Uteroglobulin-related protein 1 and severity of respiratory syncytial virus infection in children admitted to hospital
Abstract
There are several reports suggesting that genetic factors contribute to the severity of infection with the respiratory syncytial virus (RSV). Infants hospitalized with lower respiratory tract infection (LRTI) due to RSV are at a significantly increased risk for both recurrent wheezing and childhood asthma. Uteroglobin-related protein 1 (UGRP1) is a secretory protein expressed in the airways, and speculated to have anti-inflammatory activity. The presence of the −112G/A polymorphism in the UGRP1 promoter was found to have a significant correlation with asthma phenotype. Also plasma UGRP1 levels were shown to be associated both with this polymorphism and the severity of asthma. The study population consisted of 62 previously healthy infants, ≤12 months of age, who were hospitalized with RSV LRTI, and a control group of 99 healthy adults. Genotyping was performed by restriction fragment length polymorphism. UGRP1 serum levels were determined using ELISA. There were no significant differences in the overall distribution of UGRP1 −112G/A polymorphism genotypes or alleles between the hospitalized infants and healthy adults. A comparison of serum UGRP1 concentration measured at the time of admission and discharge between patients with and without the −112A allele revealed that there was no relation between the presence of the −112A allele and serum UGRP1 in hospitalized infants with RSV infection. Furthermore, there was no relationship between severity of RSV infection and genotype or serum UGRP1 concentration. These results suggest that UGRP1 does not have a major role in the development of severe RSV infection. J. Med. Virol. 83:1086–1092, 2011. © 2011 Wiley-Liss, Inc.
INTRODUCTION
Respiratory syncytial virus (RSV) is a member of the Pneumovirus genus of the family Paramyxoviridae, and is divided into two antigenic subgroups, A and B [Anderson et al., 1985; Mufson et al., 1985; Tsutsumi et al., 1988]. It is the leading cause of respiratory failure in young children, and a significant cause of morbidity and mortality in the elderly and patients receiving bone marrow and solid organ transplants [Hall, 2001]. Nearly all children are infected with RSV by 2 years of age [Glezen et al., 1986] and up to 5% of children require hospitalization for bronchiolitis, of which 2–5% require mechanical ventilation [Leader and Kohlhase, 2002]. Risk factors for severe RSV disease in infants and young children include bronchopulmonary dysplasia, premature birth, congenital heart disease, and underlying defects in immune or pulmonary function [Henrickson et al., 2004]. Furthermore, RSV infection in early childhood is strongly associated with the subsequent development of asthma or airway hyper-responsiveness [Singh et al., 2007].
Even in a single outbreak of RSV disease, the severity of infection varies markedly, and morbidity fluctuates from season to season. Whether specific strains within each subgroup are associated with the severity of illness remains unclear [Martinello et al., 2002; Fodha et al., 2007]. In addition, host response factors are speculated to be important in determining disease severity [Openshaw and Tregoning, 2005]. There are several reports suggesting that genetic factors linked to the innate immune genes, immunoglobulin heavy G2 chain, surfactant protein A(SP-A), SP-B, SP-D, interleukin-4 (IL-4), IL-4 receptor, IL-6, IL-8, IL-9, IL-10, IFN-γ, TGF-β1, and PGI2, contribute to the severity of the illness and lead to bronchial obstructive disease due to RSV infection [Hull et al., 2001; Choi et al., 2002; Lahti et al., 2002; Lofgren et al., 2002; Gentile et al., 2003; Hoebee et al., 2003, 2004; Aurivillius et al., 2005; Janssen et al., 2007; Puthothu et al., 2007; Hashimoto et al., 2008].
