PEDIATRICS Vol. 120 No. 2 August 2007, pp. e410-e415 (doi:10.1542/peds.2006-3283)
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ARTICLE |
Cytokine Profiles in the Respiratory Tract During Primary Infection With Human Metapneumovirus, Respiratory Syncytial Virus, or Influenza Virus in Infants
a Fundacion INFANT, Buenos Aires, Argentina
b Department of Pediatrics, School of Medicine
c Departments of Molecular Microbiology and Immunology
f International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland
d Division of Infectious Diseases, Hospital Materno Infantil San Isidro, Buenos Aires, Argentina
e National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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OBJECTIVES. We characterized the T helper cytokine profiles in the respiratory tract of infants infected with influenza virus, human metapneumovirus, and respiratory syncytial virus to examine whether these agents elicit similar cytokine responses and whether T helper type 2 polarization is associated with wheezing and severe disease.
METHODS. A prospective study of infants who were seeking medical help for acute upper and/or lower respiratory tract infection symptoms for the first time and were found to be infected with influenza, human metapneumovirus, or respiratory syncytial virus was performed. Respiratory viruses were detected in nasal secretions with reverse transcriptase-polymerase chain reaction assays. The study was performed in emergency departments and outpatient clinics in Buenos Aires, Argentina. T cell cytokine responses were determined in nasal secretions with immunoassays and reverse transcriptase-polymerase chain reaction assays.
RESULTS. Influenza elicited higher levels of interferon-
, interleukin-4, and interleukin-2 than did the other agents. Human metapneumovirus had the lowest interferon-/interleukin-4 ratio (T helper type 2 bias). However, no association was found between T helper type 2 bias and overall wheezing or hospitalization rates.
CONCLUSIONS. These findings show that viral respiratory infections in infants elicit different cytokine responses and that the pathogeneses of these agents should be studied individually.
Key Words: human metapneumovirus • influenza • respiratory syncytial virus • T helper
Abbreviations: RSV—respiratory syncytial virus • hMPV—human metapneumovirus • IFN—interferon • IL—interleukin • Th—T helper
Viral respiratory infections are the most frequent cause of hospitalization during the winter for infants and young children throughout the world.1 Clinical manifestations include symptoms of upper respiratory tract infection, such as cough and rhinorrhea, and symptoms of lower respiratory tract infection, such as wheezing, croup, and pneumonia.1 Although different viruses can infect the respiratory tract, no symptom is pathognomonic for a specific agent. In fact, all viruses seem to be able to elicit any of the clinical manifestations and to cause disease ranging from mild to severe.2 The similar clinical manifestations in infants infected with different agents have led to a unifying hypothesis postulating that greater severity of illness during respiratory infections is associated with higher levels of certain respiratory cytokines, particularly those mediating T helper (Th) type 2 responses.3–5
This Th2 hypothesis is supported in part by evidence of increased severity of disease during respiratory syncytial virus (RSV) infection in infants with gain-of-function polymorphisms in the interleukin (IL)-4 gene and higher levels of interferon (IFN)- being associated with protection from severe respiratory illness.3,6 In addition, Th2 bias is a frequent outcome measure in animal models of RSV disease.7,8 However, little is known about the role of Th2 cytokines in infants infected with agents other than RSV, such as human metapneumovirus (hMPV) and influenza virus, which may elicit similar diseases through different mechanisms. In fact, differences in the levels of innate cytokine responses against RSV, hMPV, and influenza virus have been described.2,9 These findings suggest that cytokine profiles may reflect virus-specific stimuli, rather than responses to a common pathogenic pathway for all agents. In this study, we compared the Th cytokine profiles of previously healthy infants visiting the emergency departments or outpatient clinics of 4 hospitals because of primary infections with RSV, hMPV, or influenza. Respiratory viruses elicited virus-specific patterns of Th cytokines. Interestingly, these cytokine responses, including a Th2 bias, did not correlate with overall disease severity.
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METHODS |
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Population
Cytokine responses were studied in infants born on or after September 1, 2001, who were brought to the outpatient clinics/emergency departments of participating institutions (Hospital Materno Infantil San Isidro, Hospital Boulogne, Hospital Frances, and Centro Periferico La Cava) in Buenos Aires to receive medical attention for acute upper respiratory tract infection and/or lower respiratory tract infection symptoms for the first time between June and September 2002.2 In the Southern Hemisphere, the period between June and August constitutes the respiratory infection season.2,10 Birth on or after September 1, 2001 was chosen as the cutoff date for enrollment because the date coincided with the end of the season in the previous year.
