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In adults and children with RSV infection, a polymorphism in the IL-6 promoter at position −174 predicted illness magnitude. Also, polymorphisms in the IL-10, TNFα and INFγ genes were associated with immune responsiveness and the frequency of complications. Here, the effect of these polymorphisms on illness and seroconversion during infection with rhinovirus type 39 (RV39) was evaluated.
Seventy-two adults were genotyped for the selected polymorphisms, experimentally exposed to RV39 and followed for infection, seroconversion and symptoms/signs of illness. Regression analysis was used to determine if these polymorphisms predicted seroconversion and illness magnitude in 57 infected subjects.
The low production IL-6 (−174, C/C) phenotype was associated with greater symptom magnitudes and the INFγ (+874) phenotype predicted the frequency of seroconversion. No relationship between the IL-10 or TNFα polymorphisms and any measured outcome was documented. IL-6 protein measured in nasal wash fluids of 51 subjects was positively correlated with symptom magnitude but was independent of the IL-6 (−174) genotypes representing high and low production phenotypes.
These results document significant associations between the IL-6 (−174) and INFγ (+874) gene polymorphisms and specific responses to experimental RV39 infection. For the IL-6 (−174) polymorphism, the results replicate those for RSV infection.
A cold-like illness (CLI) is the most common disease affecting humans. The majority of CLIs is caused by an upper respiratory tract infection (vURI) with any of a number of viruses including rhinovirus (RV), Respiratory Syncytial Virus (RSV), influenza virus, coronavirus, and enterovirus, among others [1, 2]. While usually self-limited, CLIs cause significant morbidity and are known precipitants of complications in the sinuses, lungs and middle ears .
During a vURI, a CLI is usually diagnosed based on the perceived significance of the expressed signs and symptoms of illness. The viral symptom/sign complex (vSSC) is a summary measure of the degree of illness as defined by the magnitudes and durations of those symptom/sign elements typically associated with a vURI . The vSSC elements are similar for the different causal viruses [1, 5] and are believed to represent the overt expression of host immune/inflammatory responses to a vURI . This communality suggests that these viruses provoke their vSSCs and cause CLIs via activation of similar immune/inflammatory pathways.
Cytokines are known to orchestrate and coordinate the host immune/inflammatory responses during a vURI [7, 8]. For example, assays of serial nasal wash specimens from adult subjects experimentally infected with RV, RSV, influenza virus and coronavirus document consistent patterns of nasal secretion Il-1, IL-6, IL-8 and IL-10 production that temporally track the vSSC and respond appropriately to interventions and factors that modulate the vSSC [4, 9–16].
In the population of individuals with a vURI, the vSSC is variable such that all vURIs are not associated with a CLI  and past studies documented a variety of factors that modulate the vSSC and, consequently, CLI expression . Regarding the latter, one study of natural RSV infection in infants  and a follow-up study of experimental RSV infection in adults  reported that the C/C genotype of a single-nucleotide polymorphism in the IL-6 promoter at position −174 was associated with a greater vSSC. The primary hypothesis tested in the present study is that this IL-6 genotype is associated with a greater vSSC and a more prolonged CLI during experimental infection with a second vURI virus, RV type 39 (RV39). Because the RSV studies also documented effects of polymorphisms in the IL-10, IFNγ and TNFα genes on immune responses and complication expression, a secondary hypothesis was that these polymorphisms also affect the host responses to RV39 infection [19, 20].
Healthy adult subjects were recruited by advertisement. The protocol, study methods and potential risks were explained to presenting subjects and written Informed Consents for study participation and HIV screening were obtained from those wishing to participate and a general physical examination was done and a health history taken. If not excluded for medical reasons, standard demographic information was collected; blood was drawn for cell counts, clinical chemistries and assays of serum RV39 neutralizing antibody titer and HIV antibodies; urine was collected for clinical urinalysis, and a buccal swab was collected for DNA isolation.
