Acute bronchiolitis from respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection and the major source of hospitalization in infants.1,2 It has been demonstrated that infants who developed RSV bronchiolitis are at significantly higher risk for recurrent wheeze and subsequent asthma.3 Several epidemiologic studies have shown that infants hospitalized for bronchiolitis during an epidemic season may have different phenotypes and endotypes. In particular, older infants, with a higher predisposition to asthma are hospitalized during the nonpeak months whereas younger infants with a more severe disease are hospitalized during the peak months.4,5
The active interaction among viral and host factors is essential to determine the development of respiratory sequelae, and in this regard, a predominant role may be played by the immune system. The production of effector cytokines depends on both the viral determinants and host’s characteristics.6 Conflicting reports showed that infants with RSV bronchiolitis may have a predominant type-1 or type-2 immune response.6–14 Moreover, it is still controversial whether a defective or exaggerated T-helper cells (Th)1 and Th2 response may reflect the genetic heterogeneity of infants with bronchiolitis or may depend on the specific virus that causes bronchiolitis.6–14 In this line, we recently demonstrated a predominant Th2 polarization in human rhinovirus bronchiolitis infants as compared with RSV infants.15 More information on the immunologic response of infants with bronchiolitis would help physicians draw up effective individualized therapies for bronchiolitis.
Starting from previous data on different bronchiolitis phenotypes,5 in the present study we tested the hypothesis that the type-1/type-2 balance differs between infants hospitalized during the peak months and those during the nonpeak months. To exclude the possible influence of a specific virus, we enrolled only infants hospitalized for RSV sole bronchiolitis. To this aim, we evaluated the ongoing systemic immune response by measuring the frequencies of CD4+ T cells producing interferon (IFN)-γ and interleukin (IL)-4 (Th1 and Th2, respectively) and of CD8+ T cells producing IFN- γ (Tc1).
MATERIALS AND METHODS
Of 290 unrelated full-term previously healthy infants consecutively hospitalized with bronchiolitis over 2 consecutive epidemics (November 2016 to April 2017 and October 2017 to March 2018), we detected RSV from nasopharyngeal washing in 132. Among them, we excluded 13 infants with coinfections, and we were able to collect blood sample from 90/119 infants with RSV sole. This study population belongs to an Italian cohort of term, non-low birth weight, healthy infants hospitalized with bronchiolitis at the Paediatric Department, “Sapienza” University of Rome [Brome (Bronchiolitis in Rome)]. Bronchiolitis was strictly defined as the first episode of acute lower airway infection, with respiratory distress and diffuse crackles on auscultation in infants less than 12 months of age. In line with confidentiality requirements, the database was anonymized, and the ethic committees of Policlinico Umberto I approved the study (Prot. 107/12) after the informed consent was obtained from infants’ parents.
At admission to the hospital, a clinical severity score ranging 0–8 was assigned to each infant according to respiratory rate, oxygen saturation on room air (SaO2), presence of retractions, and ability to tolerate oral feeding, as previously described.2 Days of disease were calculated as the time span from the occurrence of respiratory symptoms (as reported by the parents) to the hospitalization (days of disease) and to the blood collection (days of disease to blood collection). Breast-feeding at the hospital admission was defined as a dichotomous variable (yes or no).
Following the criteria of the previously published study,5 we counted as peak months, the month with the higher rate of hospitalization (January) together with the previous (December) and the following (February) months. Accordingly, we divided infants as follows: hospitalized during the peak months and during the nonpeak months (Fig. 1A).
As a sensitivity analysis with similar numbers, we also calculated the epidemic starting from the first to the last hospitalized infant and we divided them in quartiles. Infants in the first and the fourth quartiles were considered as hospitalized outside the epidemic and those in the second and the third quartiles were considered as hospitalized in the central epidemic (Fig. 1B).
Nasopharyngeal washing was collected from each infant within 24 hours of hospitalization and handled as previously described.15 RSV and 13 other respiratory viruses were detected using a panel of reverse transcription-polymerase chain reaction (PCR) or nested PCR assays, as reported.16 RSV sole infants were enrolled, and nasopharyngeal washings were sequenced in the second hypervariable half of the glycoprotein G gene to assign the genotype.17 A TaqMan-based real-time PCR technique for RSV-RNA quantification was performed as previous described.18 Viral load values, expressed as the number of copies per mL of nasopharyngeal washing, were analyzed as median among groups or log10 transformed.
