Impaired Immune Response in Severe Human Lower Tract... : The Pediatric Infectious Disease Journal (original) (raw)
Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory infection (ALRI) in infants.1,2 Almost all infants become infected with RSV during the first 2 years of life, and infants younger than 6 months of age are at the highest risk to develop severe illness. The mean hospitalization rate for RSV disease is less than 5% per year and the mortality rate of hospitalized infants is 0.5% to 1%.3–7 In Chile, RSV is the most frequent cause of ALRI causing yearly winter outbreaks with a fatality rate of 0.1%.8,9
Certain clinical conditions including premature birth, chronic pulmonary disease, congenital heart disease, the presence of home-smokers, low levels of maternal RSV antibodies, and immunodeficiency increase the risk of developing severe RSV disease.2 Although almost 80% of healthy infants are infected with RSV in the first year of life, less than 5% of them require hospitalization. The risk factors described so far can not explain all cases of severe RSV bronchiolitis. The pathogenesis of illness related to RSV infection is being studied, especially since the formalin-inactivated vaccine experience,10 and the role of the host immune response is being intensively investigated.
In experimental studies, mice previously sensitized to RSV surface proteins followed by RSV infection can induce T helper type 1 (TH1) and type 2 cytokine (TH2) responses.11 Mice with type 2 cytokine responses developed severe disease, whereas those with type 1 responses show increased viral clearance and reduced immune damage.12 Similar immune mechanisms have been proposed for RSV infection in infants with bronchiolitis, in which both viral replication and lymphocyte immune responses are critically involved in the pathogenesis.13–16 However, for other authors, the nature of whole mediator response remains unknown.17–21 In the last 10 years, natural killer (NK) cells have also emerged as an important cell type in controlling viral infections.22–26 There are a few reports about influenza virus infections, but there are no previous reports on the complex network of NK cells and RSV infection in infants.27–29
Previous authors have argued that the white cell count in RSV bronchiolitis is a preponderance of neutrophils.30,31 O'Donnell and Carrington32 has seen a decrease in circulating lymphocytes. It has been suggested that this reduction in lymphocyte count may be due to redistribution to the lung.33 However, lymphopenia is also known to occur in other viral illnesses, including measles, severe acute respiratory syndrome, and Ebola.34–36 Recently, it has been proposed that mechanisms of apoptosis induced by RSV infection could be responsible for the decrease in peripheral lymphocytes.37
With this background, we hypothesized that severe RSV disease in infants younger than 6 months of age is associated with low numbers of circulating lymphocytes, NK cells, and low concentrations of plasma cytokines, which could be related to an impaired immune response. We therefore compared the in vivo plasma cytokine concentrations, the numbers and phenotype of peripheral blood lymphocytes and NK cells between previously healthy infants less than 6 month of age hospitalized with their first proven RSV-ALRI and age-matched healthy infants. Blood cell counts and cytokine concentrations in RSV-infected infants were also analyzed according to clinical disease severity defined by an objective scoring system.
MATERIALS AND METHODS
Subjects
We have established a surveillance for community-acquired respiratory viruses in children <2 year of age, admitted for ALRI to the Roberto del Rio Children's Hospital, in Santiago of Chile since 1988 as previously described in detail.38–40 For the purpose of this study, previously healthy, term infants, between 28 days of life and 6 months old, with a normal weight at birth, hospitalized in a regular ward or intensive care unit for community acquired RSV-ALRI, were consecutively enrolled into the study during the winter seasons of 2005 and 2006. Patients were enrolled during the first 3 days of respiratory symptoms (nasal discharge, cough, or respiratory distress) and ALRI was confirmed by clinical signs of respiratory distress with crackles or wheezing, or hyperinflation in a chest radiograph. Age- and sex- matched patients evaluated for minor elective surgeries and with no clinical and virologic evidence of respiratory viral infections (RSV, adenovirus, influenza, or parainfluenza virus) and with no previous history of respiratory pathology were included as controls during warmer seasons, when in Chile viral respiratory infection rate are very low.8,9 The following exclusion criteria for patients and controls were observed: (i) previous hospitalization for any cause, (ii) primary or secondary immunodeficiency, (iii) prematurity, (iv) bronchopulmonary dysplasia, (v) mechanical ventilatory support, (vi) congenital heart disease, and (vii) any previous respiratory disease, including common cold and acute otitis media. The study protocol was approved by the Institutional Review Boards of the Roberto del Río Hospital and the Faculty of Medicine, University of Chile. Informed and signed consent was obtained from the parents of all study participants.