The outpatient clinics and emergency department of each of the participating institutions function jointly as one consultation service, in which children receive all of their outpatient care. Exclusion criteria included primary or secondary immune suppression, abnormal swallowing, and orofacial malfunctions. Written, witnessed, informed consent was obtained from the parents or guardians. The institutional review boards of all participating institutions approved the protocol.
On arrival at the outpatient clinic/emergency department, infants were screened by pediatric nurses using predefined clinical criteria, as described.2 Criteria for hospitalization included an oxygen saturation level of <93% in room air. Pediatricians visited the wards and ICUs daily, to monitor whether infants who had been enrolled in the study at an earlier time had been admitted to the hospital subsequently.
Detection of Viruses and Cytokines in Nasal Secretions
Nasal washes were obtained by gently flushing the infants' nostrils with 4 mL of sterile saline solution (AnalytiCals; Chemit Argentina, Buenos Aires, Argentina). Secretions were divided into aliquots, immediately snap-frozen with the use of dry ice, and stored at –80°C until diagnostic evaluation or cytokine determinations. hMPV was detected with reverse transcriptase-polymerase chain reaction using primers against a conserved region of the hMPV nucleocapsid protein.2 RSV subgroups A and B and influenza virus types A and B were detected by using the Hexaplex assay (Prodesse, Waukesha, WI). Cytokines associated with Th bias were measured in all infants infected with hMPV, RSV, or influenza virus, with the use of bead array assays (Becton Dickinson, Franklin Lakes, NJ) to detect IL-4 and IL-2 (detection limit: 2 pg/mL) and IFN- (detection limit: 7 pg/mL) and an ultrasensitive immunoassay for human IL-13 (detection limit: <10 pg/mL; BioSource, Carlsbad, CA). Measurements of IFN- were confirmed through reverse transcriptase-polymerase chain reaction by using a TaqMan gene expression assay (Applied Biosystems, Foster City, CA).
Statistical Analyses
Comparisons used Fisher's exact test (2-tailed), analysis of variance, or Mann-Whitney U test as appropriate. Correlations were analyzed by using Pearson's coefficient, a measure of the correlation of 2 variables on the same subject. P < .05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Frequency of Infections and Symptoms in the Population
As described in our previous study, 373 infants were enrolled for participation.2 The infants' mean age at consultation was 156 days (range, 5–363 days); smoking at home (231 infants; 62%) and breastfeeding (278 infants; 75%) were common. During the study,
1 viral pathogen was found for 116 (31%) of the 373 infants. hMPV was detected in 22 cases (6%) and RSV in 46 cases (12%; RSV-B in 30 cases and RSV-A in 16 cases). Influenza virus was detected in 19 of the samples (5%; 18 samples with influenza type B).2 In addition, 20 infants (5%) were infected with parainfluenza virus type 3 and 1 each with parainfluenza virus type 1 and type 2. Infants infected with influenza presented at an older age (mean ± SD: 221 ± 86 days) than did those infected with hMPV (161 ± 79 days; P = .02) or RSV (155 ± 78 days; P = .02). No differences were observed in the frequency of breastfeeding or smokers at home. Durations of symptoms before presentation were similar in all groups for upper respiratory tract infection (mean ± SD: influenza virus: 4.1 ± 2.3 days; RSV: 5.4 ± 3.8 days; hMPV: 4.2 ± 2.6 days; not significant) and lower respiratory tract infection (influenza virus: 0.4 ± 1.1 days; RSV: 1.4 ± 2.4 days; hMPV: 0.8 ± 1.1 day; not significant). Wheezing was less frequent in infants infected with influenza (4 of 19 infants), compared with RSV (24 of 46 infants; P = .02) or hMPV (13 of 22 infants; P = .02). No differences in the frequency of hospitalizations were detected.
Among infants infected with RSV, no differences in epidemiologic or clinical manifestations were observed between those exposed to subgroups A and B. Wheezing (RSV-A: 7 of 16 infants; RSV-B: 19 of 30 infants; not significant) and hospitalization (RSV-A: 4 of 16 infants; RSV-B: 6 of 30 infants; not significant) rates were similar for the 2 groups.