Seventy-two, healthy (by history, physical examination, cell counts, blood chemistry, urine profile and negative HIV antibodies), susceptible (RV39 serum antibody titer ≤4), adult (18–55 year old) subjects were enrolled. Two days before admittance to cloister, blood was drawn for repeat assay of serum RV39 neutralizing antibody titer. Subjects were then admitted to the cloister site where they were confined to an isolated floor of a hotel for 6 days (Day 0=24 hour pre-exposure period, Days 1–5=sequential 24 hour post-exposure periods). On Day 0, female subjects had a urine pregnancy test and excluded if the results were positive as were subjects presenting with a CLI. At the end of Day 0, each subject was inoculated with RV39 by direct instillation of course drops to the nasal mucosa at an estimated dose of approximately 100 Tissue Culture Infective Dose-50% . On each day of cloister, all subjects had a general physical and an Ears, Nose and Throat examination, they completed a symptom diary, nasal mucociliary clearance function was assessed, nasal secretion production was measured, and a nasal wash was done with samples aliquoted and frozen at −70°C for later assay. Throughout, subjects were not permitted to take prescription or over-the-counter medications with the exceptions acetaminophen dispensed by study personnel and birth control. At the end of Day 5, subjects were interviewed by a physician, provided with specific instructions for follow-up and discharged from cloister. On or about study Day 28, a convalescent blood sample was drawn. The protocol and Informed Consents were approved by the IRBs at the University of Pittsburgh and Carnegie Mellon University.
The challenge virus was a safety-tested strain of RV39. RV39 serum neutralizing antibodies at screening, on Day -2 and on Day 28 were assayed by a standard 2-fold dilution method with titers reported as reciprocals of the final dilution . Seroconversion was defined as a 4-fold increase in titer between Days -2 and 28. A sample of nasal wash fluid from each subject on each study day was placed (3:1; v:v) in a cryovial containing 4X concentrated viral collecting broth and frozen at −70°C. For RV39 detection, the sample was thawed, 0.2 ml of the mixture was inoculated into tubes of human embryonic lung fibroblast cells and the cells observed for RV cytopathic effect . Infection was defined as detection of the challenge virus on any of Days 1–5. A variable representing infection duration, “days shed”, was constructed as the sum of days on which RV39 was isolated.
Genomic DNA was isolated from the buccal cells using a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA), amplified using a QIAGEN Repli-g Whole Genome Amplification Kit (GIAGEN), and quantified with a Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA) . The primer and probe sequences for the cytokine genotype assays were based on previous publications; IL-6 (−174) , TNFα (−308) and IL-10 (−1082, −819, −592) , and INFγ (+874) . Genotyping was performed with a TaqMan® Genotyping Master Mix (Applied Biosystems Inc, Foster City, CA, USA) following a published protocol . The 7300 System SDS v1.4 Software (Applied Biosystems Inc.) was used for instrument control, automated data collection and genotype assignment.
Phenotypes corresponding to in vitro cytokine production by stimulated lymphocytes [24, 28–33] were assigned to each genotype using the convention published by Gentile and colleagues . Specifically, for IL-6 (−174) the G/G and G/C genotypes were assigned the high and C/C the low production phenotype; for TNFα (−308) the G/A or A/A genotypes were assigned the high and G/G the low production phenotype; for INFγ (+874) the T/T genotype was assigned the high, T/A the intermediate and A/A the low production phenotype, and for IL-10 (−1082, −819, −592) the GCC/GCC haplotype was assigned the high, GCC/ACC or GCC/ATA the intermediate and ACC/ACC, ACC/ATA or ATA/ATA the low production phenotype. For INFγ (+874) and IL-10 (−1082, −819, −592), phenotypes were coded as 1 = “low production”, 2 = “intermediate production” and 3 = “high production” and for TNFα (−308) and IL-6 (−174) phenotypes were codes as 1 = “low production” and 2 = “high production”.
One aliquot of the nasal wash fluid collected on each day from 65 subjects was thawed and assayed for IL-6 using a commercially available ELISA (BioSource, Camerillo, CA) . The lower detection limit for the assay was 0.1 pg/ml. For each subject, a summary variable for IL-6 production was constructed as the log of the baseline adjusted IL-6 concentrations summed over the 5 post-exposure days of cloister. Summed values ≤0 were assigned a value of log(.1) = −1.