Viral Studies
A first positive RSV nasopharyngeal aspirates (NPA) sample was obtained at the emergency room at admission, and analyzed using an immunofluorescence assay (IFA). A second fresh sample of NPA was obtained from each patient during the first 24 to 48 hours after admission. For each enrolled healthy control, 1 NPA sample was also obtained. For NPA samples, IFA and virus isolation for RSV, influenza, parainfluenza, and adenovirus were conducted immediately as described elsewhere.38–40 We also performed a real time polymerase chain reaction for detection of RSV in cases and healthy infants. Total RNA of NPA was extracted by the guanidinium thiocyanate-phenol-chloroform method.41 Reverse transcription was performed with F gene primer (5′TGTCTAACTATTTGAACA 3′, nucleotides 844–861 of F gene of long strain) in a Perkin Elmer Gene Amp PCR System 2400. A fragment of the N gene with specific primers42 was amplified by real time PCR in a Light Cycler 1.5 instrument (Roche).
Blood Sample Collection
A total of 5 mL of whole blood was collected into EDTA tubes, 24 to 48 hours after admission. The samples were kept cold, and transferred to the laboratory within 1 hour. Blood was centrifuged at 1.500 rpm for 10 minutes at 4°C and plasma was collected and frozen at −80°C until analysis.
Blood Cell Phenotypes
White blood cell count was performed and lymphocyte subsets composition from 1 mL of whole blood was determined using a panel of monoclonal antibodies to detect cell surface antigens by flow cytometry. Monoclonal antibodies, CD3-FITC/CD4-PE, CD3-FITC/CD8-PE, CD3-FITC/CD19-PE, CD3-FITC/CD16 + 56-PE, CD45-TC, CD94-PE (Beckman-Coulter, Florida) and CD45RA-TC, CD45RO-FITC, CD25-FITC, HLA-DR-TC, CD57-FITC, CD56-TC y CD94-PE (Caltag, Invitrogen), were used. A 3-color staining in whole blood was used for the analysis of cell subsets and immune activation markers.43 Acquisition and analysis was performed with a Coulter Epics-XL flow cytometer, System II software (Coulter Corporation, Florida). Cell subsets were gated on the basis of side-scatter versus logCD45 fluorescence and analyzed for fluorescence intensity in a log scale.
Cytokines and Chemokine Quantification
Plasma cytokines and chemokines concentrations were measured from blinded samples using commercial enzyme-linked immunosorbent assay for interferon (INF)-γ, interleukin (IL)-17 (R&D Systems), and tumor necrosis factor (TNF)-α, IL-6, IL-10, IL-13, IL-8 (Biosource, Invitrogen). For TNF-α, IL-10, IL-13, and IL-8, an ultrasensitive assay was used. The lower limits of detection were as follows: INF-γ (8 pg/mL), TNF-α (0.09 pg/mL), IL-6 (2 pg/mL), IL-10 (0.2 pg/mL), IL-13 (0.5 pg/mL), IL-17 (15 pg/mL), and IL-8 (5 pg/mL).
