Abstract

Background

   The adenoids act as a reservoir of bacterial pathogens and immune
   molecules, and they are significantly involved in children with otitis
   media with effusion (OME). As an essential carrier of intercellular
   substance transfer and signal transduction, exosomes with different
   biological functions can be secreted by various types of cells. There
   remains significant uncertainty regarding the clinical relevance of
   exosomes to OME, especially in its pathophysiologic development. In
   this study, we will seek to determine the biological functions of
   exosomes in children with adenoid hypertrophy accompanied by OME
   (AHOME).

Methods

   The diagnostic criteria for OME in children aged 4–10 years include a
   disease duration of at least 3 months, type B or C acoustic immittance,
   and varying degrees of conductive hearing loss. Adenoidal hypertrophy
   is diagnosed when nasal endoscopy shows at least 60% adenoidal
   occlusion in the nostrils or when nasopharyngeal lateral X-ray shows
   A/N > 0.6. Children who meet the indications for adenoidectomy surgery
   undergo adenoidectomy. Peripheral blood, nasopharyngeal swab, and
   adenoid tissue will be collected from patients, and the exosomes will
   be isolated from the samples. Following the initial collection,
   patients will undergo adenoidectomy and peripheral blood and
   nasopharyngeal swabs will be collected again after 3 months.

Expected results

   This study aims to identify differences in exosomes from preoperative
   adenoid tissue and peripheral blood samples between children with AHOME
   and those with adenoid hypertrophy alone. Additionally, it seeks to
   determine changes in microbial diversity in adenoid tissue between
   these groups.

Conclusions

   The findings are expected to provide new insights into the diagnosis
   and treatment of OME, to identify novel biomarkers, and to enhance our
   understanding of the pathophysiology of OME, potentially leading to the
   development of innovative diagnostic and therapeutic approaches.

   Keywords: Exosome, Otitis media with effusion, Adenoid hypertrophy

Background

   Otitis media, also known as middle ear inflammation, is a spectrum of
   diseases, including acute otitis media, chronic suppurative otitis
   media, and otitis media with effusion (OME). OME was first described by
   Politzerzai in 1867 as a non-suppurative inflammatory disease in the
   middle ear characterized by hearing loss caused by loss of transduction
   of sound waves in the middle ear due to the presence of middle ear
   effusion [[32]1]. OME is very common and is one of the main causes of
   hearing loss in children, and about 80% of children suffer one or more
   episodes of OME by 10 years of age [[33]2]. However, because of its
   inconspicuous symptoms, the incidence and prevalence are difficult to
   establish accurately, which is a significant obstacle to the prevention
   and treatment of OME. Treatment for OME includes enhancement of middle
   ear ventilation and drainage, removal of middle ear fluid, infection
   management, and treatment of related conditions. For children with OME
   without high-risk factors, there should be a 3-month watchful waiting
   period after diagnosis [[34]3]. Routine systemic use of antibiotics and
   hormones is not recommended, and there is a lack of long-term evidence
   for the efficacy of nasal corticosteroids and antihistamines. Current
   therapies, whether surgical or non-surgical, can temporarily alleviate
   ear cavity aeration, but the disease's natural course, long-term
   prognosis, and relapse rate remain unclear. For tympanostomy tubes
   [[35]4], most of the important considerations are the impact of OME on
   children's social interactions and learning ability. There are no
   uniform recommendations for the indication of adenoidectomy in national
   guidelines, and the age of the child and whether there is a recurrence
   are important factors to consider.

   OME has a complicated etiology that can be caused by several factors
   [[36]5, [37]6]. Dysfunction of the Eustachian tube, which balances the
   air pressure within and outside the middle ear and provides a cleansing
   function to avoid retrograde infection, is typically thought to play a
   major role in the etiology of OME [[38]7], and such dysfunction can be
   caused by adenoid hypertrophy, chronic sinusitis, or a deviated nasal
   septum. OME can also be caused by bacterial infection [[39]8, [40]9],
   and contemporary detection techniques, such as polymerase chain
   reaction (PCR), have been used to demonstrate the existence of
   bacterial DNA in patients' middle ear secretions.

