Alveolar macrophages support interferon gamma-mediated viral clearance in RSV-infected neonatal mice
© Eichinger et al. 2016
Received: 17 July 2015
Accepted: 15 September 2015
Published: 5 October 2015
Poor interferon gamma (IFNγ) production during respiratory syncytial virus (RSV) is associated with prolonged viral clearance and increased disease severity in neonatal mice and humans. We previously showed that intra-nasal delivery of IFNγ significantly enhances RSV clearance from neonatal lungs prior to observed T-lymphocyte recruitment or activation, suggesting an innate immune mechanism of viral clearance. We further showed that alveolar macrophages dominate the RSV-infected neonatal airways relative to adults, consistent with human neonatal autopsy data. Therefore, the goal of this work was to determine the role of neonatal alveolar macrophages in IFNγ-mediated RSV clearance.
Clodronate liposomes, flow cytometry, viral plaque assays, and histology were used to examine the role of alveolar macrophages (AMs) and the effects of intra-nasal IFNγ in RSV infected neonatal Balb/c mice. The functional outcomes of AM depletion were determined quantitatively by viral titers using plaque assay. Illness was assessed by measuring reduced weight gain.
AM activation during RSV infection was age-dependent and correlated tightly with IFNγ exposure. Higher doses of IFNγ more efficiently stimulated AM activation and expedited RSV clearance without significantly affecting weight gain. The presence of AMs were independently associated with improved RSV clearance, whereas AM depletion but not IFNγ exposure, significantly impaired weight gain in RSV-infected neonates.
We show here for the first time, that IFNγ is critical for neonatal RSV clearance and that it depends, in part, on alveolar macrophages (AMs) for efficient viral clearing effects. Early reductions in viral burden are likely to have profound short- and long-term immune effects in the vulnerable post-natally developing lung environment. Studies are ongoing to elucidate the pathologic effects associated with early versus delayed RSV clearance in developing neonatal airways.
KeywordsNeonate RSV Lung IFNγ Alveolar macrophage
Neonates, more than older children and adults have an increased risk for bronchiolitis and pneumonia, due in part, to the anti-inflammatory environment that supports post-natal lung development. Respiratory syncytial virus (RSV) is the most common respiratory virus in neonates and young children worldwide causing an estimated 3.4 million lower respiratory tract infections and approximately 200,000 deaths annually . Nearly all children are infected with RSV by three years of age, but not all children develop severe disease [2, 3]. In addition to known risk factors associated with disease progression, including low birth weight, premature birth, and congenital heart disease,  recent clinical studies by Devincenzo’s group and others have tied high RSV titers to increased severity of disease and/or length of hospital stay [5–8].
The conditions present when a T cell encounters antigen for the first time are essential to subsequent immunity, often determining the nature of the response to infection. Alveolar macrophages (AMs) play an important role in establishing these initial conditions through cytokine production, recruitment of lymphocytes, and antimicrobial activity. However, these functions vary significantly in neonates compared to older children and adults and are often further influenced by invading pathogens. Though infants are prone to Th2-type cytokine responses left over from fetal life, they are capable of Th1-type immunity . Other factors known to contribute to neonatal Th2 immunity are low levels of mature T cells and IFNγ, which when exposed to high burdens of viral antigen lead to Th2 immunity, producing such cytokines as IL-4, IL-5, and IL-13 [10, 11]. By regulating the cytokine environment through early, in vivo delivery of IFNγ, studies in neonatal models of murine leukemia virus have shown that immune responses can be shifted to a Type I pathway with enhanced viral clearance .
Interferon gamma (IFNγ) is a potent type II interferon known to stimulate direct antimicrobial effects among macrophages including the up-regulation of antigen processing and presentation . IFNγ-stimulated macrophages secrete TNFα, IL-12 and other inflammatory cytokines, which facilitate the trafficking of T cells and NK cells to the site of inflammation . Though its limited production in the early months after birth is thought to protect post-natal lung development,  elevated levels of IFNγ in the serum are associated with milder RSV disease, whereas detection of Th2-type cytokines, IL-4 and IL-5, are typically associated with greater disease severity [15–17]. Polymorphisms in IFNγ gene expression during neonatal RSV infection are linked to increased severity of illness, duration of stay in the intensive care unit (ICU), and frequency of otitis media . Moreover, influenza- versus RSV-infected neonates produce markedly more IFNγ, suggesting that reduced production is likely linked, at least in part, to RSV infection . Autopsy data of lung sections taken from human neonates who died with severe RSV infection show insufficient recruitment of Th1 and Th2 cytokine-producing lymphocytes, including CD4 and CD8 T cells as well as natural killer (NK) cells [19, 20], suggesting that impaired recruitment of IFNγ-producing cells are associated with increased disease severity.