Severity of RSV Disease
To distinguish infants admitted with severe RSV-ALRI, we designed a scoring system, which was applied prospectively during hospitalization. The factors included in the scoring system and definitions are shown in Table 1. The geometric mean and median score values for all RSV patients were 6.75 and 7.0, respectively. The median score value of 7 or greater was used to define severe RSV-ALRI infection and scores less than 7 were considered as mild or moderate disease. There were no differences in our ability to identify patients with severe disease by using this scoring system versus the Tal score previously published for bronchiolitis (P = 0.592).44
Clinical Scoring System
Statistical Analysis
Statistical Analysis was performed using the SigmaStat program (version 3.5). χ2 and Mann-Whitney Rank Sum test were used for sex and age comparisons, respectively. To compare cytokines and blood cell surface antigen data between infants with moderate versus severe RSV infection and healthy controls the nonparametric Kruskal-Wallis method (ANOVA on Ranks) was applied. To compare the results adjusted for age, linear regression analysis was conducted among the 3 groups of patients, using healthy infants as a control group (STATA 10.0 program). Speerman Correlations analyses were performed between length of supplemental oxygen, maximal fractional inspired oxygen (FIO2) required, and immunologic data (Sigma Stat 3.5). P < 0.05 was considered significant.
RESULTS
Demographic and clinical data are presented in Table 2. Sixty-nine (92%) RSV-infected patients was receiving exclusive breast-feeding at the time of hospitalization. There were no differences in sex, or age between infected and healthy infants, nor between children with severe and moderate RSV infection. The implemented scoring system enabled us to identified 37 infants with severe disease and 38 with moderate disease. All items included in the scoring system were significantly different between the 2 groups of RSV patients when the median score 7.0 was applied. The most representative data of respiratory distress was the need of increasing concentration of oxygen (FIO2) when oxygen saturation was less than 95% in room air by pulse oximetry. The duration of oxygen administration was correlated with the duration of hospitalization (Spermman correlation: 0.792, P < 0.0001) and FiO2 requirement (Spermman correlation: 0.610, P < 0.0001), (data not shown). To confirm RSV infection from the initial IFA obtained in the emergency room, a second fresh NPA sample was obtained for each patient for IFA, virus isolation and PCR assay within the first 24 to 48 hours of admission. PCR confirmed RSV infection in 92% of the cases, IFA in 56%, and virus isolation in 36%. In healthy controls, RSV was not detected by any method.
Demographic and Clinical Features of the Study Subjects
Immune Cell Populations
Blood lymphocyte counts were measured in all RSV-infected infants and in 19 healthy controls. There was not enough blood for a complete analysis for NK subsets cells in 4 controls. Absolute cells count and subset percentages of peripheral blood of healthy infants were comparable with those published by Shearer et al.45 There was a trend for lower median absolute blood cell counts of CD3+, CD4+, and CD19+ subsets in subjects with severe RSV than in controls (mentioned in Table, Supplemental Digital Content 1, https://links.lww.com/A1348). CD8+ T cell counts were significantly lower in infants with RSV infection than in healthy controls (P = 0.03). Although the regression coefficient of CD8 was lower in severe patients than in healthy individuals, these differences were not significant in the crude coefficients nor in adjusted coefficients by age, (P = 0.089 and P = 0.123) (Table, Supplemental Digital Content 2, https://links.lww.com/A1349). However, the linear regression test in RSV-infected infants for crude and adjusted coefficients for age, showed significant differences among infants with moderate and severe RSV (P = 0.038 and P = 0.042; data not shown). Moreover, there was a significant inverse correlation between the length of supplemental oxygen requirement and CD8+ cells count (P = 0.025) and a negative trend among maximal FIO2 administered and these T cells (P = 0.059) (Fig., Supplemental Digital Content 3, https://links.lww.com/A1350).
