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. 2014 Sep 4;178(1):94–101. doi: 10.1111/cei.12389

Divergent mucosal and systemic responses in children in response to acute otitis media

D Verhoeven 1, M E Pichichero 1
PMCID: PMC4360199  PMID: 24889648

Abstract

Acute otitis media (AOM), induced by respiratory bacteria, is a significant cause of children seeking medical attention worldwide. Some children are highly prone to AOMs, suffering three to four recurrent infections per year (prone). We previously determined that this population of children could have diminished anti-bacterial immune responses in peripheral blood that could fail to limit bacterial colonization in the nasopharynx (NP). Here, we examined local NP and middle ear (ME) responses and compared them to peripheral blood to examine whether the mucosa responses were similar to the peripheral blood responses. Moreover, we examined differences in effector cytokine responses between these two populations in the NP, ME and blood compartments at the onset of an AOM caused by either Streptococcus pneumoniae or non-typeable Haemophilus influenzae. We found that plasma effector cytokines patterned antigen-recall responses of CD4 T cells, with lower responses detected in prone children. ME cytokine levels did not mirror blood, but were more similar to the NP. Interferon (IFN)-γ and interleukin (IL)-17 in the NP were similar in prone and non-prone children, while IL-2 production was higher in prone children. The immune responses diverged in the mucosal and blood compartments at the onset of a bacterial ME infection, thus highlighting differences between local and systemic immune responses that could co-ordinate anti-bacterial immune responses in young children.

Keywords: acute otitis media, adaptive immune responses, mucosal cytokines, otitis-prone children, Streptococcus pneumonia

Introduction

Streptococcus pneumoniae (Spn) and non-typeable Haemophilus influenzae (NTHi) frequently colonize the NP mucosa of infants and reflux through the Eustachian tubes to cause acute otitis media (AOM), usually at the onset of an upper respiratory viral infection 1–3. In previous work we have shown that children can be divided into two groups: prone to repeated AOM, who exhibit at least three middle ear infections within a 6-month time span or four infections within a 12-month time span despite tympanocentesis and pathogen-directed antibiotic treatment until the age of 7 years, or non-infection-prone children (non-prone), who exhibit no or infrequent middle ear infections 4–7.

We have shown previously that infection-prone children have diminished adaptive immune responses to Spn and NTHi compared to non-prone children, including lower mucosal and systemic Spn-specific antibodies and lower CD4 T cell responses in the blood 8–10. However, T cell responses in peripheral blood could be dissimilar from mucosal CD4 T cell responses in this population of children. Potential differences in cytokine secretion from these cells could affect the level of resistance/protection afforded from Spn and NTHi migration into the middle ear to cause frequent AOMs. In this study we have focused on differences in the T cell-associated cytokine responses in the NP and ME mucosal compartment and blood in infection-prone and non-prone children.

Differences in T cell-associated mucosal cytokine responses during NP colonization or middle ear infection versus the blood could impact upon the ability of the child host to clear NP colonization, the first step in disease pathogenesis. Mucosal memory T cells participate in the vanguard of protection from respiratory bacterial pathogens because they are capable of facilitating an early and vigorous antibody response to contain dissemination of bacteria 11. The Eustachian tubes that drain fluid from the ME space into the NP are particularly enriched in mucosal memory T cells. However, the ME mucosa is largely devoid of immune cells, and may rely on cells refluxed from the NP or on trafficking from the peripheral blood to mount an anti-bacterial response in the ME during AOM 12–14.

In this study, we sought to determine whether compartmental responses, reflected in the cytokine profile of activated CD4 T cells, differed by comparing the cytokines in NP, ME and systemic peripheral blood in infection-prone and non-prone children. We found that the middle ear cytokine response mirrored those of the nasal mucosa versus the peripheral blood, suggesting that proximal mucosal sites may better predict the quality of the middle ear response than peripheral blood.