In mouse models of RSV infection, local delivery of IFNγ has demonstrated both acute and long-term benefits in neonatal RSV infection. These include faster viral clearance as well as protection against RSV-mediated airway hyper-responsiveness (AHR) [21, 22]. This study aimed to determine if AMs can be therapeutically stimulated in a neonatal mouse model of RSV infection using inhaled IFNγ to enhance initial viral clearance, promote T cell immunity, and improve functional outcomes. Our lab has published data showing that neonatal mice produce negligible IFNγ in response to RSV with a corresponding delay in viral clearance compared to IFNγ-producing adults . We further show that activation of AMs and pulmonary DCs are significantly enhanced upon delivery of intranasal IFNγ with expedited RSV clearance early in infection without eliciting weight loss commonly associated with the systemic delivery of IFNγ [23, 24]. Despite enhanced activation of innate immunity following i.n. IFNγ, the recruitment of CD4 and CD8 T cells was unexpectedly reduced in RSV-infected neonatal mice compared to age-matched controls .
Materials and methods
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed at The University of Pittsburgh Division of Laboratory Animal Resources. These animal experiments were approved by The University of Pittsburgh Institutional Animal Care and Use Committee (IACUC), approved protocol number 14023340 and mice were handled according to IACUC guidelines. All efforts were made to minimize animal suffering.
Mice and viral preparation
Pathogen-free breeder BALB/cJ breeder mice and IFNγ receptor knock out (IFNγR KO) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 5–7 weeks of age and maintained in pathogen-free facilities. Females were bred as previously described,  and pups from the resultant pregnancies were treated experimentally at 2–7 days of age, as mice less than 7 days of age are considered neonates. Additional pathogen-free BALB/cJ female and male mice were purchased from The Jackson Laboratory at 8 weeks of age for experimental and control purposes as described below. Line 19 RSV was provided by Dr. Martin Moore, Emory University, Atlanta, GA. RSV line 19 and viral lung titers were prepared as previously described . Briefly, RSV was passed through 4 rounds of plaque purification; after a single plaque was isolated, it was propagated in HEp-2 cells (American Type Culture Collection), then titered by standard hematoxylin-eosin (H&E) plaque assay, as previously described . Viral stocks were snap-frozen and stored at −80 °C. Viral stocks and HEp-2 cell lines were routinely monitored for mycoplasma and other contaminants using the Plasmo Test Mycoplasma Detection Kit (InvivoGen) and the LookOut® Mycoplasma PCR Detection Kit according to manufacturer’s instructions. Lung titers were determined, as previously described, within one month of sterile removal and storage of infected lungs at −80 °C .
Clodronate liposome delivery
To deplete alveolar macrophages, clodronate liposomes (Clip) (5 μl/g of 5 mg/ml suspension) were administered intranasally (i.n.) to 2-day-old mice under 2 % isoflurane anesthesia beginning day −1 post-infection, then daily until the mice were euthanized for sample collection. On days 1, 3, and 5 when IFNγ was co-administered, the doses of CLip and rIFNγ were separated by ≥ 6 h.
Interferon gamma treatment
Recombinant-murine interferon gamma (IFNγ) (16 ng/g or 60 ng/g, Peprotech, NJ) was administered i.n. to 2–7 day old mice under 2 % isoflurane anesthesia on 1, 3, and 5 days post-infection. The total volume administered to adults was 50 μl and 10–20 μl to neonates. For the pharmacokinetic experiment, a single dose of 16 ng/g was administered i.n. to RSV-naïve mice at time zero and they were subsequently euthanized using 100 % isoflurane at 0, 0.5, 1, 2, 4, 6, 8, 12, 18, 24 and 48 h post-dose.
Neonatal mice were infected with 5 × 105 pfu/g to 3 × 106 pfu/g of RSV L19 as previously described;  mock infected controls received identical volumes of cell lysate (supernatant from lysed cells that remained uninfected) or PBS. Animals were anesthetized prior to infection using 2 % isoflurane. Left lung was collected and snap frozen in alcohol/dry ice for subsequent quantification of lung titers, as previously described . Bronchoalveolar lavage (BALF) and first wash were harvested and stored separately for flow cytometry or cytokine analysis, respectively. Right lungs were collected and immediately processed for analysis using flow cytometry or cytokine analysis as described below.
Real-time polymerase chain reaction
Left lungs were snap frozen in liquid nitrogen for qRT-PCR as previously described . Briefly, mRNA was harvested using Mini Qiagen Kit (Life Technologies, NY) and quantified using a NanoDrop® spectrophotometer (Invitrogen, NY). The mRNA was reverse transcribed to cDNA using a Superscript III First-strand synthesis Supermix for qRT-PCR kit (Life Technologies, NY) and quantified on a 7500 ABI Fast RTPCR system (Life Technologies). Pre-mixed Taqman primer and probes using a Fam/Tamara reporter/quencher combination were purchased from ABI specific for Gob5A. Results are represented as a relative increase from media only or from media + IFNγ, using the delta, delta ct method indicating fold change over the house keeping gene (GAPDH). Data are compared to control wells treated with media only using a paired T-test for 3–4 wells per group per mouse.