The total number of NK cells and NKT cells were measured in 60 RSV-infected patients and in 19 control infants (Table, Supplemental Digital Content 4, https://links.lww.com/A1351). Likewise, NK subsets were measured in 60 RSV-infected infants and in 15 controls. Most median cell counts for NK subsets were lower in RSV patients than in the control group. In children with RSV infection, we found absolute lower counts and significant lower percentage of NK cells expressing CD94 antigen (absolute cell count: P = 0.06; % of cells: P < 0.001). The linear regression analysis showed significant differences between severe and healthy infants (P = 0.045) and a negative trend in the coefficients adjusted for age (P = 0.069) (Table, Supplemental Digital Content 2, https://links.lww.com/A1349). In nonactivated CD57 NK cells, which express the CD94 antigen, lower absolute cell count (P = 0.046), and less percentage of cells (P < 0.001) were found. The lowest values on those cells were found in patients with severe RSV infection. These differences were confirmed in the regression analysis, where a negative trend was higher in severe infants (P = 0.056), (Table, Supplemental Digital Content 2, https://links.lww.com/A1349). Likewise, there was a negative trend in the correlation between these type of cells and the length of supplemental oxygen requirement (P = 0.055), (Fig., Supplemental Digital Content 3, https://links.lww.com/A1350). The median cell count of activated NK cells, CD57+, which express CD94+ antigen, was also lower in RSV patients than in control infants, especially in severe cases, but no significant differences were observed. In contrast, RSV-infected infants showed high values of activated NK cells, CD57+, that do not express CD94 antigen (CD57+CD94−), compared with controls. While the differences in the absolute values were not significant, the analysis of the percentage of cells showed that RSV infants had a higher proportion of this type of cells, more than twice that in the control group (P = 0.004). The linear regression analysis adjusted by age, showed that these differences were significant for both critically ill as well as moderately ill patients (P = 0.032; P = 0.036).
Cytokine Concentrations
Almost all cytokines measured were detected in the subjects tested, except IL-6 and IL-17. IL-6 was detected in blood samples from 35 of 47 (74.5%) infants with RSV infection and in blood samples from only 6 of 18 (33.4%) healthy controls. IL-17 was detected in blood samples from 10 of 67 (14.9%) patients with RSV infection, but was not detected in healthy children.
Plasma cytokines and chemokine concentrations from RSV infected patients showed no significant differences compared with controls, except for the concentrations of IL-8, IL-13, and IL-17 (Table, Supplemental Digital Content 5, https://links.lww.com/A1352). Infants with RSV infection showed lower values of IL-13 than did controls (P = 0.014). But the linear regression analysis adjusted for age showed that this difference was due to higher concentrations of Il-13 in infants between 4 and 6 months of age (P = 0.523), (Table, Supplemental Digital Content 2, https://links.lww.com/A1349). IL-8 was present in higher concentrations in the patients with RSV infection compared with controls (P = 0.024). This difference was greater between infants with severe disease and controls (P < 0.05; Dunn test) and linear regression for crude and adjusted coefficients for age confirm these differences (P = 0.031 and P = 0.042). There were also a significant correlations among these cytokines and the length of supplemental oxygen (P = 0.0297) and maximal FIO2 required (P = 0.0116). IL-17 was not detected in healthy infants. Thus, the only possible comparison was made between RSV-infected children with moderate and severe disease. The largest concentrations of this cytokine were detected in patients with moderate disease (P = 0.033). The linear regression analysis confirmed the trend of negative coefficients in severe RSV infants compared with moderate cases and discarded the influence of age. Also there was a significant difference in the correlation between IL-17 quantification and maximal FIO2 required (P = 0.0427). No differences were observed between RSV children undergoing to mechanical ventilation versus not ventilated infants, possibly due to the low number of patients who expressed IL-17.
Follow-Up Evaluation for Wheezing
Children with RSV infection were followed for a year after discharge to determine the rate of wheezing after RSV infection. We defined wheezing by the presence of 3 or more episodes of wheezing documented by a physician. Follow-up was obtained in 51 (68%) of 75 infants with RSV infection; 22 of them had had severe RSV infection, and 29 had had moderate disease. A total of 22 (43.1%) infants developed wheezing; 13 (59%) of them belonged to the group with severe disease and 9 (31%) to the group with moderate disease (P = 0.045). No differences were observed between the immune data collected during the acute RSV infection and the development of wheezing at the 1 year follow-up. New hospitalization for ALRI was documented in 8 of 75 (10.6%) RSV-infected patients, 6 of them during the next month after discharge.