Patients and methods

Study population

The study was approved by the Rochester General Hospital Research Subjects Review Board and written informed consent was obtained for participation and all procedures. Sixteen children with current AOMs were studied, along with eight healthy controls (without current viral upper respiratory infection (URI) or AOM) (Table 1). Children were enrolled at age 6 months from a private paediatric middle-class practice (Legacy Pediatrics in Rochester, NY) and followed prospectively until 30 months of age. In the current study, NP samples were used from prone and non-prone children, each approximately 9–18 months of age, who experienced AOM caused by Spn or NTHi, representing both infection-prone and non-prone children. Children within the study were all seen at the onset or the next day after symptoms of AOM occurred. All had a current clinical viral URI and AOM, as diagnosed by their paediatrician. NP samples of infection-prone and non-prone children with normal responses from healthy control children (baseline) of the same age (without clinical viral URI or AOM) were compared. In both groups of children, ME fluid (MEF) was obtained by tympanocentesis to ensure the diagnosis of AOM, identify otopathogens, provide therapeutic benefit of drainage of bacteria and to provide samples for study 15. NP secretions from all study subjects were obtained by washing the NP, as reported previously 9,16. Bacterial identification in MEF and NP washes was accomplished as reported previously 4.

Table 1.

Patient demographics

Median age (age range) Prone Non-prone
11·9 months (9–18 months) 13·5 months (12–18 months)
Median number of AOM events 4·5 2
Household smoking? No No
Breastfed to 6 months of age? Yes Yes
Prior NTHi colonization? Yes Yes
Prior Spn colonization? Yes Yes
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AOM = acute otitis media; NTHi = non-typeable Haemophilus influenzae; Spn = Streptococcus pneumoniae.

Nasal cytokine assays

Protein levels from NP washes were assayed by BCA assay (Pierce Scientific, Rockford, IL, USA) and equilibrated for total protein (1 mg/ml) using phosphate-buffered saline (PBS). A 1-ml sample was then incubated overnight with a membrane cytokine array (48 cytokines) and processed according to the manufacturer's directions (Ray Biotech, Norcross, GA, USA). Arrays were developed with Trueblue solution (KPL, Gaithersburg, MD, USA) and calculated by EC software using an inverse image of the array. Data was calculated using the manufacturer's software (Ray Biotech).

Plasma cytokine assays

Frozen plasma obtained from blood was assayed for cytokine responses by ElisaMax (Biolegend, San Diego, CA, USA), as per the manufacturer's instructions, using an average of the duplicate for each plasma (using three dilutions of plasma) for each patient. Positive dilutions were calculated ×2 the standard deviation of the negative controls for a cut-off value. Cytokine concentrations were then calculated by the standard curves from the positive controls within each kit. All dilution data-points used for concentration calculations fell within the mid-range of the standard curve.

RNA extraction and quantitative reverse transcription–polymerase chain reaction (qRT–PCR)

RNA was isolated from total cell extracts from Trizol (Invitrogen, Carlsbad, CA, USA)-lyzed MEF cell pellets, according to the manufacturer's directions, followed by DNase and column purification (Qiagen, Gaithersburg, MD, USA). RNA was then reverse-transcribed (from mRNA using Superscript III) and 30 ng of cDNA was amplified with Sybergreen (Biorad, Hercules, CA, USA) using glyceraldehyde 3-phosphate dehydrogenase (G3PDH) as a calibrator or CD3, interferon (IFN)-γ, interleukin (IL)-2 or IL-17a primers as designed using PrimerBank (Harvard, Massachusetts, MA, USA) 17–19. Data were calculated by the ΔΔCT method and expressed as fold change between groups.

T cell cytokine assays

Frozen peripheral blood mononuclear cells (PBMCs) were thawed and stimulated with two Spn proteins [pneumococcal choline binding protein A (PcpA) or pneumococcal histidine triad protein D (PhtD)], that we have used previously in this study population 10, and anti-CD28 antibody (1 μg/ml; BD Bioscience, San Jose, CA, USA) overnight followed by 5-h incubation in the presence of brefeldin A 10. Briefly, 1 μg of either recombinant PcpA or PhtD (obtained from Sanofi Pasteur, Lyon, France) were added to PBMCs at the start of the incubation. Cells were surfaced-stained for CD4, CD3, CD45RA and CD69. Cells were then fixed in 1% paraformaldehyde (BD Biosciences), washed and permeabilized using 1X Fix/Perm buffer (BD Biosciences) followed by intracellular staining with antibodies for IFN-γ, IL-2, IL-17 and tumour necrosis factor (TNF)-α (Biolegend). Gating for cytokine-positive cells were set based on unstimulated and Staphylococcus enterotoxin B (SEB)-stimulated cells. Data were acquired on a LSRII with doublet discrimination and analysed with FlowJo software (Treestar, Ashland, OR, USA).