Flow cytometry was used to evaluate surface protein expression in BALF and lung digest (LD) as previously described . Right lungs were enzyme digested as previously described . Briefly, lung tissue was minced, incubated with DNase and collagenase for 1 h at 37 °C, then pushed through a 70 micron mesh screen. RBCs were then removed from both BALF and LD with a hypertonic lysis buffer, cells were counted, and non-specific binding was blocked with anti-CD16/32 (BD Biosciences). Staining was performed with murine-specific fluorochrome-conjugated antibodies and fixed in 0.5–1 % paraformaldehyde prior to analysis with an LSRII or LSR Fortessa flow cytometer (BD Biosciences) within 12 h. Within the population of large granular cells, CD11b-PerCpCy™5.5 and CD11c-APC (BD Biosciences, San Jose, CA) or CD11c-PECy7 (Biolegend, San Diego, CA) were compared in dot plot quadrants, AMs were defined as CD11c + CD11b-, and activation of AMs was determined by the expression of major histocompatibility complex (MHC) class II (MHC II; I-ad)-FITC.. Data was analyzed using FlowJo software (Tree Star Inc. Ashland, OR).
At 8 days post-infection (dpi), right lungs were gravity filled (25 cm from meniscus to catheter) with 10 % formalin after flushing the respiratory system with phosphate buffered saline (PBS). Lungs were preserved for at least 48 h in 10 % formalin at 4 °C. The lungs were paraffin-embedded and stained processed at the Transplant Pathology Research Laboratory of University of Pittsburgh (Pittsburgh, PA). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and periodic acid-Schiff (PAS) stains were used to identify cells undergoing apoptosis and mucus accumulation, respectively. Slides were examined and quantified by two individuals blinded to treatment group. PAS-staining was scored according to previously published methods .
Collection and processing of biological samples
Right lungs were collected, weighed and immediately snap-frozen in liquid nitrogen. Samples were stored at -800 C until they were processed for protein quantification and cytokine analysis as previously described . Briefly, frozen lungs were homogenized in cold Tissue Protein Extraction Reagent (T-PER, Thermo Scientific) and protease inhibitor (HALT Protease Inhibitor Cocktail, Thermo Scientific) (1 mL T-PER + 10 μl HALT protease inhibitor cocktail used for every 100 μg of frozen lung tissue), then centrifuged at 9000 g for 10 min at 40 C. The supernatant was collected and total protein was quantified by bicinchoninic acid assay (BCA assay, Thermo Scientific Pierce) per manufacturer’s instructions. The remaining supernatant was stored at −80 °C for cytokine analysis. IFNγ concentrations in lung homogenate and first wash samples were analyzed by a murine multiplex cytokine kit (Bio-Rad, Hercules, CA). Homogenized lung samples were diluted to a total protein concentration of 500 μg/ml using a 1:1 mixture of T-PER and sample diluent (provided by manufacturer). Samples of first wash were added directly to the plate without dilution. The plates were read using a Luminex® 200™ Total System machine (Luminex Corp, Austin, Tx); data was analyzed using the LDS1.7 Software.
Lung IFNγ concentration-time data after a single i.n. IFNγ dose was analyzed by non-compartmental analysis (NCA). The terminal elimination rate constant, kel, was estimated by log-linear regression of at least three time points visually assessed to be in the terminal phase of each lung concentration-time plot. The terminal phase (elimination) half-life, t1/2, was calculated as ln2/kel. The area under the lung concentration versus time curve, AUC0→∞, was also calculated. Using the linear trapezoidal rule,  AUClast→∞ was extrapolated to infinity by calculating Clast/kel. The AUC from time zero to infinity was calculated as the sum of AUClast and Clast/kel. Other pharmacokinetic parameters calculated include IFNγ total body clearance, estimated as Dose/AUC. The estimated neonatal dose capable of achieving adult AUC values was calculated by dividing the adult AUC0→∞ by neonatal total body clearance. The new neonatal dose was then calculated on a per gram basis by dividing dose by the average weight of a 3–5 day-old neonate.
Data are expressed as the mean ± SEM of at least three mice per group. Statistical analysis was performed using GraphPad Prism 5 software (La Jolla, CA). A two-way ANOVA was used to compare differences among data collected at multiple time points between ≥ 2 neonatal groups or between neonatal groups receiving multiple treatments followed by a Bonferroni post-test. For analysis of multiple groups of neonates at a single time point a 1-way ANOVA was used with a Tukey-Kramer post-test or a Kruskal Wallis test for nonparametric data. A two-way repeated measures ANOVA was used to compare differences in weight gain for neonates in the AM depletion study.