Thirteen vSSC elements were evaluated by self-completed diary on each study day (4 point scale: none, mild, moderate, severe). These represented four symptom domains: nasal symptoms (rhinorrhea, nasal congestion and sneezing), throat symptoms (sore throat and cough), general symptoms (headache, malaise, chills, sweats and fever) and complication symptoms (earache, chest congestion and sinus pain). For each element, the baseline score at day 0 was subtracted from the score on each of the 5 post-challenge days. Summary variables for each domain and for the total symptom score were calculated as the sum over all days of the baseline-adjusted scores for all contained elements (negative summed values were assigned a value of 0). The daily diary included a question as to the presence/absence of a “cold”. A continuous variable, “cold days”, was constructed by summing the “yes” responses over Days 1–5.
On each cloister day, subjects expelled their nasal secretions into pre-weighed tissues and sealed the tissues in plastic bags of known weight. At the end of each 24 hour period, the bags with expended tissues were weighed, and the secretion weight (i.e. nasal secretion production) was determined by subtraction . Nasal mucociliary clearance function was measured by placing approximately 20 μL of a dyed saccharin solution bilaterally onto the anterior aspect of the inferior turbinate using a calibrated pipette. Nasal mucociliary clearance time was calculated as the difference between the time when the solution was placed and the time when the subject reported a sweet taste . Summary nasal secretion production and mucociliary clearance time variables were calculated as the baseline-adjusted sum of the values over Days 1 through 5. Because these summary variables were not normally distributed, the data for both were log transformed and values ≤0 were assigned as log(.1) = −1.
The 9 continuous outcome variables were the log of the summary nasal secretion weight, mucociliary clearance time and IL-6 concentration; the summary nasal, throat, general, complication and total symptom scores, and cold days. The presence/absence of seroconversion was treated as a dichotomous outcome variable. Potential predictor variables consisted of the IL-6, IL-10, TNFα and INFγ phenotypes, day -2 RV39 titer, virus shedding days, age in years, race (coded: white=1, non-white=2) and sex (coded: female=1, male=2). To determine if these variables were significant predictors of each continuous outcome variable, multiple regression analysis was used. To determine if these variables were significant predictors of seroconversion, logistic regression was used. The relationships between the log of the baseline-adjusted IL-6 protein concentration and the continuous outcome variables were evaluated using simple linear regression. All statistical procedures were done using the NCSS 2000 Statistical Package (Kaysville, Utah, USA). Unless otherwise indicated, the format mean±standard deviation is used in the data presentation.
Eight of the 72 enrolled subjects had a serum RV39 titer >4 on study day -2 and were defined by protocol as non-susceptible, 5 subjects did not have RV39 recovered on any day and were defined as uninfected (no “wild” viruses were isolated from any nasal secretion sample), and buccal samples for cytokine genotype analysis were not available for 2 subjects. The data for these 15 subjects were eliminated from the data set.
Of the remaining 57 subjects, 31 were male, 36 were white and the mean age was 30.3±10.7 years. The data for demographics, symptoms/signs, virology assays and genotype data for INFγ (+874) and TNFα (−308) were complete for all subjects. The genotype assay for IL-6 (−174) and the haplotype assay for IL-10 (−1082, −819, −592) failed in 1 and 3 subjects, respectively. IL-6 protein concentrations in nasal secretions collected on each study day were available for 51 of these subjects.
None of the cytokine genotype distributions deviated significantly from the Hardy-Weinberg equilibrium. The distribution of cytokine phenotypes for TNFα was 12 high, 45 low; for IL-6 was 47 high, 9 low; for INFγ was 9 high, 24 intermediate, 24 low, and for the IL-10 haplotype was 16 high, 21 intermediate, 17 low. The number of subjects shedding virus on 1, 2, 3, 4 and 5 days was 4, 6, 12, 24 and 11, respectively. The Day-2, RV39 antibody titer was 1 (defined as a titer of <2), 2 and 4 in 40, 15 and 2 subjects, respectively. Thirty-four (60%) subjects seroconverted. The mean cold days was 1.68±1.79 days, the mean summary nasal, throat, general, complication and total symptom scores were 9.39±7.32, 4.75±4.45, 3.67±4.84, 3.11±4.84 and 20.91±17.85, respectively, the mean summary log clearance time and mucus weight were .99±.69 log(min) and .61±75 log(grams), respectively, and the mean summary log IL-6 concentration was 1.85±.93 log(pg/ml).