Statistical analysis

Data were analysed by two-tailed Student's t-tests with and without log-transformation (non-normal distribution) of the data or Mann–Whitney U-test, and results with a P-value less than 0·05 considered significant.

Results

Infection-prone children have lower frequencies of pathogen-specific memory CD4 T cells in the systemic compartment during infection than non-prone children

To assess differences in the CD4 T cell peripheral blood response between infection-prone and non-prone children, we compared the differential cytokine expression patterns after ex-vivo recall with PhtD and pneumococcal surface protein A (PspA)–Spn protein antigens studied previously 8,20–22. We found lower IFN-γ and IL-2 responses from stimulated CD4 T cells in infection-prone compared to aged-matched non-prone children to PhtD antigen (Fig. 1a,b and Table 2), consistent with our earlier report for Spn 10. We also found low production of IL-17a in PBMCs which was not significantly different between infection-prone and non-prone children, again consistent with our earlier report 10. Similar results were obtained with stimulation by OMP26 (NTHi) stimulation (Table 2). The low cytokine recall patterns in both groups of children are explained in that we have shown previously that young children have significantly lower T cell recall responses to all Spn or NTHi antigens compared to adults 23.

Fig 1.

Fig 1

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Prone children have lower peripheral blood T cell pools to bacterial antigens. We assessed the peripheral blood recall responses of CD4 T cell responses (CD3+CD4+) to pneumococcal histidine triad protein D (PhtD) of children exposed to Streptococcus pneumoniae (Spn) as percentage of total CD4 T cells. (a) Representative dot-plots are shown for both prone and non-prone children using Staphylococcus enterotoxin B (SEB) control; or (b) PhtD-stimulated. Cytokine+ cells were also CD45RA–. P < 0·05 for PhtD between prone and non-prone children for interleukin (IL)-2 and interferon (IFN)-γ responses. Percentages shown are the total percentage of cytokine-positive memory CD4 T cells. (n = 8 each group). Gating of cell populations was similar to a prior study 10.

Table 2.

Summary of memory CD4 T cell recall responses to bacterial antigens

IFN-γ IL-2
Prone Non-prone Prone Non-prone
PhtD (Spn) 0 002 ± 0 002 0 012 ± 0 005 0 005 ± 0 005 0 03 ± 0 011
PspA (Spn) 0 001 ± 0 001 0 009 ± 0 003 0 01 ± 0 007 0 041 ± 0 009
IFN-γ IL-2
Prone Non-prone Prone Non-prone
OMP26 (NTHi) n.d. 0 048 ± 0 015 0 025 ± 0 005 0 07 ± 0 05
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All comparisons P < 0 05 for all antigens. Values shown are average % ± standard deviation. IL = interleukin; IFN = interferon; PhtD = pneumococcal histidine triad protein D; PspA = pneumococcal surface protein A; NTHi = Haemophilus influenzae; n.d. = not detected.

Divergent T cell associated cytokines detected in the plasma of children during infection

We studied the plasma cytokine patterns and levels from infection-prone and non-prone children at the onset of a Spn AOM infection to further validate the impaired effector T cell responses in infection-prone children. The plasma cytokine patterns were consistent with those observed in peripheral blood memory CD4 T cell recall experiments. Specifically, we found significantly lower levels of IFN-γ and IL-2 in the plasma of infection-prone children compared to non-prone children, and again low IL-17a in both infection-prone and non-prone children (Fig. 2).

Fig 2.

Fig 2

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Plasma cytokines concentrations correlate with the T cell memory pool recall functions. We assessed the plasma concentrations of interferon (IFN)-γ, interleukin (IL)-2 and IL-17a by enzyme-linked immunosorbent assay (ELISA) from prone and non-prone children with a current Streptococcus pneumoniae (Spn)-induced acute otitis media (AOM). Healthy controls were included for comparison (n = 8 each group, mean ± standard deviation).

Nasal mucosal effector cytokine levels in children at onset of an infection

Because mucosal sites are enriched with memory T cells and have enhanced antigen-specific T cell trafficking during infection, we assessed differences in the effector cytokine levels in nasal secretions of infection-prone and non-prone children at onset of AOM with Spn. We utilized a membrane array rather than enzyme-linked immunosorbent assays (ELISAs), as we found it to be more sensitive due to the larger volumes of the collected sample and it allowed for the simultaneous detection of many effector cytokines. We found higher expression of IL-2 in infection-prone children but similar expression levels of IFN-γ and IL-17a compared to non-infection-prone children (Fig. 3).