   The microbiome plays a crucial role in airway inflammation and
   homeostasis. The different microenvironments in the upper airway –
   including the anterior nostrils, the nasal cavities, the sinuses, the
   nasopharynx, the Eustachian tubes, the middle ear cavities, the oral
   cavity, the oropharynx, and the larynx – host specific microbial
   communities [[41]10]. Bacterial biofilms have been found in the middle
   ear secretions using fluorescence confocal microscopy [[42]11],
   suggesting that the formation of bacterial biofilms may also be
   associated with the pathogenesis of OME [[43]12]. Inflammatory
   reactions also play an indispensable role in the pathogenesis of OME
   [[44]13], and it has been suggested that OME is related to the
   nasopharyngeal inflammatory and immunological response. Exudates rich
   in proteins and inflammatory mediators are secreted by the inflammatory
   response, which generates cytokines. The dilation of blood vessels in
   response to cytokines improves gaseous exchange in the middle ear and
   lowers endotympanic pressure [[45]14]. The middle ear cavity remains
   relatively stable, except for the tympanic membrane, making the pars
   tensa the most vulnerable area due to its lack of a fibrous layer, and
   the pars tensa is frequently the primary site of retraction. If the
   drop in endotympanic pressure persists, the tympanic atelectasis will
   lead to injury of the pars tensa, eventually leading to complete
   atelectasis of the tympanic membrane. Long-term middle ear inflammation
   causes cellular differentiation and an increase in the number of mucous
   cells, which leads to a gradual increase in exudate that fills the
   middle ear cavity. The decrease in upstream pressure in the middle ear
   due to mucus adhering to the Eustachian tube makes it more difficult to
   drain the mucus, which aggravates OME even further. In addition,
   biofilm production on mucosal membranes has been observed in OME
   [[46]15], in which biofilms are created by bacteria creating an
   "anchor-like" microcolony that colonizes an adherent matrix containing
   polysaccharides, nucleic acids, and proteins [[47]16]. Therefore,
   biofilms on the surface of adenoid tissue are an excellent candidate
   for exploring the pathogenesis of OME [[48]11, [49]12].

   The detection of exosomes in these tissues to explore the
   pathophysiology of OME has already been performed, with peripheral
   blood primarily used in the study of OME and adenoid tissue primarily
   used in the study of adenoid hypertrophy. Exosomes were first described
   in the late 1960s by Bonucci and Anderson [[50]17], and exosome-related
   research accelerated greatly in the 1980s [[51]17]. Exosomes are the
   smallest extracellular vesicles (EVs), with a diameter of 30~200 nm,
   and they contain multiple cargo types, including protein, DNA, mRNA,
   and miRNA, and they have an endosomal origin [[52]17, [53]18] and can
   be found in different bodily fluids [[54]19]. Receptor cells take up
   exosomes through endocytosis to transfer their cargo. The critical
   functions of exosomes are cell-cell communication, immune modulation,
   extracellular matrix turnover, stem cell differentiation,
   neovascularization, and cellular waste removal [[55]20]. Proteins and
   miRNA contained in exosomes can regulate cell physiology and can modify
   the microenvironment around target cells, indicating that exosomes have
   potential as diagnostic and therapeutic tools [[56]21].

   Exosomes have been found to have pathological roles in a variety of
   diseases, including heart disease [[57]22], neurodegenerative diseases
   [[58]23, [59]24], cancer [[60]25, [61]26], and inflammation
   [[62]27–[63]29], and many studies have shown that exosomes may be
   crucial in regulating inflammation-based cells. For example, repeated
   injection of exosomes isolated from the peripheral blood of mice fed a
   high-fat diet into mice on a regular diet causes the accumulation of
   activated immature CD11b^+Ly6ChiLy6G^– bone marrow cells in the liver,
   thus promoting obesity-related diseases such as fatty liver on top of
   causing chronic inflammation [[64]27]. Exosome numbers are
   substantially greater in lipopolysaccharide-treated cells compared to
   control cells, as seen in electron microscope images [[65]30]. Exosomes
   can also be used as inflammatory bowel disease (IBD) biomarkers, and
   their source can be hosting cells, food, or microorganisms [[66]31]. It
   has been found that samples from cases with malignant common bile duct
   stenosis contain significantly higher levels of EVs than bile samples
   from healthy controls [[67]29]. Both ANXA1 and PSMA7, which are present
   in exosomes released by intestinal epithelial cells and oral mucosal
   cells, are expressed at significantly different levels in IBD patients
   and healthy controls [[68]32, [69]33], suggesting that they might be
   promising biomarker candidates for early detection of IBD. In the field
   of otolaryngology, EVs play an important role, for example, in head and
   neck cancer metastasis [[70]34], lymphocyte regulation [[71]35],
   angiogenesis [[72]36], microenvironment remodeling [[73]37, [74]38],
   and drug resistance [[75]39, [76]40]. Related studies have shown that
   disease-associated bacterial products drive the differential expression
   of miRNAs in human middle ear epithelial cells in the OME model
   [[77]41]. Elevated levels of cysteine protease inhibitors-1 and -2 in
   EVs [[78]42] in patients with chronic rhinosinusitis (CRS) may serve as
   markers of CRS and predict disease phenotype, while EVs may also lead
   to polyp formation by upregulating pappalysin and serpins [[79]43]. A
   previous study found that keratinocyte-derived exosomal miR-17 led to
   the upregulation of fibroblast protein expression, promoted osteoclast
   differentiation, and led to bone destruction, which are hallmarks of
   acquired cholesteatoma [[80]44]. Inner ear cells release Hsp70-carrying
   exosomes in response to heat stress, and these interact with TLR4 on
   hair cells to provide a protective effect [[81]45]. Although limited
   research has been performed to date, studies have shown an important
   role for EVs in various otolaryngological conditions [[82]46].
   Continued research into the characterization of EVs in ENT diseases
   will help develop new diagnostic biomarkers and therapeutic targets and
   will provide clinicians in the field of ENT with a new set of tools.