Whole Blood Stimulation
Children’s peripheral whole blood was collected directly into Na-Heparin-containing vacutainers (BD Biosciences, San Jose) at median 7 days of disease (range: 3–16 days) and processed within 1 hour as described.19 Briefly, whole blood (300 μL) was diluted 1:1 with RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA), then was either left unstimulated or stimulated with Staphylococcal enterotoxin B (SEB), at 1 μg/mL (Sigma-Aldrich, St. Louis, MO). Blood was incubated at 37°C overnight (in RPMI), and Brefeldin-A (Sigma-Aldrich), at 10 μg/mL, was added after 5 hours to inhibit cellular secretion. Whole blood cultures were then harvested and red blood cells lysed with FACS lysing (BD Biosciences). The remaining white cells were extensively washed with FACS buffer (PBS containing 0.5% BSA and 0.09% NaN3) and stained with Live/Dead Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific). Cells were then fixed by 1% PFA for 15 minutes at room temperature and permeabilized using FACS buffer and 5% Saponin.
Staining, Acquisition and Analysis
Preparation of the samples for flow cytometric analysis was performed as described.19 Briefly, cells were incubated with a panel of fluorochrome-conjugated monoclonal antibodies (mAbs) for 30 minutes at 4°C. The composition of panel was: anti-CD3-FITC (clone UCHT1), anti-CD8-Alexa Fluor 700 (clone RPA-T8), anti-IL-4 antigen-presenting cell (clone MP4-25D2) (all from BD Biosciences), anti-IFN-γ-PerCPCy5.5 (clone4S.B3) (Biolegend, San Diego, CA), anti-CD14-eFluor450 (clone 61D3) and anti-CD19-eFluor450 (clone HIB19) (both from eBioscience, Thermo Fisher Scientific). Saponin (5%) was added to the antibody mix. Cells were finally resuspended in 250 μL 1% paraformaldehyde and acquired on a Gallios Flow Cytometer (Beckman Coulter, Miami, FL). Percentages of viable cells were consistent throughout the study (data not shown). Cells were then washed and acquired by flow cytometry in a Gallios Flow Cytometer (Beckman Coulter). Stained samples were acquired with a standard stopping gate set at 50,000 CD3+ T cells. Compensation was performed using Versa Comp Antibody Capture Beads (Beckman Coulter) individually stained with each fluorophor and compensation matrices were calculated with Kaluza Analysis 1.3 software (Beckman Coulter, Miami, FL). An example of data from flow cytometry with a description of the gating strategy is shown in the Figure, Supplemental Digital Content 1, http://links.lww.com/INF/D669.
The frequencies of Th1, Th2 and Tc1 cells were measured for each infant. The Th2 polarization index was calculated as the ratio between Th2 and Th1 cells.
We run for categorical variables, Pearson's χ2 test and for numerical variables Mann-Whitney U or Kruskal-Wallis rank test for comparison in 2 groups. Spearman’s ρ coefficient was calculated to assess the correlation between percentages of T cells producing studied cytokines and RSV load. We considered statistically significant a P < 0.05. Statistical analysis was performed using the SPSS software (version 23.0; SPSS Inc., Chicago, IL).
Ninety infants (Caucasian: 87.8%, 43 males, median age: 73.5 days, age range: 14–266 days) with RSV bronchiolitis were hospitalized for a median of 5.5 days (range: 2–11 days), with a median severity score of 4 (range: 1–8). Infants had median 94% SaO2 (range: 80%–99%) and 40 (44.4%) required oxygen supplementation. Fourteen (15.6%) infants were hospitalized in intensive care unit only during the epidemic peak but none required intubation or invasive ventilator support.
Of the 90 samples positive for RSV alone, 84 were successfully genotyped by sequencing (93.3%): 48 were attributed to RSV subtype A (57.1%) and 36 to RSV-B (42.9%). The qRT-PCR assay from 35 infants who had enough residual sample for assay showed that RSV load ranged from 4192.0 to 28.7 × 109 copies/mL (median: 14.8 × 105 copies/mL). Viral load did not differ between RSV-A (N = 19, Log10 = 6.78 copies/mL) and RSV-B (N = 16, Log10 = 6.34 copies/mL).