Signaling through the IFNγR is required for efficient viral clearance
Age-dependent activation of AMs can be enhanced by i.n. IFNγ
The effects of i.n. IFNγ administration can be seen in the dot plots and histograms representing 7 dpi BALF samples. Large cells are characterized as CD11c + b- in mock-infected (Fig. 2d) and RSV-infected neonates (Fig. 2f). Neonatal AMs did not respond to RSV as evidenced by the lack of MHCII class II expression following infection (Fig. 2e, g). In contrast, RSV-infected neonates treated with i.n. IFNγ showed a marked shift in airway cells from CD11c + CD11b- to a larger population of cells that were CD11c + CD11b+, a population shown to have greater antigen presentation in the context of MHC class II expression and often characterized as dendritic cells (DCs) (Fig. 2h). In the RSV+/IFNγ + group, large cells that remained CD11c + b- also efficiently upregulated MHC class II+ expression (37 %) compared to PBS-treated controls (3 %) (Fig. 2i).
Pharmacokinetic differences result in age-dependent AM activation
IFNγ dose responsiveness correlates with improved AM activation and enhanced viral clearance
To determine the effect of IFNγ dose on total lung macrophage (LM) activation in RSV-infected neonatal lungs, the activation profile of neonatal LMs from LD was examined. At each time point tested, the 60 ng/g dose increased LM activation significantly more than pups treated with 16 ng/g or PBS alone (Fig. 4c). LMs from mice receiving 16 ng/g also significantly increased LM activation at 7 and 10 dpi compared to PBS-treated mice (Fig. 4c) but not at 4dpi, suggesting a stronger activation signal may be required for initial neonatal LM activation.
To determine the functional effect of optimizing i.n. IFNγ, RSV clearance was quantified by viral plaque assay. By 4 dpi, RSV clearance was greatest in the 60 ng/g group followed by the 16 ng/g of IFNγ compared to PBS; however, only the group receiving 60 ng/g demonstrated enhanced RSV clearance at 7 dpi (Fig. 4d). By 10 dpi, 80 % (4/5) of animals in both IFNγ groups had undetectable viral loads while only 50 % (2/4) of neonates in the PBS group had similarly undetectable virus.
Low dose IFNγ is protective in the RSV-infected neonatal airway
Depletion of neonatal alveolar and lung macrophages using clodronate liposomes
IFNγ and AMs play a significant role in eradicating RSV from neonatal airways
At 8 dpi, CLip (+)/IFNγ (−) animals had the highest viral burden which was significantly higher than both CLip (−)/IFNγ (−) and CLip (−)/IFNγ (+) animals (Fig. 8b). However, viral titers of CLip (+)/IFNγ (−) animals were not significantly higher than CLip (+)/IFNγ (+) animals demonstrating that the viral clearing effects of IFNγ was lost by 8 dpi in the absence of AMs. In fact, treatment with IFNγ accounted for only 5 % of the variability observed. The presence or absence of AMs played a critical role in RSV clearance, accounting for 43 % of the variability. Regardless of IFNγ treatment, neonates whose AM populations were eliminated with CLip had RSV viral titers that were nearly a log fold higher than CLip (−) neonates at 8 dpi.
The potential for biological toxicity resulting from either CLip or IFNγ treatment was analyzed by calculating the neonates’ ability to gain weight over time. Interestingly, CLip treatment but not IFNγ significantly impaired the ability of neonates to gain weight (Fig. 8c). Visual inspection of the charted daily weights revealed a separation of the groups beginning at 4 dpi. Regardless of IFNγ treatment, the daily percent change in weight began to slow in CLip (+) neonates when compared to CLip (−) neonates beginning at 4 dpi and continued until the final time point. At 7 dpi, both CLip (+) groups demonstrated significantly impaired weight gain when compared to CLip (−)/IFNγ (−) neonates.
Since the formalin-inactivated RSV vaccine trials of the late 1960’s [22, 23], RSV-mediated pathology has been tightly linked to the host immune response [6, 13–15, 33]. Studies showing that ribavirin, a drug that blocks viral replication, does not effectively improve clinical outcomes such as hospital length of stay or time on mechanical ventilation [27, 38] has encouraged theories supporting RSV-mediated immunopathology. However, mounting evidence indicates that viral clearance kinetics plays an important role in disease severity [3, 4]. A recent study involving RSV-infected children < 2 years of age, demonstrated that a faster rate of RSV clearance was independently associated with shorter hospitalization . Due to the fact that RSV replicates several times less efficiently in mouse models than in humans, debates continue over whether immature host immunity versus viral load contributes to severity of disease [4, 7].