The uteroglobin-related protein 1 (UGRP1) gene, officially known as secretoglobin 3A2 (SCGB3A2), encodes a homodimeric secretory protein of 10 kDa [Niimi et al., 2002a]. It was identified originally as a downstream target gene for the homeodomain transcription factor, thyroid-specific enhancer-binding protein (T/EBP), also known as thyroid transcription factor 1 (TTF1) or NKX 2.1[Niimi et al., 2001]. T/EBP regulates the expression of thyroid- and lung-specific genes, the latter of which includes SP-A, AP-B, AP-C, and Clara cell secretory protein (CCSP) [Bohinski et al., 1994; Bruno et al., 1995; Kelly et al., 1996; Ray et al., 1996; Niimi et al., 2002a]. The human UGRP1 gene is located on chromosome 5q31–32, in an area that contains one or more genes that might play a role in airway inflammation associated with atopic asthma [Niimi et al., 2002b]. These genes include a number of proinflammatory cytokines such as IL-3, -4, -5, -9, and -13[Postma et al., 1995; Ruffilli and Bonini, 1997; Bleecker, 1998; Cookson and Moffatt, 2000; Ober and Moffatt, 2000]. In the murine lung, UGRP1 is expressed mainly in the airway epithelial cells, and is an early molecular marker for non-ciliated bronchial epithelial cells (Clara cells)[Reynolds et al., 2002]. Clara cells secrete CCSP abundantly, which modulate lung inflammatory and immune responses, including allergic asthma and RSV infection [Chen et al., 2001; Wang et al., 2001; Wang et al., 2003; Johansson et al., 2009]. These findings suggest that UGRP1 may play a significant physiological role in the lung. Niimi et al. [2002b] previously reported the presence of a G to A polymorphism at −112 bp in the human UGRP1 gene promoter, and that the −112A allele is responsible for a 24% reduction in promoter activity compared to that for the wild-type −112G allele. This polymorphism shows a significant association with the asthma phenotype in the adult Japanese population. Inoue et al. [2008] reported that plasma UGRP1 levels were associated with both the G-112A UGRP1 gene promoter polymorphism and the severity of asthma. However, little is known about its relationship to respiratory tract viral infection.
The purpose of the present study was to determine the association between the −112G/A polymorphism in the UGRP1 promoter or serum UGRP1 level and the severity of RSV infection.
METHODS
Study Subjects
The study population was composed of 62 previously healthy infants, who were enrolled in the study at the time of their admission to Fukushima Medical University Hospital with RSV lower respiratory tract infection (LRTI). The enrolment period extended throughout three RSV epidemic seasons. Ninety-nine healthy adults served as a control population to assess the distribution of the −112G/A polymorphism in the UGRP1 promoter. In the infants infected with RSV, serum was collected at the time of hospitalization and at discharge to measure UGRP1 levels. All study subjects were of the same ethnic background (Japanese). A scoring system [Rodriguez et al., 1997] was used to determine the severity of the RSV infections (Table I, Supporting Information). The same trained observers evaluated each patient twice daily during the course of their hospital stay using this scoring system, and each patient was then reviewed by the director of pediatrics. Informed consent from one or both parents was obtained for all infants included in the study as well as from the healthy adult subjects. This study was carried out under the auspices of the Human Research Committee in accordance with the Guideline on Human Experiments at Fukushima Medical University.
| Clinical characteristic | Male (N = 29); mean ± SD or median [IQR] | Female (N = 33); mean ± SD or median [IQR] | P-value* |
|---|---|---|---|
| Gestational age (weeks) | 38.3 ± 1.7 | 38.5 ± 1.6 | 0.74 |
| Birthweight (g) | 3010 ± 280 | 2945 ± 430 | 0.28 |
| Age (months) | 5.8 ± 3.9 | 6.6 ± 3.6 | 0.32 |
| Illness days | 8.0 [8.0–9.0] | 9.0 [7.0–10.5] | 0.73 |
| LRTI score | 3.69 [3.4–3.5] | 3.50 [3.0–4.0] | 0.81 |
| Respiratory score | 1.70 [1.30–2.25] | 1.80 [1.50–2.30] | 0.38 |
| WBC (×103/mm3) | 11.5 ± 5.2 | 11.2 ± 3.5 | 0.67 |
| CRP (mg/dl) | 0.22 [0.09–1.01] | 0.54 [0.23–1.66] | 0.08 |
| Body temperature (°C) | 38.1 ± 0.87 | 38.2 ± 0.79 | 0.89 |
- Illness days, days from the beginning of symptoms until discharge from hospital; respiratory score, the highest score during hospitalization; WBC, white blood cell; CRP, C-reactive protein; body temperature, measured at the time of hospitalization.