Enrolled infants were at their first episode of RSV-related disease and in presence of low or absent pre-existing RSV memory T-cell pool, thus the ongoing effector T-cell responses were measured by polyclonal stimulation. SEB was preferred to other polyclonal T cells stimulant since it covalently binds major histocompatibility complex (MHC) and t cell receptor and triggers proliferation and cytokine responses in a way very similar to physiologic t cell receptor triggering, requiring the presence of antigen-presenting cells. Analyzing T cells expression according to the month of hospitalization, we found that the percentage of not treated (nt) Th2 cells was higher in infants hospitalized in November and March than in December, January and February. The frequencies of Th1 cells were similar in infants hospitalized throughout the epidemic season, while the percentage of Tc1 cells was higher in infants hospitalized in December, January and February than in March and April (Fig. 2). We found higher Th2 index in infants hospitalized in March, April and November than in December, January and February; the stimulation with SEB highlighted differences in the pattern of the ongoing immune response (Fig. 3).
Th2 index did not correlate with age of the infants (nt r: −0.005, P = 0.96; SEB r: −0.18, P = 0.08). No significant correlation was found between percentages of T cells producing studied cytokines and RSV load (Th2 cells: nt r: −0.034, P = 0.8; SEB r: −0.65, P = 0.7; Th1 cells: nt r: −0.05, P = 0.8 and SEB r: −0.9, P = 0.6; Tc1 cells: nt r: −0.03, P = 0.9; SEB r: −0.06, P = 0.7). Moreover, all these parameters were not significantly different when considering separately RSV-A and -B cases (data not shown).
We divided infants according to the time of hospitalization as follows: 71 (78.9%) during peak months and 19 (21.1%) during nonpeak months. Infants hospitalized during nonpeak months were less frequently breast-fed (P = 0.012), had higher SaO2 (P = 0.036) and had a significantly higher eosinophils count (P = 0.003) than infants hospitalized during peak months. Clinical variables, such as severity score, day of disease, length of hospital stay and white blood cells count, were similar in the 2 groups. The RSV genotypes were equally distributed among infants hospitalized during and outside the epidemic (Table 1). Infants hospitalized during the nonpeak months had a slightly higher viral load value (N = 8, mean Log10 = 7.49 copies/mL) than infants hospitalized during the peak months (N = 27, mean Log10 = 6.31 copies/mL, P = 0.12 by Kruskal-Wallis rank test).
Infants Hospitalized During the Nonpeak Months Have a Higher Th2 Polarization
Infants hospitalized during peak months showed higher frequencies of CD4 T cells as compared with infants hospitalized during nonpeak months, accordingly the frequencies of CD8 T cells were higher in the latter group (Table 2). Infants hospitalized during the nonpeak months had a higher percentage of SEB Th2 cells (P < 0.0001) and a higher SEB Th2 polarization (P = 0.001) than infants hospitalized during the peak months (Table 3; Fig. 3B). We transformed the SEB Th2 index in tertiles and we found that the higher tertile was more frequent (68.4% vs. 23.9%) and the lower tertile was less frequent (15.8% vs. 38.0%) in children hospitalized during the nonpeak months than in infants hospitalized during the peak months (P = 0.001).
When we analyzed both seasons separately, we found consistent results (see Tables, Supplemental Digital Content 2, http://links.lww.com/INF/D670 and 3, http://links.lww.com/INF/D671).
As a control analysis, we also compared infants hospitalized in the central (N = 44) and outside (N = 42) the epidemic. Infants hospitalized outside epidemic had a higher percentage of SEB Th2 (P = 0.006) and a higher SEB Th2 polarization (P = 0.012) than infants hospitalized in the central epidemic.
The main finding of the present study is that in vivo Th2 polarization was higher in infants with RSV bronchiolitis hospitalized during the nonpeak months compared with infants hospitalized during the peak months, thus demonstrating the presence of at least 2 different endotypes in infants during a single RSV bronchiolitis epidemic. To our knowledge, this is the first study where frequencies of systemic Th1, Th2 and Tc1 cells in infants < 12 months hospitalized for RSV sole bronchiolitis were analyzed. Insofar as adaptive immunity was suggested to differ according to causative organism,6,15 we studied the different patterns of response only in infants with RSV sole bronchiolitis. From our results, we may assume that the observed differences reflect infant’s intrinsic characteristics.