Logistic regression identified the INFγ (+874) phenotype as the only significant predictor of the frequency of seroconversion (β= −1.42; Std. Error of β=.63; Z-value=2.23, P-value=.024; where β is the odds ratio per unit change in the predictor). Seroconversion rates for the low, intermediate and high production INFγ phenotypes were: .79, .54 and .22, respectively.
For each continuous outcome variable, the statistical results for the multiple regression analysis are presented in Table I which lists only those predictors with an associated p-value <.1. Age was not a significant predictor of any variable, but race (whites>blacks) was a significant predictor of the throat symptom score and sex (males>females) was a significant predictor of cold days, throat symptom score and total symptom score, and a marginally significant predictor of the log secretion weight. Higher baseline RV39 titer was a significant predictor of fewer cold days, and lesser nasal, throat and total symptom scores and a marginally significant predictor of a lesser complication symptom score. Greater virus shedding days was a significant predictor of greater cold days, greater log secretion weight, and greater summary nasal, throat, complication and total scores. The low production IL-6 (−174) phenotype was a significant predictor of greater cold days and greater general and total symptom scores and a marginally significant predictor of greater nasal and complication symptom scores. Neither the TNFα (−308) or IL-10 (−1082, −819, −592) phenotype was significantly associated with any outcome variable.
The figure shows the baseline-adjusted mean daily symptom scores in each of the 4 domains and total symptoms for groups defined by the low and high IL-6 (−174) phenotypes. With the exception of the throat symptom score, the mean scores for the other domains and for total symptoms were greater on most days in the group defined as the low (i.e. C/C genotype) production phenotype when compared to the group defined as the high (i.e. C/G, G/G genotypes) production phenotype.
The multiple regression analysis was over-specified for the contribution of all possible predictors to the log baseline-adjusted, total IL-6 protein concentration. Limiting the possible predictors to the cytokine phenotypes, days shed and baseline RV39 titer documented no significant effect of any variable on that outcome. The mean values of that outcome variable for the low and high production IL-6 (−174) phenotypes were 1.84±1.31 and 1.83±.86 log (pg/ml); the between-group difference was not significant (P=.99, 2-tailed, Student’s t test). The Figure shows the mean log IL-6 concentration as a function of study day for the two groups defined by the IL-6 (−174) phenotype. The two curves were closely approximated with no obvious differences on any study day. The log of the baseline-adjusted, total IL-6 protein concentration was positively correlated with cold days (r=.50, p<.001), and the summary nasal (r=.54, p<.001), throat (r=.37, p=.008), general (r=.37, p=.007), complication (r=.36, p=.009) and total (r=.50, p<.001) symptom scores, and log secretion weight (r=.26, p=.061).
There is a vast literature relating cytokine polymorphisms to various inflammatory and infectious diseases, but few studies examined the possible role played by these polymorphisms in modulating the various expressions of a vURI. One study of RSV infection in hospitalized infants reported that the low production IL-6 (−174) phenotype was associated with greater illness severity and a study of experimental RSV infection in adults reported that the low production IL-6 (−174) phenotype was associated with a greater vSSC. The results of the present study for experimental RV39 infection in adults are consistent with those findings and support our primary hypothesis. Specifically, the low production IL-6 (−174) phenotype predicted a greater vSSC and greater symptom scores for most symptom domains in the RV39 infected subjects with the effect realized across all post-exposure days (See Figure). While there was no effect of the IL-6 (−174) phenotype on the two objective illness measures, nasal secretion production and mucociliary clearance time, these outcome variables were not predicted by the IL-6 (−174) phenotype in adults experimentally infected with RSV .
The results for two other studies support an effect of the IL-6 (−174) on vURI expression as reflected in the frequency of a common vURI complication, otitis media . Specifically, in a cross-sectional study, Patel and colleagues reported that the low production IL-6 (−174) phenotype was significantly more frequent in children with a history of recurrent acute otitis media when compared to children without a history of otitis media , and, in a longitudinal study of young children, Alper and colleagues reported that the low production IL-6 (−174) phenotype was a significant predictor of the otitis media coincidence rate for RV infections .