Fig 3.

Fig 3

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Effector cytokine concentrations in the nasal mucosa differ from those of the plasma. Interferon (IFN)-γ, interleukin (IL)-2 and IL-17a concentrations were determined by membrane array from nasal washes from prone and non-prone children with a current Streptococcus pneumoniae (Spn)-induced acute otitis media (AOM). Healthy controls were included for comparison (n = 8 each group, mean ± standard deviation).

We also analysed for cytokines that are responsible for T cell expansion and survival that could affect cytokine concentrations. The nasal mucosa of infection-prone children contained lower expression of insulin-like binding protein 4 (IGFBP-4) and IL-7 compared to non-infection-prone secretions (Fig. 4). No statistical difference was detected with insulin-like growth factor-1 (IGF-1).

Fig 4.

Fig 4

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Cytokines associated with T cell turnover/proliferation differ between prone and non-prone children. Insulin-like growth factor binding protein-1 (IGFBP-1), insulin-like growth factor binding protein-4 (IGFBP-4), insulin-like growth factor-1 (IGF-1) and interleukin (IL)-7 were determined by membrane array from nasal washes of prone and non-prone children with an active acute otitis media (AOM). Background cytokines from healthy controls were subtracted from both groups (n = 8 each group, mean ± standard deviation).

Middle ear fluid cytokine profiles diverge from blood but are similar to the NP

We compared the RNA expression of IFN-γ, IL-2 and IL-17a in cell pellets obtained from the MEF. We utilized qRT–PCR rather than protein ELISAs, as our study protocol called for cell pellets to be placed in Trizol; this allowed for better calibration of cytokine differences due to the highly divergent but uniformly low protein concentrations (non-cellular fraction) in MEF of children. Using qRT–PCR to determine the inverse ratio of delta CT to RNA levels, we found that the MEF RNA cytokine expression levels mirrored the cytokine protein levels in the NP mucosa (Fig. 5a). Specifically, we found no statistical difference between infection-prone and non-prone children in IFN-γ or IL-17a transcript levels, while the levels of IL-2 responses in infection non-prone children were statistically lower than prone children (Fig. 5b).

Fig 5.

Fig 5

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Cytokine transcripts in the middle ear suggest a mucosal rather than a systemic adaptive immune response profile in the prone child. (a) ΔCT values are shown for each cytokine and CD3 from middle ear fluid (MEF) samples for prone and non-prone patients. Bars show average ΔCT for each group. (b) Cytokine transcript profiles were determined by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) from cell pellets obtained from fluid drained during a tympanocentesis from prone and non-prone children with acute otitis media (AOM)caused by either Streptococcus pneumoniae (Spn)-induced or non-typeable Haemophilus influenzae (NTHi) (prone children n = 7, non-prone children n = 7, mean relative fold change ± standard deviation). One sample from each group was excluded for low RNA levels. P-values are for fold changes after normalization.

The middle ear has enhanced infiltration of neutrophils during AOM, as evidenced by removal of highly neutrophil-laden material during tympanocentesis; however, the number of T cells in MEF obtained from tympanocentesis are few 24 but more highly proliferative than those in the Eustachian tubes during experimental infection 20. As we detected higher levels of IL-2 in the middle ear of children, we hypothesized that there might be higher CD3+ T cell infiltration in the MEF of prone children. Therefore, we determined the relative number of T cells in MEF indirectly by assessing the levels of CD3 RNA detected in infection-prone and non-prone children at onset of AOM. However, we found similar low levels of CD3 mRNA in the middle ear of infection-prone and non-prone children (Fig. 5a,b).

Discussion

The mechanisms driving recurrent infections in the middle ear of young children has been an area of investigation for our group. After more stringently defining an ear infection-prone population 4–7,25, we determined previously that such children generate lower antibody levels, fewer memory B cells and fewer memory T cells in response to antigens expressed by the two most common otopathogens – Spn and NTHi – at onset of infections 11,12,26,27. Here we studied differences in the immune response of children in three compartments: blood, NP and middle ear. We found that the blood diverged from the NP and middle ear with respect to effector cytokine levels with children who experienced infrequent infections, displaying higher IFN-γ/IL-2 responses in the peripheral blood but lower or similar responses in the ME and NP compartment, respectively. The results suggest a discontinuous immune response with respect to the systemic and mucosal compartments' responses to middle ear infection.