We previously reported that i.n. IFNγ reduces the burden of RSV L19 in neonatal mice . We further showed a preponderance of AMs in RSV-infected neonatal compared to adult airways throughout infection . Based on our studies and those in human neonates demonstrating the dominance of AMs during RSV infection [19, 20], we tested the hypothesis that i.n. IFNγ enhances RSV clearance through activation of immature neonatal AMs. Using IFNγRKO mice, we showed that IFNγ significantly contributes to viral clearance in neonatal mice. To determine how immature AMs correlate with poor IFNγ exposure, we infected neonates at 2–4 or 7 days of age and analyzed AM activation in association with IFNγ production. In mice infected at 2–4 days of age, virtually no IFNγ production was detected which was consistent with an absence of AM activation . Only when IFNγ was given exogenously did AM activation increase in this age cohort. However, when neonates were infected at 7 days of age, AMs became activated during RSV infection, though it was significantly delayed and reduced compared to RSV-infected adults. Moreover, it was preceded by a fleeting, but significant increase in IFNγ that was not observed in the younger cohort of mice indicating that in the neonatal lung, AM exposure to IFNγ was likely important for their activation.
Based on these age- and IFNγ-mediated increases in AM activation, we sought to better understand and immunokinetically manipulate the relationship between IFNγ exposure and AM activation in an effort to hasten viral clearance and in turn, minimize RSV-mediated pathology. Specifically, we examined the relationships between IFNγ exposure and its correlation with mucus production and apoptotic cellular debris among RSV infected neonates treated with i.n. IFNγ or PBS controls. We previously showed that expression of the suppressive cytokine TGF-β1, is upregulated in the lungs of neonatal compared to adult mice . Therefore, in our immunokinetic analysis, we targeted a higher IFNγ exposure in neonatal animals than is typically observed, in anticipation of a lower AM activation threshold brought about, not only by RSV itself, but by elevated levels of TGF- β1. Additionally, it was assumed that neonates are equally efficient in their IFNγ signaling pathway as compared to adults, once IFNγ binds its receptor on AMs. This assumption was challenged by Marodi et al. who showed that IFNγ receptor-mediated signaling in neonatal macrophages harvested from cord blood compared to those from adult macrophages were reduced in regard to Candida killing, as well as in the release of superoxide anion . By performing a pharmacokinetic analysis in which both neonates and adults received the same weight-based dose of IFNγ, we showed that the IFNγ AUC of adults was 3.6 times larger than that of neonates. Larger IFNγ exposure in the adults resulted in expedited and enhanced MHC class II expression on AMs in adults compared to neonates. The large age-based differences in IFNγ exposure was not a result of differential IFNγ metabolism but was most likely an artifact of the neonate’s larger lung to body size ratio.
Taking this into consideration, a non-compartmental model analysis was used to determine a new neonatal dose of IFNγ that would achieve more adult-level AUCs. We predicted the new neonatal IFNγ dose of 60 ng/g would not only expedite viral clearance, but would also modulate associated risk factors linked to skewed Th2 pathology, such as mucus production. The data presented here shows the capacity of IFNγ to induced AM activation and RSV clearance in a clear, dose-dependent manner without provoking weight loss. To determine the extent to which AMs contributed to RSV clearance, we then depleted neonatal AMs using clodronate liposomes, which further demonstrated that AMs significantly contribute to viral clearance in the neonatal lung.
Low levels of IFNγ in the BALF are consistently associated with greater disease severity in neonates  yet it is unclear what IFNγ’s protective role is. Consistent with this finding is data published by Cohn et al. demonstrating that mucus production is inhibited by IFNγ in an adult mouse model  and our previously published data showing greater mucus production in RSV-infected neonatal mice in which there is minimal IFNγ production compared to RSV-infected adult mice . Consistent with previous findings, these data show here for the first time, reductions in goblet cell formation (as indicated by PAS staining) in RSV+ neonatal lungs treated with inhaled IFNγ (16 ng/g and 60 ng/g) when compared to untreated RSV+ pups. Mounting evidence continues to support the role of IFNγ as a regulator of mucus production [34–36]. Determining its precise mechanism in decreasing mucus production during neonatal RSV infection is an ongoing focus of research in our laboratory.