- * Mann–Whitney U-test.
Inclusion Criteria
Infants were enrolled if they were healthy previously, ≤12 months of age, and hospitalized for bronchiolitis and/or pneumonia with nasal wash specimens determined to be positive for RSV antigen using immunochromatography. Patients were excluded if they had known or suspected cardiopulmonary disease, premature birth with a gestational age of <36 weeks, immunodeficiency disease (including human immunodeficiency virus infection), known serum IgA deficiency, renal failure, or previous reaction to blood products. Exclusion criteria also included the receipt of blood or blood products in the preceding 60 days, an established diagnosis of reactive airway disease, and apnea without evidence of LRTI on presentation. Infants with a concomitant bacterial infection at or during hospitalization were not excluded.
Attending physicians performed physical examinations and clinical assessments, and were responsible for medical care, including intravenous fluid administration, nebulization treatments, supplemental oxygen, mechanical ventilation, and/or the prescription of steroids or antibiotics. The decision to discharge patients was also made by the attending physician. Data documented by the attending physician was used to calculate the LRTI and Respiratory scores for each infant. The Respiratory score is the sum of the component scores for respiratory rate, O2 saturation, and physical findings divided by 3. The LRTI score consists of the sum of scores for wheezing, rales, and retractions divided by 2 (see Supporting Information).
Genotyping
Genomic DNA was extracted from total blood using a QIAamp DNA Blood Mini Kit (Qiagen, Inc., Valencia, CA). Genotyping was performed by restriction fragment length polymorphism. The UGRP1 −112G/A polymorphism was examined with the use of a PCR fragment amplified using the forward primer 5′ GCA AGG GTT TAT GCA AGA GG 3′ and reverse primer 5′AGT GTG ATG GCT GCT TTT GCA C 3′. PCR was performed in a final volume of 20 µl containing 5 ng genomic DNA, 4 pmol of each primer, 1 µl dNTP-Mix (2 mmol), 2 µl 10× PCR Buffer II, and 0.2 µl (1U) TaKaRa EX Taq HS (TaKaRa, Japan). The PCR conditions were as follows: denaturing at 94°C for 1 min, then 40 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, and final incubation at 72°C for 7 min. The −112G/A polymorphism was identified by loss of the HphI restriction site (for the G allele, 110, 37, 70 bp fragments; for the A allele, 147 and 70 bp fragments). The PCR products were digested with 5 U of HphI (New England BioLabs, Inc, Beverly, MA) at 37°C for 1 hr, electrophoresed on a 4% agarose gel and visualized using ethidium bromide staining and ultraviolet transillumination. The accuracy of the genotyping was confirmed using the direct sequencing of PCR products from 20 subjects selected randomly from among the infants infected with RSV and the controls.
Quantitation of UGRP1
UGRP1 serum levels were determined using ELISA with two monoclonal antibodies against human UGRP1, MoAb 4G10 and MoAb 5B1, as described previously [Inoue et al., 2008]. A linear standard curve was obtained between 9.6 and 1,250 pg/ml of UGRP1. All assays were performed in duplicate.
Statistical Data Analysis
Clinical and demographic variables were expressed as median and interquartile range (IQR) or mean and standard deviation (±SD) for the distribution of continuous variables or the number and percentage of categorical variables, respectively. Continuous variables were compared using Mann–Whitney U-test by gender or −112G/A genotype, and categorical variables were compared using Chi-square tests by −112G/A genotype or allele. UGRP1 concentrations were log transformed to meet linear regression criteria of normality of the residuals. Spearman's correlation coefficient (rho) was used to assess bivariate correlations among continuous variables. A value of P < 0.05 was considered to be statistically significant.
