.
Collection of bronchoalveolar lavage (BAL) fluid for cells and supernatants and the harvesting of lung tissues were carried out as previously described (
11). For the preparation of lung mash supernatants, lungs were homogenized through 100-μm-mesh-size cell strainers (BD Pharmingen) and washed through with a 1-ml volume of RPMI 1640 five times. Following centrifugation, this supernatant was retained for enzyme-linked immunosorbent assay (ELISA) analysis. After the removal of the supernatants from all tissues, cells were treated with ACK lysing buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) for 5 min and then DNase I (40 μg/ml)-collagenase (50 μg/ml) in RPMI 1640 (with 10% fetal calf serum) for 5 min at room temperature to remove clumps. Finally, they were resuspended in RPMI 1640. Cell viability was assessed by trypan blue exclusion, and total cell numbers were counted by use of a disposable multiwell hemocytometer (Immune Systems, United Kingdom). Airway cells were differentiated by hematoxylin-eosin staining of samples of cells from BAL fluid.
Flow cytometry.
Prior to staining, cells were blocked with CD16/32 (Fc Block; BD). For surface staining, antibodies against the surface markers CD4, CD8, CD3, CD69, CD11b, CD11c, MHC class II (MHC-II), CD27, and DX5 (BD) were added at a 1:100 dilution for 30 min on ice. Gating for lymphocytes was determined by back gating on CD3/CD8 double-positive cells. For the detection of intracellular cytokines, cells were incubated with 50 ng/ml phorbol myristate acetate, 500 ng/ml ionomycin, and 10 μg/ml brefeldin A for 4 h at 37°C. Samples were permeabilized with 0.5% saponin in phosphate-buffered saline (PBS) for 10 min. Anticytokine antibodies (anti-IFN-γ, anti-IL-4; BD) or isotype controls were added for a further 20 min at room temperature. Cells were analyzed on a CyAn ADP flow cytometer (Dako), with data on at least 50,000 events being collected.
Inflammatory mediators.
The levels of inflammatory mediators in the BAL fluid were measured. In the time course experiment (depicted in
Fig. 1), IL-4, IL-5, IL-6, tumor necrosis factor alpha (TNF-α), CXCL1, CCL5, and IFN-γ were measured by use of a Luminex kit (Millipore), IFN-α was measured by ELISA using capture and detection antibodies (PBL; Interferon Source), and CCL11 was measured by use of an ELISA DuoSet system (R&D Systems). In subsequent studies, IFN-γ and IL-4 were measured by use of the ELISA DuoSet system (R&D Systems). The limits of detection were as specified by the manufacturer.
Statistical analysis.
Results are expressed as the mean ± standard error of the mean (SEM); statistical significance was calculated by analysis of variance, followed by Tukey tests when there were 3 groups and t tests for the comparison of 2 groups, using GraphPad Prism software.
DISCUSSION
Here we show that neonatal RSV infection primed for pathogenic innate and adaptive immune responses on RSV reinfection in adults. Following reinfection of neonatally infected mice, there was a wave of proinflammatory cytokines followed by an influx of highly activated, proinflammatory NK cells. NK cell depletion reduced disease, suggesting that NK cells are important in promoting pathology in this model. The depletion of macrophages by clodronate liposome treatment during secondary infection significantly reduced cellular recruitment to the lungs and weight loss, indicating that they have a role in the long-term effects of neonatal RSV infection.
Macrophages are a major producer of proinflammatory cytokines in the initial stages of RSV infection (
14), and it is likely that they promote inflammation in the lung via their local cytokine and chemokine production. Interestingly, the inflammatory role of macrophages on challenge of neonatally primed mice contrasted with the role of macrophages during secondary challenge of adult primed mice, where they are thought to act in an immunosuppressive capacity (
17). Alveolar macrophages from infants have been shown to be immature (
18), and the Th2 environment of the neonatal lung may switch macrophages from the classical to the alternatively activated pathway, which is associated with RSV disease (
19). The failure to activate macrophages properly may also lead to airway occlusion with cellular debris, contributing to RSV disease (
20). We have previously observed differences in antigen-presenting cell populations after adult or neonatal infection and that neonatal treatment with CpG improved the outcome on RSV reinfection associated with a switch to Th1 responses (
16).
NK cells play an important role in the defense against lung infection (
21). We and others have previously observed that NK cells can contribute to RSV disease (
9,
10). It has been proposed that both mouse and human NK subsets are distinguishable through surface levels of CD11b and CD27 (
12). CD27
+ NK cells display greater effector function, a lower activation threshold, and a higher responsiveness to chemokines (
12). Importantly, these two subsets also display distinct localizations, with CD27
+ NK cells being restricted to the systemic/lymphoid compartment, while usually only CD27
− NK cells are found in the periphery (
12). On day 4 after infection, there was a rapid accumulation of CD11b
+ CD27
+ NK cells and even some CD11b
− CD27
+ NK cells in the mice primed as neonates, a feature not seen in either naive mice or mice primed as adults, where the majority of NK cells remained the expected CD11b
+ CD27
− subset. These activated CD27
+ NK cells produce high levels of IFN-γ, which may further contribute to the disease seen (
10). The role of NK cells was investigated using anti-asialo-GM1 treatment, which does have off-target effects on both basophils (
13) and CD8 T cells (
22), but in our study, we observed significantly reduced NK cell numbers but not reduced CD8 T cell numbers, as we have observed in other studies (
9), and basophil depletion had no effect on disease. These studies suggest that NK cells contribute to the long-term effects of neonatal RSV infection.
Interestingly, in contrast to our findings in secondary RSV infection following neonatal sensitization, the depletion of NK cells during adult RSV infection was reported by others to make no difference to the viral load but to lead to the development of a Th2 response (
23). In influenza virus infection, NK cells were implicated in promoting pathology in the lung (
24). Thus, NK cells play contrasting roles depending on the immunological context of the infection.
In summary, our study highlights the potential of two components of innate immunity, natural killer cells and alveolar macrophages, to modulate immune responses to infection and dictate the severity of secondary disease. Based on these findings, we propose the following mechanism of the effects of secondary RSV reinfection on neonatally infected mice. Neonatal infection with RSV not only fails to produce a protective antibody response (
25) but also primes an inflammatory cellular response. Following viral reinfection, macrophages rapidly produce proinflammatory cytokines, which cause recruitment of inflammatory cells, including NK cells, which in turn further promote pulmonary inflammation. While much attention has been paid to promoting and modulating specific T cell responses to infection, progress in novel treatments for RSV disease are hampered by incomplete understanding of innate and adaptive immunity in early life. Therapeutic targeting of innate immunity may provide a novel, broad-based approach to reducing the severity of not only primary lung infections but also secondary disease; for example, neonatal treatment with CpG improved the outcome on RSV reinfection (
16). Further study of the innate response may enrich our understanding of the link between neonatal RSV infection and subsequent wheeze in later childhood.