A second objective of this study was to explore the effect of TNFα (−308), INFγ (+874) and IL-10 (−592, −819, −1082) polymorphisms on the host responses to RV39 infection. These polymorphisms were shown to affect other host responses during RSV infection  and the rate of vURI complications [19, 23, 37]. For the outcomes examined in this study, only the INFγ (+874) polymorphism was shown to have a significant effect, with its phenotype being an inverse predictor of the rate of seroconversion during RV-39 infection. In adults experimentally infected with RSV, the TNFα (−308) phenotype was directly related to the frequency of seroconversion while the INFγ (+874) phenotype was indirectly related to the frequency of subjects developing a 2-fold increase in nasal RSV-specific sIgA antibody titers . The lack of significant effects of the other cytokine polymorphisms in the present study may be explained by their primary effects in previous studies on vURI complications such as the frequencies of otitis media and pneumonia [19, 23, 37], events that were not observed in the present study.
From these results, there is sufficient evidence to advance the hypothesis that the IL-6 (−174, C/C) genotype (low production phenotype) is associated with up-regulated inflammatory responses to a vURI as reflected in the vSSC and the frequency of expressed complications. While the mechanism(s) by which that polymorphism exerts these effects remains elusive given the complex and poorly understood temporal interactions among the various cytokines and other inflammatory chemicals in provoking the vSSC and other expressions of a vURI , the simplest hypothesis can be rejected. Specifically, as reported in this and past studies [12, 14, 16], higher nasal IL-6 concentrations were positively correlated in both phase and magnitude with the expressed vSSC leading to the expectations that the high production IL-6 (−174) phenotype would predict higher nasal IL-6 concentrations and consequently greater illness magnitudes. However, neither of these expectations were supported by the experimental data, i.e. no significant relationship was demonstrated between the IL-6 (−174) phenotype and local IL-6 nasal secretion concentration for either experimental RV39 or RSV infection in adults , and for all studies reviewed, the low (not high) IL-6 (−174) production phenotype predicted greater illness and complications [19, 20, 23, 37].
In interpreting these results, it should be recognized that the cytokine phenotypes were assigned based on in vitro results for stimulated cytokine release by leukocytes which may [28, 30, 38] or may not relate to in vivo cytokine production in the nasal mucosa, blood [39, 40] and local secretions  during a vURI. Moreover, there are a large number of polymorphisms in the IL-6 and other cytokine genes that were not assayed in the present study but have the potential to interact with the assayed polymorphisms to influence cytokine production [33, 41–44] and past studies showed that the local production of specific cytokines was influenced by polymorphisms in the genes for other cytokines (e.g. the TNF α (−308) polymorphism predicted nasal secretion IL-6 and IL-8 protein concentrations) . These observations emphasize the difficulties of predicting cytokine production for any biological compartment in response to a vURI using the genotype-phenotype associations for a given cytokine polymorphism based on in vitro studies.
In conclusion, the present study documents significant effects of certain cytokine polymorphisms on selected expressions of RV39 infection. Limitations of the study include the relatively few cytokine polymorphisms assayed, the assay of only IL-6 protein in nasal secretions, and the restriction of outcomes to seroconversion, provoked symptoms and two objective measures of illness. Also, because only adults were studied where the frequency of vURI complications is low, the effects of these cytokine polymorphisms on complication frequency could not be assessed. Nonetheless, it is highly likely that continued study of cytokine polymorphisms within the settings of natural and experimental vURIs will yield important information regarding the genetic basis for CLIs, vURIs and vURI complications.
The authors thank Dr. Ellen Mandel, Dr. Sancak Yuksel, James Seroky, Ellen Conser, Wesley Barnhart, Julianne Banks, Brendan Cullen-Doyle and Amy Seroky for their assistance in screening and evaluating the study subjects before, during and after the cloister phase of the study.
1We acknowledge that none of the authors have a conflict of interest regarding any of the materials presented in the manuscript.
2This research was supported by grants from the National Institute of Allergy and Infectious Disease (AI066367), the Pennsylvania Department of Health through a Commonwealth Research Enhancement grant, the John D. and Catherine T. MacArthur Foundation’s Network on Socioeconomic Status and Health, and the Eberly Research Endowment to the Department of Pediatric Otolaryngology, Children’s Hospital of Pittsburgh.
3The results of this study have not been presented at any society meetings either national or international.