Eliciting of Higher Levels of IFN- and IL-4 in Nasal Secretions by Influenza Virus Than by RSV or hMPV
Several reports examined the role of IFN-, a prototypic Th1 cytokine, in respiratory infections.11–13 In a previous study, we reported higher levels of IL-12 in nasal secretions of infants infected with influenza virus or RSV, compared with those infected with hMPV.2 Therefore, we hypothesized that IL-12 levels would affect production of IFN-.14 Levels of IFN- were higher in infants infected with influenza, compared with RSV (P = .03) (Fig 1A). No difference in IFN- levels was observed between infants infected with RSV or hMPV (not significant) (Fig 1A). The greater production of IFN- was confirmed with measurements of mRNA IFN- levels by using reverse transcriptase-polymerase chain reaction; influenza mRNA was detected earlier than that of other viruses (mean detection cycle for viral/ß-actin mRNA: influenza: 1.06; hMPV: 1.19; RSV: 1.22).
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Subsequently, we compared production of IL-4, a prototypic Th2 cytokine, in nasal secretions of infants infected with the 3 viruses. Again, IL-4 levels were higher in the respiratory tract of infants infected with influenza than in those infected with RSV or hMPV (compared with RSV, P = 0.01; compared with hMPV, P = 0.04) (Fig 1B). No differences in IL-4 levels were detected between the latter 2 groups (Fig 1B). Similarly, only infants infected with influenza virus (5 of 19 infants; 26%) had detectable levels of the T cell cytokine IL-2 in nasal secretions. A fourth group of infants, infected with parainfluenza virus type 3, presented low levels of IFN-
(mean ± SD: 6.0 ± 2.3 pg/mL); only 2 infants had detectable levels of IL-4, and none produced detectable IL-2 (data not shown).
IL-13 is a Th2 cytokine that has been associated with acute wheezing in experimental models of respiratory disease.15 The relationship between specific agents, acute wheezing, and IL-13 levels was of interest to us, because wheezing is a cardinal manifestation of respiratory disease in infants.1–3,6 In this population, a greater frequency of wheezing was reported previously for infants infected with RSV or hMPV, compared with influenza.2 However, no difference in IL-13 levels was detected in nasal secretions of infants infected with any of these agents (not significant for all comparisons) (Fig 1C). Interestingly, infants infected with RSV-A versus RSV-B who presented with similar symptoms and disease severity did not differ in IFN- (Fig 2A), IL-4 (Fig 2B), and IL-13 (data not shown) production in the respiratory tract.
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Th2 Polarization in the Respiratory Tract of Infants Infected With hMPV
After higher levels of IFN-
(Th1) and IL-4 (Th2) were detected in nasal secretions of infants infected with influenza, compared with other viruses, we investigated the relationship between IFN- and IL-4 levels in the 3 groups, as an indicator of the Th balance in the immune response.14 Infants infected with hMPV had lower IFN-/IL-4 ratios (comparatively greater Th2 bias) than did those infected with RSV (P = .02) or influenza virus (P < .01) (Fig 3). No differences were observed between infants infected with influenza and RSV (not significant). These results are in agreement with our previous observation of decreased IL-12 production (less Th1) in the respiratory tract of hMPV-infected patients.2
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Lack of Correlation Between Cytokine Levels and Overall Disease Severity in Viral Respiratory Infections
In our study, infants were infected with influenza at an older age, compared with RSV or hMPV.2 Therefore, the greater production of IFN-
in infants with influenza could reflect an age-dependent immune maturation toward a Th1 bias during the first year of life.16 However, no correlation was observed between age at the time of infection and IFN- production (not significant) (Fig 4A) or IFN-/IL-4 ratios (not significant) (Fig 4B). As discussed above, no differences in the duration of symptoms before presentation, which can potentially affect the cytokine response, were detected between groups.