   In this study, we are collecting exosomes from peripheral blood and
   adenoid tissue following adenoidectomy and have been evaluating the
   microbial modifications in the biofilm on the adenoid surface to better
   understand the pathophysiology of OME. Thus, this study will highlight
   topics for future research and give scientific insight into the
   development of drugs for treating OME.

Methods

Study design

   The study design for the current prospective trial is described in Fig.
   [83]1.

Fig. 1.

   [84]Fig. 1
   [85]Open in a new tab

   Technology Road map. A The grouping and sample collection procedure. B
   The evaluation of exosomes and the examination of microbial alterations
   in the samples. Abbreviation: AHOME group, adenoid hypertrophy
   accompanied by OME; AH group, adenoid hypertrophy alone

Patient and public involvement

Group

   The children will be divided into two groups: the adenoid hypertrophy
   accompanied by OME (adenoid hypertrophy accompanied by OME, AHOME)
   group and the adenoid hypertrophy alone (adenoid hypertrophy, AH)
   group.

   The biofilm-positive rate in patients with adenoid hypertrophy was
   shown to be 35.5% in the study by Szalmás et al. [[86]47]. The odds
   ratio of the AHOME group compared to the AH group is projected to be
   1.25, and a chi-square test was used to estimate the positive rate of
   biofilm in the AH group with a statistical power of 80% and an alpha
   level of 5%, resulting in sample sizes of 122 for the AHOME group and
   the AH group.

   A total of 244 subjects (n = 122 in the AHOME group and n = 122 in the
   AH group) will be recruited from the Eye & ENT Hospital of Fudan
   University and from the local community. After obtaining informed
   consent, nasopharyngeal swabs, peripheral blood, and adenoid tissue
   samples will be collected.

Inclusion and exclusion criteria

   Patients who meet the following inclusion criteria will be considered
   eligible for the AH group:
    1. Aged between 4 and 10 years old.
    2. Nasal endoscopy showing adenoidal occlusion in 60% of the nostrils
       or nasopharyngeal lateral X-ray showing A/N > 0.6.

   Patients diagnosed with AH will also meet at least one of the following
   inclusion criteria to be considered eligible for the AHOME group:
    1. Diagnosis of OME with a disease course of at least 3 months.
    2. An acoustic immittance of type B or C.
    3. The presence of conductive hearing loss with PTA (the mean hearing
       threshold at 500 Hz, 1000 Hz, and 2000 Hz) ≥ 30 dB.

   Patients who meet any of the following exclusion criteria will be
   considered ineligible:
    1. One or more episodes of otitis media in the previous one year.
    2. Rhinitis (acute or chronic rhinosinusitis or allergic rhinitis).
    3. Any immunologic diseases, any intrinsic diseases of the hearing
       system, or any anatomical or physiological defects of the ear.

Surgery and specimen collection

   All of the enrolled children will be eligible for adenoidectomy.
   Peripheral blood and nasopharyngeal swabs will be collected before
   surgery, and adenoid tissue samples will be collected during surgery.
   Blood samples and nasopharyngeal swabs will be collected again 3 months
   after surgery to monitor the changes in exosome profiles over time and
   to assess the long-term effects of the surgery on the exosome-mediated
   immune response and microbial alterations.

Isolation and detection of exosomes

   Exosomes in the different samples will be extracted by
   ultracentrifugation. The sample will be collected in a 15 mL tube and
   then centrifuged at 500 × g for 10 min, and the supernatant will be
   collected in a new tube. The supernatant will then be centrifuged at
   2,000 × g for 15 min at 4 °C, and the resulting supernatant will be
   transferred to a new centrifuge tube. This supernatant will be
   centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant will be
   collected and filtered through a sterile 0.22 μm filter (Thermo Fisher,
   Invitrogen). Exosome isolation reagent (Thermo Fisher, Invitrogen) will
   be mixed with the supernatant at a ratio of 1:2 and incubated overnight
   at 4 °C. The next day, the supernatant will be centrifuged at
   10,000 × g for 60 min at 4 °C to pellet the exosomes. The supernatant
   will be removed completely, and the pellet will be resuspended in PBS
   and stored at –80 °C. Transmission electron microscopy will be used to
   observe the morphology and size of the exosomes, and NanoSight particle
   tracking will be used to accurately and quickly detect the distribution
   of vesicle sizes as a whole. Western blot and flow cytometry will be
   used to verify the purity and presence of exosomes with the commonly
   used exosome markers CD63, CD81, and CD9.