RESULTS
Clinical Characteristics
Sixty-two infants, including 29 males (46.7%) and 33 females (53.3%), were hospitalized with RSV LRTI between January 2003 and January 2005. The following clinical characteristics were examined: birth-weight, gestational age, age in months, illness days (i.e., the number of days between the onset of symptoms and discharge from hospital), LRTI score, respiratory score, C-reactive protein level, white blood cell count, and body temperature (Table IS). There were no significant differences by gender in any factor at the time of hospitalization; therefore, subsequent analyses were not stratified by gender.
Genotypic Distribution of the −112G/A Polymorphism in the UGRP1 Gene
The frequencies of the −112G/A polymorphism in the infant infected with RSV and control groups are listed in Table II. These genotype distributions were confirmed to fit to the Hardy–Weinberg distribution. In patients infected with RSV, there were no significant differences in the overall distribution of genotypes (P = 0.114) or alleles (P = 0.08) between males and females. There were also no significant differences in the overall distribution of UGRP1 −112G/A polymorphism genotypes (P = 0.419) or alleles (P = 0.121) between the infants hospitalized with RSV LRTI and the 99 healthy adults.
| Hospitalized infants | Control | |||
|---|---|---|---|---|
| Male N | Female N | Total N (%) | Total N (%) | |
| Genotype | ||||
| A/A genotype | 2 | 0 | 2 (3.2) | 2 (2.0) |
| G/A genotype | 13 | 10 | 23(37.1) | 28 (28.3) |
| G/G genotype | 14 | 23 | 37 (59.7) | 69 (69.7) |
| Total | 29 | 33 | 62(100) | 99(100) |
| Allele type | ||||
| A | 17 | 10 | 27 (21.8) | 60 (30.3) |
| G | 41 | 56 | 97 (78.2) | 138 (69.7) |
| Total | 58 | 66 | 124 (100) | 198 (100) |
- N for genotype-based analysis is the number of infants, whereas for allele-based analysis N is the number of alleles (i.e., twice the number of subjects).
Serum Concentration of UGRP1 and Clinical Characteristics Determined by −122G/A Polymorphism Genotype
The serum UGRP1 concentration was measured in patients hospitalized due to RSV infection. The UGRP1 concentration at the time of admission and discharge were 176.4 [IQR97.8–392.6] pg/ml and 154.4 [IQR106.4–352.6] pg/ml, respectively (P = 0.210). Patients were divided into two subgroups by allele, with one subgroup having at least one −112A allele (G/A or A/A genotype) and the other possessing the wild-type (G/G genotype). At the time of admission, the UGRP1 concentration in patients with and without the −112A allele was 175.4 [IQR56.1–532.4] pg/ml and 159.0 [IQR102.1–504.5] pg/ml, respectively (P = 0.61). At the time of discharge, the UGRP1 concentration in patients with and without the −112A allele was 133.2 [IQR71.5–548.8] pg/ml and 156.2 [IQR115.9–352.3] pg/ml, respectively (P = 0.57).These data suggest that there was no relation between −112A allele and serum UGRP1 in infants hospitalized with RSV infection. Furthermore, there were no significant differences in any clinical factors between patients with and without the −112A allele (Table III).