A significantly lower CD4 T cell Spn-specific memory/effector response in the peripheral circulation of infection-prone versus non-prone children, as seen previously 10, was confirmed here, reflecting a lower capacity to mount a vigorous effector response in the infection-prone child. In this report we add further evidence regarding a CD4+ T cell deficit in otitis-prone children by showing a difference in the effector response of IFN-γ and IL-2 in the plasma of infection-prone versus non-prone children. The higher infection rates of otitis-prone children would lead one to believe that they exhibit higher T cell responses to protein antigens shared between strains, but this was not the case. This further highlights that the prone child has immune deficits that fail to increase recall responses after additional exposure to antigens upon additional infections.

The NP mucosal compartment cytokine levels diverged from the responses in the peripheral blood, but were similar to those in the middle ear. These data are consistent with our earlier report 16 that MEF contains predominantly secretions from the NP, and suggests limited migration of immune cells or transudation of immune effectors from blood. IL-2 production plays a significant role in driving inflammatory responses during experimental AOM infection of animal models 28–30. Therefore, detection of higher expression of IL-2 transcripts in the NP and middle ear of infection-prone children may well reflect higher inflammatory conditions in this population. High production of IL-2 could also drive higher regulatory T cell (Treg) production 21 and inhibit recruitment of CD4 and CD8 T cells to the NP and middle ear of infection-prone children. While our assays utilized strong stimulators that induce release of a robust IL-2 response, poor antigen-presenting cell (APC) stimulation could play a role in the low IL-2 recall responses of antigen-specific memory CD4 T cells that we detected. Importantly, we cannot rule out differences in APC/T cell interactions that could also influence the amount of T cell stimulation and effector responses, and is a focus of future studies.

We measured similar levels of IFN-γ and IL-17a in infection-prone and non-prone children in NP secretions and MEF, although we understand that secretion of these cytokines is not restricted simply to CD4 T cells. They are secreted by a diversity of cells including macrophages and neutrophils, respectively. In light of the differing clinical outcomes, i.e. frequent recurrent infections versus few infections in infection-prone versus non-prone children, respectively, we speculate that the source of IFN-γ may differ in the two populations. Higher IFN-γ in the infection-prone child may derive from macrophages that could drive enhanced swelling in the nasal mucosa and middle ear and contribute to enhanced pathogenesis 22,31. While we saw only a trend for increased IFN-γ in the MEF of infection-prone children, unchecked IFN-γ levels are known to exacerbate Spn disease pathogenesis in murine studies of PspA vaccination 32 or experimental meningitis 33. IL-17a has been shown to correlate with protection from Spn NP colonization 32,34. Similarly, IL-17a in infection-prone children may derive from an exuberant neutrophil migration to the NP and MEF, whereas for non-infection-prone children the levels may reflect protective CD4 T cell responses. We intend to study these hypotheses in future work. IGF-1 and IL-7 both play a role in proliferation and T cell homeostasis 26,27,35, and both were significantly lower in nasal washes of infection-prone versus non-prone children. IGFBP-1 and -4 both play a role in competing with IGF for receptor binding. The divergent expression of both IGFBP-1 and -4 suggests that prone children may have defects in their ability to regulate T cell proliferation that needs to be explored further. These data suggest that a divergence in potential protection from activation-induced apoptosis could exist in infection-prone and non-prone children. Future studies will examine cytokine mechanisms mediating the expansion and/or survival of T cells in both populations with respect to lower antigen-specific CD4 T cells.

In conclusion, we compared the differential cytokine response to respiratory bacterial infections during an active AOM event in young children. Although we had to optimize the assays for each specific tissue investigated due to differences in study design and sensitivity to noise, comparisons between compartments between infection-prone and non-infection-prone rather than between compartments was possible. As in our previous work, divergence between infection-prone and non-infection-prone children was observed, suggestive of different capacities of these child populations to respond to NP colonization and middle ear infections.

Acknowledgments

This work was supported by R01DC008671 awarded to M. E. P. We would like to thank Karin Pryharski and Sheldon Perry for technical assistance and Robert Zagursky PhD for editorial assistance.

Disclosure

Authors have no conflicts to declare.

Author contributions

D. V. designed the study, performed experiments and wrote the manuscript. M. E. P. designed the study and wrote the manuscript.

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