RSV non-structural proteins, NS1 and NS2, have been shown to impair early cellular apoptosis. It is speculated that this is advantageous to the virus; allowing RSV to spread from one cell to another throughout the airway . Other data support the idea that RSV sensitizes cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by upregulating the expression of death-receptors 4 and 5 . The sensitization is tempered however by early increases in Mcl-2, a member of the anti-apoptotic Bcl-2 family, which acts to delay the effects of TRAIL-mediated signaling and delays apoptosis in infected epithelial cells . We reasoned, then, that IFNγ, a well-established and appreciated inducer of cellular apoptosis  may be associated with less severe RSV disease clinically , due to its facilitation of apoptosis thereby limiting the spread of the virus. To test the hypothesis that IFNγ interferes with RSV-mediated impairment of apoptosis, we examined the effect of i.n. IFNγ on apoptosis in RSV infected neonatal mice. Surprisingly, RT-PCR data showed that i.n. IFNγ elicited no change in caspase 3 or 8 expression by 4 or 7 dpi in RSV infected neonates treated with 60 ng/g of IFNγ compared to PBS-treated controls (Unpublished data), despite enhanced viral clearance. However, significant reductions in TUNEL-positive cells in groups receiving i.n. IFNγ suggest possible enhanced clearance of apoptotic cells rather than IFNγ-mediated changes in apoptosis. Studies are ongoing to determine the role of IFNγ in mitigating the clearance of apoptotic cellular debris in the neonatal airway.
We postulate that IFNγ may be augmenting mechanisms of viral resistance in airway epithelial cells that dramatically reduce the viral burden at the onset of infection. IFNγ treatment was independently associated with significantly lower viral titers at our earliest time point, 4 dpi, suggesting that IFNγ treatment reduced the initial viral burden. High levels of IFNγ exposure prior to RSV infection have been shown to dramatically inhibit RSV replication in adult BALB/c mice . Additionally, exposure of a human airway epithelial cell line (A549) to IFNγ for 48 h prior to RSV infection reduced 2-day post-infection viral titers when compared to IL-4 treated or control cells. This reduction was associated with the upregulation of antiviral proteins, such as IFIT1 and Mx1 . Ongoing studies are investigating the contributions of IFNγ-treated airway epithelial cells to reduced RSV infectivity.
Finally, to our knowledge this is the first study, using a neonatal animal model, to examine the role of AMs in the clearance of RSV through the i.n. administration of CLip. We demonstrated that AMs play a significant role in RSV clearance and that delayed viral clearance is associated with reduced weight gain in a neonatal murine model of RSV. Although AM-mediated regulation of weight gain requires further investigation, impaired weight gain in CLip versus PBS-treated pups highlights the importance of AMs during neonatal RSV infection. Mice do not develop clinical symptoms of RSV but a failure to appropriately gain weight suggests that the animals experienced detrimental effects from dramatically reduced viral clearance. As previously mentioned, clinical data from human neonates < 2 years of age found that faster RSV viral clearance was associated with shorter hospitalizations . Future studies will investigate the histological effects of AM depletion in RSV-infected neonatal mice. We hypothesize that neonatal mice depleted of AMs have an abundance of necrotic debris and mucus obstructing their airways.
We have provided phenotypic and functional analysis of the role of IFNγ in neonatal RSV infection and furthered the understanding of AMs’ role in neonatal disease. These studies demonstrated the age-dependent development of AMs and the direct correlation of IFNγ in the neonatal AM response during RSV infection. Specifically, neonatal AMs display acute IFNγ dose responsiveness whereby higher doses of IFNγ are capable of overcoming the delay between IFNγ exposure and AM activation observed with lower IFNγ doses. Histological analysis of neonates infected with RSV and treated with IFNγ revealed the crucial role that it plays in reducing mucus production and decreasing the number of apoptotic cells in the airway. Lastly, AMs play a significant role in RSV clearance. Impaired RSV clearance as a result of AM depletion correlates with poor weight gain in neonatal mice. These findings warrant further investigation into the AM-dependent and independent effects of IFNγ in neonatal RSV disease.
Together these data indicate that early IFNγ exposure is critical to both reductions in viral burden and prevention of severe pathology in the neonatal airway. These data further demonstrate that i.n. IFNγ plays a critical role in reducing RSV burden early in infection and that neonatal AMs are crucial in coordinating efforts to eliminate RSV and return neonatal lungs to homeostasis.
This work was supported by the National Institutes of Health through grant numbers KL2TR000146, R03 RHD080874A, A University of Pittsburgh, School of Pharmacy internal merit award, and The University of Pittsburgh, Central Medical Research Fund (to K.M.E.).