| Clinical characteristic | G/G (N = 37); mean ± SD or median [IQR] | G/A or A/A (N = 25); mean ± SD or median [IQR] | P-value* |
|---|---|---|---|
| Gestational age (weeks) | 38.4 ± 1.7 | 38.4 ± 1.5 | 0.84 |
| Birthweight (g) | 2,990 ± 374 | 2953 ± 360 | 0.87 |
| Age (months) | 6.3 ± 3.6 | 6.0 ± 3.9 | 0.62 |
| Illness days | 8.0 [7.5–9.0] | 9.0 [8.0–11.0] | 0.13 |
| LRTI score | 3.5 [3.0–4.0] | 3.5 [3.0–3.75] | 0.64 |
| Respiratory scorec | 1.80 [1.40–2.30] | 1.70 [1.50–2.25] | 0.67 |
| WBC (×103/mm3) | 11.5 ± 4.6 | 11.0 ± 3.9 | 0.88 |
| CRPc (mg/dl) | 0.48 [0.23–1.29] | 0.21 [0.08–1.38] | 0.15 |
| Body temperature (°C) | 38.2 ± 0.79 | 37.8 ± 0.84 | 0.15 |
| A-UGRP1 (pg/ml) | 159.0 [102.1–504.5] | 175.4 [56.1–532.4] | 0.61 |
| D-UGRP1 (pg/ml) | 156.2 [115.9–352.3] | 133.2 [71.5–548.8] | 0.57 |
- Illness days: days from the beginning of symptoms until discharge from hospital; respiratory score, the highest score during hospitalization; A-UGRP1, serum level of UGRP1 at admission; D-UGRP1, serum level of UGRP1 at discharge; WBC, white blood cell; CRP, C-reactive protein; body temperature, measured at the time of hospitalization.
- * Mann–Whitney U-test.
Relationship Among Clinical Characteristics and Serum UGRP1 Concentration
The serum UGRP1 concentration at the time of hospitalization and discharge was examined in relation with illness days, physical findings, LRTI score, respiratory score, C-reactive protein level, white blood cell count, and body temperature (Table IV). No correlations were observed between serum UGRP1 concentration and any of the above factors.
| Clinical characteristic | rhoa | P-value* |
|---|---|---|
| A-UGRP1 (pg/ml) | ||
| Illness days | 0.014 | 0.923 |
| LRTI score | −0.076 | 0.549 |
| Respiratory score | −0.017 | 0.890 |
| WBC (×103/mm3) | −0.085 | 0.516 |
| CRP (mg/dl) | −0.074 | 0.577 |
| Body temperature (°C) | −0.236 | 0.060 |
| D-UGRP1 (pg/ml) | ||
| Illness days | −0.071 | 0.610 |
| LRTI score | −0.096 | 0.445 |
| Respiratory score | 0.032 | 0.798 |
| WBC (×103/mm3) | −0.110 | 0.398 |
| CRP (mg/dl) | −0.028 | 0.836 |
| Body temperature (°C) | 0.163 | 0.197 |
- Illness days, days from the beginning of symptoms until discharge from hospital; respiratory score, the highest score during hospitalization; A-UGRP1, serum level of UGRP1 at admission; D-UGRP1, serum level of UGRP1 at discharge; WBC, white blood cell; CRP, C-reactive protein; body temperature, measured at the time of hospitalization.
- a Spearman correlation coefficient (rho).
- * Spearman rank analysis.
DISCUSSION
This is the first study aimed at determining an association between UGRP1 and the severity of RSV infection. However, the results of this study indicate that there is no association between the UGRP1 −112G/A polymorphism or serum UGRP1 concentration and the severity of RSV infection in hospitalized children.
The severity of illness and presence of LRTI in association with cases of RSV infection are likely to be the result of both the immune response to virus and direct viral effects on the lung and epithelium. Previous studies revealed that both innate and adaptive immune responses are critical factors in disease severity in cases of RSV [Oshansky et al., 2009]. Thus, the host immune response is an important determinant of the severity of RSV-induced illness. Therefore, following lung injury, resolution of inflammation is required for the recovery of normal tissue function and tissue homeostasis.
A significant correlation was reported between the −112G/A polymorphism in the UGRP1 promoter and the asthma phenotype in the adult Japanese population [Niimi et al., 2002b]. However, there were no significant differences in the overall distribution of UGRP1 −112G/A polymorphism genotypes or alleles between the infants hospitalized with RSV infection and the healthy controls. On the other hand, in a previous study, plasma UGRP1 levels were shown to be affected by the −112/G/A polymorphism in the UGRP1 promoter [Inoue et al., 2008]. The mean plasma UGRP1 levels for subjects with or without asthma, but having at least one −112A allele, was lower than that in those having the −112G allele. Luciferase reporter assays showed a 24% reduction in promoter activity in patients with the −112A allele as compared with those with the −112G allele [Niimi et al., 2002b]. However, there were no differences in serum UGRP1 levels between those with or without the −112A allele among the infants hospitalized with RSV LRTI. Furthermore, there were no relationships between the severity of RSV infection and either genotype or serum UGRP1 concentration. This indicates that there are differences in mechanisms between RSV-related LRTI in childhood and asthma in adults, though RSV infection in early childhood is strongly associated with the subsequent development of asthma or airway hyper-responsiveness.