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Finally, airway reactivity and severity in viral respiratory infections have been associated with Th2 polarization of the immune response, attributable to both excess production of Th2 cytokines and decreased production of IFN-
.3,6 However, no association between IFN-/IL-4 ratios and acute wheezing (mean ± SEM: wheezing: 5.6 ± 0.5; no wheezing: 5.9 ± 0.4; not significant) or hospitalization (hospitalized: 5.7 ± 0.7; outpatient: 5.7 ± 0.4; not significant) was detected in our population (Fig 4, D and F). Similarly, no association was identified between IFN- levels and wheezing (wheezing: 10 ± 1.5 pg/mL; no wheezing: 11.4 ± 2 pg/mL; not significant) or hospitalization (hospitalized: 9.6 ± 1.5 pg/mL; outpatient: 11.1 ± 1.4 pg/mL; not significant) (Fig 4, C and E). In addition, no difference in IFN- levels was observed between infants of smoking and nonsmoking parents (smoking: 10.7 ± 2.2 pg/mL; nonsmoking: 10.9 ± 3.2 pg/ mL; not significant) and between breastfed and bottle-fed infants (breastfed: 10.8 ± 2.3 pg/mL; bottle-fed: 11 ± 3.4 pg/mL; not significant). IFN-/IL-4 ratios were also unaffected by smoking or breastfeeding (not shown). |
DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This article characterizes the Th cytokine profiles in nasal secretions of infants infected with influenza, RSV, or hMPV. These profiles do not correlate with wheezing or severity of illness in the population and highlight the disadvantage of advancing a unifying Th-related hypothesis to explain the pathogenesis of all viral respiratory illnesses. According to these and other observations,2,9 studying the mechanisms of clinical manifestations common to different agents requires determination of the effects of each virus individually, because certain cytokines may promote wheezing or severity in association with one virus but not others.
The role of IFN- in respiratory infections has been a long-standing source of interest.3,11–13,17 Although some studies identified an association between IFN- production and protection,3,11,17 others did not.13 The majority of the literature on IFN- and respiratory viruses focused on the effects of this cytokine during RSV infection and found responses to be decreased, compared with infections with other viruses.13,17 In agreement with these observations, infants infected with RSV in our studies had high levels of innate inflammatory cytokines2 but low adaptive responses, including IFN- levels. Conversely, infants infected with hMPV had low levels of both innate and adaptive responses, with a greater Th2 bias than those infected with RSV or influenza. Our observations are in agreement with findings of Th2 polarization in hMPV murine models18–20 and with the postulated role of hMPV as a trigger for asthmatic exacerbations.19,21 Interestingly, this is (to our knowledge) the first description of the Th profile elicited by hMPV in the respiratory tract of infants. Finally, influenza elicited higher levels of cytokines than did the other viruses. A role for high cytokine levels in severe cases of influenza associated with the H1N1 1918 and H5N1 viruses has been postulated.22,23 Our findings suggest that even mild cases of influenza virus type B infection lead to high levels of cytokine production, compared with responses elicited by other viruses. Importantly, despite the predilection of this virus for relatively older infants, cytokine responses, as in a previous study of several agents,17 neither correlated with age at infection nor were explained by delays in seeking medical help.
Intuitively, it would seem that assessments of levels of cytokines in infants with lower respiratory tract infection symptoms would benefit from samples obtained from the lower airways. Because it was obviously impossible to obtain tracheal aspirates in this population, only nasal washes were obtained. However, several observations indicate that our findings are biologically meaningful. These reassuring observations include the virus specificity of different Th profiles, the similar patterns in responses to RSV subgroups A and B, the Th2 bias in hMPV-infected infants that matches previous observations in animal models,18–20 the confirmation of lower IFN- levels in RSV-infected infants,13,17 the biological plausibility of greater IFN- levels in the group with higher IL-12 levels,2 and a previous report of correlation between upper and lower respiratory tract Th cytokine responses.24 Unfortunately, our study lacked power to analyze the clinical role of cytokines in association with individual viruses. It is also possible that other (unmeasured) cytokines may play a role common to the pathogeneses of all viral respiratory diseases in infancy.
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CONCLUSIONS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Respiratory viruses elicit different cytokine profiles in the respiratory tract. Influenza virus was associated with increased cytokine responses, and hMPV caused a greater Th2 bias than did other agents. However, no association was found between IFN-
or Th bias and wheezing or disease severity. These findings indicate that a unifying mechanism of illness for respiratory viruses is unlikely; therefore, the study of their pathogeneses should be virus specific.
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ACKNOWLEDGMENTS |
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This work was supported by a National Institute of Environmental Health Sciences contract between Johns Hopkins University and Fundacion INFANT and by National Institute of Allergy and Infectious Diseases grant AI-054952 (to Dr Polack). Dr Kleeberger was supported by the intramural research program of the National Institute of Environmental Health Sciences.
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FOOTNOTES |
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Accepted Apr 2, 2007.
Address correspondence to Fernando P. Polack, MD, Johns Hopkins University, 615 N Wolfe St, E5202, Baltimore, MD 21205. E-mail: fpolack{at}jhsph.edu
The authors have indicated they have no financial relationships relevant to this article to disclose.
Drs Melendi and Laham contributed equally to this article.
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PEDIATRICS (ISSN 1098-4275). ©2007 by the American Academy of Pediatrics
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