When we analyzed infants hospitalized during nonpeak months, we found a significantly higher percentage of Th2 cells. It has been shown that immaturity of the immune system in young infants lead them to be prone to develop type 2 immune response.14 In our study, the lack of a correlation between age and type 2 response may be related to a personal immune tendency to type 2 polarizations in this group of infants infected with RSV. This finding, supported by the high number of blood eosinophils that we found in children hospitalized during nonpeak months, suggests that these infants may be prone to develop atopy. The frequencies of CD4 T cells were slightly increased in infants hospitalized during peak months reaching the statistical significance compared with nonpeak infants, probably reflecting a stronger T-lymphocytes response.
Another original result of our study is the much lower rate in the breast-feeding among nonpeak RSV-bronchiolitis cases; interestingly, these cases presented higher viral loads and type 2 polarizations. It would be tempting to speculate that less breast-feeding may have a negative impact on gut microbiota composition and in turn on the infants’ immune response. However, the effects of RSV infections on the infant gut microbiota (and vice versa) are very complex because of variation and heterogeneity in several parameters (mode of delivery, changes over time during lactation) and are of the scope of the present study.
The higher viral load in infants hospitalized during the nonpeak months, is probably linked to the higher viral shedding into respiratory tract in this group of infants, probably caused by a less effective immune response, secondary to the Th2 response. In a previous study, we demonstrated that a high RSV load at the time of admission for bronchiolitis was associated with recurrent wheezing at 36-month follow-up.20 According to these results, the high RSV load might contribute to the risk of respiratory sequelae in infants hospitalized for bronchiolitis during nonpeak months.
When we compared our findings with other studies, we found discordant data on type-1/type-2 polarization in children with RSV infection. Indeed, this may reflect the different inclusion criteria of the population studied: some authors included older children,6,7,9,11,13,21–23 and some authors selected RSV infected children irrespectively of the clinical diagnosis.6–8,10,11,21–23 A distinct characteristic of our cohort is the adherence to strict inclusion criteria in bronchiolitis diagnosis, such as age < 1 year, infants at the first episode of lower respiratory tract infection and the presence of diffuse crackles on lung auscultation. Moreover, we decided to evaluate the Th1, Th2 and Tc1 cells by the intracellular staining coupled to flow cytometry instead of cytokine production assessed by ELISA methods6,8,9,13,14,21–23 as intracellular staining enables the simultaneous detection of the specific subset of responder cells (eg, CD4+ or CD8+ T cells). Moreover, we stimulated cells with SEB instead of a combination of phorbol 12-myristate 13-acetate and ionomycin, used by others.7,10–12 This approach was preferred since SEB interacts with T-cell receptors and class II MHC Molecules. The simultaneous binding of SEB outside of the MHC on antigen-presenting cells and to T-cell receptors expressing certain Vβ results in superantigen like properties in elicting polyclonal T-cell activation and cytokine responses.
In this study, we confirmed previous epidemiologic finding from our group5 of 2 different phenotypes existing within a distinct cohort of infants hospitalized with RSV bronchiolitis: previously healthy full-term infants hospitalized during the peak months and bronchiolitis in infants with a possible genetic predisposition to atopy, hospitalized during the nonpeak months. Similarly, Carroll et al,4 proposed a different risk of early childhood asthma differentiating bronchiolitis occurring during RSV-predominate months (December to February) and rhinovirus-predominate months (May, August to September) and they estimated a 25% increased risk of respiratory sequelae in the second group. These authors, even if they did not perform viral detection during bronchiolitis, speculate that their results may support the association between rhinovirus infection (usually predominant in nonpeak months) and subsequent asthma. In a previous study,15 we demonstrated a different immune response between infants hospitalized for rhinovirus bronchiolitis and for RSV bronchiolitis. The results of the present study, on a Th2 polarization in infants with RSV sole bronchiolitis hospitalized during the nonpeak months are in line with the Carroll et al study.
This study has some limitations. First, the number of subjects is small but we recruited a very selective population and we obtained blood samples from very young infants. Second, we collected blood samples at a median 7 days after the appearance of respiratory symptoms; it would be interesting to evaluate the immune response in infants at different stages of the disease.
Finally, it remains to be determined whether the type-2 polarization demonstrated in the present study, is a transitory phenomenon. In perspective, it would be interesting to investigate whether the differences between these 2 groups of infants reflect the occurrence of respiratory sequelae.
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