We would like to thank Drs. Martin Moore and Stokes Peebles for their generosity in providing the RSV line 19 and Dr. Philip Empey for his helpful comments.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet. 2010;375:1545–55.View ArticlePubMedPubMed CentralGoogle Scholar
- Shay DK, Holman RC, Newman RD, Liu LL, Stout JW, Anderson LJ. Bronchiolitis-associated hospitalizations among US children, 1980–1996. JAMA. 1999;282:1440–6.View ArticlePubMedGoogle Scholar
- Leader S, Kohlhase K. Recent trends in severe respiratory syncytial virus (RSV) among US infants, 1997 to 2000. J Pediatr. 2003;143:S127–32.View ArticlePubMedGoogle Scholar
- Meissner HC, Long SS. Revised indications for the use of palivizumab and respiratory syncytial virus immune globulin intravenous for the prevention of respiratory syncytial virus infections. Pediatrics. 2003;112:1447–52.View ArticlePubMedGoogle Scholar
- El Saleeby CM, Bush AJ, Harrison LM, Aitken JA, Devincenzo JP. Respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children. J Infect Dis. 2011;204:996–1002.View ArticlePubMedPubMed CentralGoogle Scholar
- DeVincenzo JP, Wilkinson T, Vaishnaw A, Cehelsky J, Meyers R, Nochur S, et al. Viral load drives disease in humans experimentally infected with respiratory syncytial virus. Am J Respir Crit Care Med. 2010;182:1305–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Bagga B, Woods CW, Veldman TH, Gilbert A, Mann A, Balaratnam G, et al. Comparing influenza and RSV viral and disease dynamics in experimentally infected adults predicts clinical effectiveness of RSV antivirals. Antivir Ther. 2013;18:785–91.View ArticlePubMedGoogle Scholar
- DeVincenzo JP, El Saleeby CM, Bush AJ. Respiratory syncytial virus load predicts disease severity in previously healthy infants. J Infect Dis. 2005;191:1861–8.View ArticlePubMedGoogle Scholar
- Welliver TP, Reed JL, Welliver Sr RC. Respiratory syncytial virus and influenza virus infections: observations from tissues of fatal infant cases. Pediatr Infect Dis J. 2008;27:S92–6.View ArticlePubMedGoogle Scholar
- Sarzotti M, Gomes Mda P, Hoffman PM. IFN-gamma production in response to neuropathogenic Cas-Br-M murine leukemia virus infection. Viral Immunol. 1993;6:207–17.View ArticlePubMedGoogle Scholar
- Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol. 1994;12:635–73.View ArticlePubMedGoogle Scholar
- Butts C, Zubkoff I, Robbins DS, Cao S, Sarzotti M. DNA immunization of infants: potential and limitations. Vaccine. 1998;16:1444–9.View ArticlePubMedGoogle Scholar
- Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75:163–89.View ArticlePubMedGoogle Scholar
- Futata EA, Fusaro AE, de Brito CA, Sato MN. The neonatal immune system: immunomodulation of infections in early life. Expert Rev Anti Infect Ther. 2012;10:289–98.View ArticlePubMedGoogle Scholar
- Openshaw PJ. Antiviral immune responses and lung inflammation after respiratory syncytial virus infection. Proc Am Thorac Soc. 2005;2:121–5.View ArticlePubMedGoogle Scholar
- Hoebee B, Rietveld E, Bont L, Oosten M, Hodemaekers HM, Nagelkerke NJ, et al. Association of severe respiratory syncytial virus bronchiolitis with interleukin-4 and interleukin-4 receptor alpha polymorphisms. J Infect Dis. 2003;187:2–11.View ArticlePubMedGoogle Scholar
- Lee FE, Walsh EE, Falsey AR, Lumb ME, Okam NV, Liu N, et al. Human infant respiratory syncytial virus (RSV)-specific type 1 and 2 cytokine responses ex vivo during primary RSV infection. J Infect Dis. 2007;195:1779–88.View ArticlePubMedGoogle Scholar
- Gentile DA, Doyle WJ, Zeevi A, Howe-Adams J, Kapadia S, Trecki J, et al. Cytokine gene polymorphisms moderate illness severity in infants with respiratory syncytial virus infection. Hum Immunol. 2003;64:338–44.View ArticlePubMedGoogle Scholar
- Welliver TP, Garofalo RP, Hosakote Y, Hintz KH, Avendano L, Sanchez K, et al. Severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses. J Infect Dis. 2007;195:1126–36.View ArticlePubMedGoogle Scholar
- Johnson JE, Gonzales RA, Olson SJ, Wright PF, Graham BS. The histopathology of fatal untreated human respiratory syncytial virus infection. Mod Pathol. 2007;20:108–19.View ArticlePubMedGoogle Scholar
- Harker JA, Lee DC, Yamaguchi Y, Wang B, Bukreyev A, Collins PL, et al. Delivery of cytokines by recombinant virus in early life alters the immune response to adult lung infection. J Virol. 2010;84:5294–302.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee YM, Miyahara N, Takeda K, Prpich J, Oh A, Balhorn A, et al. IFN-gamma production during initial infection determines the outcome of reinfection with respiratory syncytial virus. Am J Respir Crit Care Med. 2008;177:208–18.View ArticlePubMedGoogle Scholar