DISCUSSION
The detailed nature of the pathogenesis of RSV infection in humans remains unknown. However, it is recognized that the type of immune response elicited by the virus plays a major role in the severity of the respiratory disease.1,10–21,46
We decided to develop a scoring system different from that normally used to systematically assess respiratory distress in hospitalized children;44,47 this system is designed to assess the patient's respiratory distress in 1 moment, to establish immediate therapeutic measures. In our analysis, we should be able to assess the entire course of each patient, to compare 2 different groups of patients, homogeneous in their demographic characteristics, but different in the behavior they showed throughout their hospitalization. The new scoring system had no differences in the classification of cases with the score of Tal44 when this latter was applied in the worst report (P = 0.592). A study was recently published that uses many of these factors to compare the severity of patients infected by RSV.48
The immune system of newborns and infants less than 6 months old is immature compared with many of the functions that reach the immune system of the adult, including RSV infection.49,50 The inability to respond properly to RSV infection has been attributed to lack of lung elasticity.51,52, and also the poor response of immunity effectors elements of both the adaptive and innate response in this period of life.53,54 Thus, we were especially careful to not include infants younger than 28 days old and older than 6 months of age and cellular subsets count obtained in our healthy population were consistent with the values obtained by other authors.45
Other factors that have been considered in severe RSV disease include viral load, the subgroup A or B, and different genotypes circulating of RSV, especially genotypic variants of the glycoprotein G. All the results are controversial and do not support a significant involvement in the evolution of severe disease.6,48,55–57 Unfortunately, in the design of this prospective study, we did not consider measuring viral load of RSV. International experience on this point is controversial.6,56 On the contrary; there is consensus that the immune response, both innate and adaptive is a key in controlling the viral infection, also in the first months of life.49
In our patients hospitalized with RSV infection, the blood cell counts of CD3+, CD4+, CD8+, and CD19+ lymphocytes were lower but not significantly different than in control subjects. The reduced number of circulating CD8+ T cells in severe hospitalized infants may reflect an impaired immunity to RSV infection, or it may be the result of an immature immune system in development. While the ratio of T cells, CD4/CD8, during the first month of life is similar to adult, these cells are functionally more immature than the T cells of the adult.49 Indeed in the neonate and very young infants there is a lower production of important cytokines such as IFN-γ and a reduced ability of the T cells–mediated cytotoxicity. In our severe infants, low count of circulating CD8+ T cells is consistent with an increased respiratory distress. Roe et al37 have proposed a mechanism of apoptosis induced by RSV as responsible for low lymphocyte counts. Low counts of peripheral CD8+ lymphocytes could also be associated with low counts of T cells in the lung. Previously, the consensus view was that an exaggerated lung immune response was directly related to the development of bronchiolitis. However, some authors, such as Welliver et al,20, have demonstrated low lymphocyte counts in lung tissue from fatal cases of RSV infection; these authors hypothesize an apoptotic mechanism.