A previous study demonstrated that the anti-inflammatory effect of IL-10 may be mediated by UGRP1 [Srisodsai et al., 2004]. In general, IL-10 is associated with the resolution of the inflammatory response process [Hawrylowicz and O'Garra, 2005] and is known to block allergic inflammation in animal models [Stampfli et al., 1999; Oh et al., 2002]. IL-10 is up-regulated during acute exacerbation of asthma induced by viral infection, particularly in the recovery phase [Nicholson et al., 1993; Johnston et al., 1995; Busse and Gern, 2005; Hawrylowicz and O'Garra, 2005]. RSV induces the expression of IL-10 and its mRNA by alveolar macrophages [Panuska et al., 1995]. IL-10 was elevated in nasopharyngeal secretions in children with RSV infection [Murai et al., 2007]. IL10 level in the lung during RSV LRTI was not examined in this study, but IL10 might influence UGRP1 induction in the lung.
The amino acid sequence of human UGRP1 has 25% identity to human CCSP, which is believed to function as an anti-inflammatory protein [Niimi et al., 2001]. The amino acid sequence similarities between UGRP1 and CCSP family proteins are significant in the signal peptide and antiflammin regions [Niimi et al., 2001]. Currently, only limited information on the functional role of UGRP1 in the lung is available. However, its similarity to CCSP, particularly in the antiflammin domain, which exhibits major anti-inflammatory and immunomodulatory activities, suggests a possible anti-inflammatory function for UGRP1. In addition, the suppression of allergic airway inflammation by an over-expression of UGRP1 in the airways has been reported in mice [Chiba et al., 2006]. These findings suggest that UGRP1 may also have an anti-inflammatory function. Based on studies using CCSP-deficient mice, Harrod et al. [1998] and Wang et al. [2003] reported that CCSP modulated lung inflammatory and immune responses to acute viral infection with adenovirus and RSV, respectively. Further, as with the UGRP1 and CCSP lung secretory proteins, the expression of SP-A, SP-B, and SP-C are regulated by a single transcription factor, T/EBP [Bohinski et al., 1994; Bruno et al., 1995; Kelly et al., 1996; Ray et al., 1996; Niimi et al., 2002a]. SP-A, along with SP-D, has an important role in the innate immune response [Griese, 2002]. SP-B and SP-C are hydrophobic and essential constituents of lung surfactant [Puthothu et al., 2007], and all surfactant proteins are generally synthesized in the endoplasmic reticulum of alveolar type II cells. There are reports that polymorphisms of SP-A, SP-B, and SP-D are associated with severe RSV infection in humans [Lahti et al., 2002; Lofgren et al., 2002; Puthothu et al., 2007]. SP- C deficient mice have also been reported to be susceptible to RSV infection [Glasser et al., 2009].
There are several potential limitations to this study that should be considered. First, the study population is small. Second, the etiology of RSV infection is complex and is likely to be multigenic. Last, sera or DNA samples from age-matched controls were not used. To conclude, the present results suggest that UGRP1 may not have a major role in the development of severe RSV infection.
Acknowledgements
We thank the members of the Department of Pulmonary Medicine, Microbiology and Pediatrics for their valuable advice and technical assistants in relation to this study. None of the authors have financial relationships with companies that have an interest in the subject matter of this manuscript. This research was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Young Scientists (B), 2003–2005, 15790534, Scientific Research (C), 2006–2008, 18591196.