- Empey KM, Orend JG, Peebles Jr RS, Egana L, Norris KA, Oury TD, et al. Stimulation of immature lung macrophages with intranasal interferon gamma in a novel neonatal mouse model of respiratory syncytial virus infection. PLoS ONE. 2012;7:e40499.View ArticlePubMedPubMed CentralGoogle Scholar
- Kling HM, Nau GJ, Ross TM, Evans TG, Chakraborty K, Empey KM, et al. Challenges and future in vaccines, drug development, and immunomodulatory therapy. Ann Am Thorac Soc. 2014;11 Suppl 4:S201–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Graham BSPM, Wright PF, Karzon DT. Primary respiratory syncytial virus infection in mice. J Med Virol. 1988;26:153–62.View ArticlePubMedGoogle Scholar
- Empey KMOJ, Stokes Peebles R, Egana L, Norris KA, Oury TD, Kolls JK. Stimulation of immature lung macrophages with intranasal interferon gamma in a novel neonatal mouse model of respiratory syncytial virus infection. PLoS One. 2012;7.Google Scholar
- Empey KM, Hollifield M, Garvy BA. Exogenous heat-killed Escherichia coli improves alveolar macrophage activity and reduces Pneumocystis carinii lung burden in infant mice. Infect Immun. 2007;75:3382–93.View ArticlePubMedPubMed CentralGoogle Scholar
- McDuffie E, Obert L, Chupka J, Sigler R. Detection of cytokine protein expression in mouse lung homogenates using suspension bead array. J Inflamm (Lond). 2006;3:15.View ArticleGoogle Scholar
- Gabrielsson J, Weiner D. Non-compartmental analysis. Methods Mol Biol. 2012;929:377–89.View ArticlePubMedGoogle Scholar
- Kurkjian C, Hollifield M, Lines JL, Rogosky A, Empey KM, Qureshi M, et al. Alveolar macrophages in neonatal mice are inherently unresponsive to Pneumocystis murina infection. Infect Immun. 2012;80:2835–46.View ArticlePubMedPubMed CentralGoogle Scholar
- Marodi L. Deficient interferon-gamma receptor-mediated signaling in neonatal macrophages. Acta Paediatr Suppl. 2002;91:117–9.View ArticlePubMedGoogle Scholar
- García C, Soriano-Fallas A, Lozano J, Leos N, Gomez AM, Ramilo O, et al. Decreased innate immune cytokine responses correlate with disease severity in children with respiratory syncytial virus and human rhinovirus bronchiolitis. Pediatr Infect Dis J. 2012;31:86–9.View ArticlePubMedGoogle Scholar
- Cohn L, Homer RJ, Niu N, Bottomly K. T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production. J Exp Med. 1999;190:1309–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Cho SH, Oh SY, Lane AP, Lee J, Oh MH, Lee S, et al. Regulation of nasal airway homeostasis and inflammation in mice by SHP-1 and Th2/Th1 signaling pathways. PLoS One. 2014;9:e103685.View ArticlePubMedPubMed CentralGoogle Scholar
- Hong JY, Bentley JK, Chung Y, Lei J, Steenrod JM, Chen Q, et al. Neonatal rhinovirus induces mucous metaplasia and airways hyperresponsiveness through IL-25 and type 2 innate lymphoid cells. J Allergy Clin Immunol. 2014;134:429–39.View ArticlePubMedPubMed CentralGoogle Scholar
- Shieh YH, Huang HM, Wang CC, Lee CC, Fan CK, Lee YL. Zerumbone enhances the Th1 response and ameliorates ovalbumin-induced Th2 responses and airway inflammation in mice. Int Immunopharmacol. 2015;24:383–91.View ArticlePubMedGoogle Scholar
- Bitko V, Shulyayeva O, Mazumder B, Musiyenko A, Ramaswamy M, Look DC, et al. Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth. J Virol. 2007;81:1786–95.View ArticlePubMedGoogle Scholar
- Kotelkin A, Prikhod’ko EA, Cohen JI, Collins PL, Bukreyev A. Respiratory syncytial virus infection sensitizes cells to apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand. J Virol. 2003;77:9156–72.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang S, Smith PK, Lovejoy M, Bowden JJ, Alpers JH, Forsyth KD. The apoptosis of neutrophils is accelerated in respiratory syncytial virus (RSV)-induced bronchiolitis. Clin Exp Immunol. 1998;114:49–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Kumar M, Behera AK, Matsuse H, Lockey RF, Mohapatra SS. Intranasal IFN-γ gene transfer protects BALB/c mice against respiratory syncytial virus infection. Vaccine. 1999;18:558–67.View ArticlePubMedGoogle Scholar
- Yamada Y, Matsumoto K, Hashimoto N, Saikusa M, Homma T, Yoshihara S, et al. Effect of Th1/Th2 cytokine pretreatment on RSV-induced gene expression in airway epithelial cells. Int Arch Allergy Immunol. 2011;154:185–94.View ArticlePubMedGoogle Scholar