There are few reports regarding the total number of NK cells in RSV-infected hospitalized infants.33 Although NK cells are present in normal values at birth, CD56+ cells count in neonatal blood are about 50% of those in adults and the cytolytic function of NK cells increases progressively during the first month of age.49 In our study, different NK cell subsets were lower in RSV patients than in the control group, but in none of the reported differences, did age have an influence. Similar findings have been described for NK cells, specifically CD56+ cells by Welliver et al20 in lung tissue from fatal cases of RSV. The numbers of NK cells expressing CD94 antigen, which is a molecule associated with the major histocompatibility complex (MHC) class Ib system,22,23 were lower in patients with RSV infection. In contrast, activated NK cells count (CD57+) that do not express this receptor increased in RSV patients. The NK cytolytic activities are controlled by a variety of receptors including CD94-NKG2 family receptors and killer immunoglobulin-like receptors, which bind to major histocompatibility complex class I molecules on target cells.58 The increase of NK cells that does not express the CD94 antigen in blood of hospitalized patients with RSV infection may be associated with more these cells in lung, which could also participate in the tissue damage, without appropriate regulatory mechanisms from other populations of NK cells or the adaptive response,59 or it may be related to a mechanism used by RSV to evade the immune response during acute infection.23 Recently, Reed et al60 observed expanded numbers of 1 type of NK cell (CD49+CD3−) in the lungs of susceptible NZB mice (New Zealand Black strains) after RSV infection compared with BALB/c control mice, that suggests that NK cells were functional and their activity perhaps exaggerated after infection in NZB mice. Also, they found a deficiency in the number of alveolar macrophages associated with dense inflammatory infiltrate and occlusion of the airway both in the lung histopathology of the NBZ mice infected with RSV and in the lungs of infants who died of RSV.
Similar to other reports, low production of proinflammatory cytokines such as INF-γ and TNF-α, unlike what happens in other viral infections, may be related to the lower T cell counts observed in these patients.20, 21, Previous studies from infants with RSV bronchiolitis indicated that in nasal samples a greater proinflammatory response is associated with less-severe bronchiolitis, while there are low concentrations of these cytokines in critically ill patients.14,20,61–64 IL-17, another proinflammatory cytokine, was not detected in healthy infants and the largest concentration was found in moderately ill patients compared with severe infants, suggesting a protective proinflammatory activity of these cytokine in infants with moderate disease, but insufficient or altered in severely ill patients.
Some authors have argued that RSV infection elicits an increased production of IL-6 and IL-10.14,15 In our patients, there was an increase in the mean concentrations of IL-6 and IL-10, but not in median values, which is why these differences were not significant. The highest concentrations of these 2 cytokines were observed in infants with severe infections. The concentrations of IL-13 were lower than those from healthy subjects. However, adjusted for age analysis demonstrated that low values of IL-13 belonged to infants among 1 to 3 month old. This cytokine is a mediator of allergic inflammation.65–66
IL-8 is a chemokine that acts on a wide range of cells and is associated with inflammatory diseases such as RSV bronchiolitis.67,68 Our results are consistent with studies in which greater concentrations of this cytokine were observed. Although the increase of IL-8 concentrations was significant in critically ill patients compared with controls, we did not observe differences among children undergoing to mechanical ventilation versus not ventilated infants as did Bont et al69 (P = 0.822), possibly due to the less number of patients on mechanical ventilation.
One limitation of the present study is that cytokines were measured only once during the first days of acute illness. Another limitation in this study is not to have considered the quantification of viral load from respiratory secretions that would have allowed correlation of clinical and immunologic data.
According to our results, we propose that in infants younger than 6 months, severe RSV infection is associated with a peripheral impaired immune response, especially during the first days of illness, that is characterized by low T-cell counts compared with healthy infants, particularly CD8+ lymphocytes, a poor expression of certain circulating proinflammatory mediators such as INF-γ, TNF-α, and IL-17 and high concentration of IL-8 chemokine in plasma, that could not effectively control the infection. In addition, we found that infants with severe RSV infection have low NK cell counts in blood, particularly those expressing the CD94 antigen, while another group of activated NK cells that did not express the CD94 antigen were in higher percentage, especially in patients with severe disease. The lack of regulation of the NK system, mediated by a poor expression of CD94-NKG2 receptors could lead to increased of lung damage, either through increased direct RSV damage on epithelial cells in the absence of an effective immune response or through apoptotic signals in the lung20,60 or in blood.32,37 Finally, the development of wheezing during one year of follow-up had no relation to these immunologic markers studied during the first days of acute infection.
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Keywords:
respiratory syncytial virus; severe infantile respiratory infection; immune response
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