High-throughput sequencing and pathway analysis

   The molecular mechanisms behind the pathogenesis of OME can be revealed
   through high-throughput sequencing, which can suggest new avenues for
   the development of clinical diagnosis and therapeutic methods. Gene
   ontology (GO) ([87]http://www.geneontology.org) and Kyoto Encyclopedia
   of Genes and Genomes (KEGG) ([88]http://www.genome.jp/kegg) analyses
   will be performed on the differentially expressed genes. GO is used to
   analyze the biological processes, molecular functions, and cellular
   components of differentially expressed genes, while KEGG is used to
   identify pathway enrichment analysis of the differentially expressed
   genes. Differentially expressed genes will be annotated using the
   Cluster Profile package in R using the criteria of |Log[2] Fold
   Change|≥ 1 and adjusted p < 0.05.

Histopathologic examination

   According to reports, the combination of hematoxylin and eosin (H&E)
   staining and Gram staining protocols is a viable method to detect the
   presence of biofilms and corresponding histopathological changes
   because H&E and Gram staining can show microstructures and various
   bacterial elements, respectively [[89]48]. Adenoid specimens acquired
   by surgical excision will be preserved in 10% (w/v) formaldehyde and
   then cut into tissue sections. Conventional H&E and Gram staining will
   be used to evaluate two successive 5 μm frozen sections. A surrounding
   polysaccharide layer and the presence of a distinctive shape and the
   presence of a gram-positive/negative micro-community of bacteria
   following light microscopy examination will be the criteria for the
   histopathological confirmation of microbial biofilms [[90]48]. Two
   independent pathologists who are blinded to the clinical diagnosis will
   evaluate both histologic staining protocols for evidence of bacteria
   and biofilms.

Detection of microbial alterations

   The 16S ribosomal RNA (16S rRNA) is a component of the 30S subunit in
   the prokaryotic ribosome. Prokaryotic 16S rRNA sequences feature nine
   highly variable regions, of which regions V4-V5 offer the highest
   specificity and provide database information for bacterial diversity
   analysis and annotation. To acquire information on the abundance of
   each species in the colonies, we will amplify the genome of the V4-V5
   region, collect sequence information using a MiSeq sequencer, and
   compare the sequences with a known bacterial genome database.

Statistical methods

   R software version 4.0.5 will be used for all statistical analyses, and
   a p-value < 0.05 will be considered statistically significant.

Expected results

    1. Exosomal differences in adenoid tissue: We expect to identify
       differences in exosomes derived from preoperative adenoid tissue
       samples between the AHOME group and the AH group.
    2. Exosomal differences in peripheral blood: We expect to identify
       differences in exosomes derived from preoperative peripheral blood
       samples between the two groups.
    3. Alterations in microbial diversity: We expect to see changes in
       microbial diversity in the adenoid tissues between the two groups.
    4. Diagnostic and therapeutic insights: We expect to gain new insights
       into the diagnosis and treatment of OME, thus contributing to the
       development of novel diagnostic and therapeutic approaches.
    5. Identification of novel biomarkers: We expect to identify novel
       biomarkers for OME, thus laying an important scientific foundation
       for the development of preventive measures, diagnostic techniques,
       and treatment options.
    6. Comprehensive understanding of OME pathophysiology: We expect to
       gain a more comprehensive understanding of the pathophysiology of
       OME, revealing downstream pathways of initiation factors that may
       contribute to OME development.

Discussion

   The patients in this study with adenoid hypertrophy are being divided
   into two distinct groups, namely those with isolated adenoid
   hypertrophy and those with coexisting OME and adenoid hypertrophy.
   Peripheral blood samples have been obtained from the participants in
   both groups, and exosomes will subsequently be isolated for further
   analysis. Nasopharyngeal swabs were used to detect microbial
   alterations. Adenoidectomies are being performed, and adenoid tissue
   samples are being collected during the procedures. Three months after
   the surgery, another round of blood and nasopharyngeal swab collection
   will take place. Exosomes will be isolated again for subsequent
   studies, mirroring the pre-surgery protocol. Our investigation will
   also encompass proteome research, exosome biomarker exploration, and
   the application of high-throughput sequencing techniques. These
   methodologies will allow us to delve into the pathophysiology of OME,
   explicitly focusing on the etiology related to inflammation.
   Ultimately, we aim to offer innovative insights into OME diagnosis and
   treatment.

   OME differs from acute otitis media in that it lacks the evident signs
   and symptoms of an acute infection [[91]3], but once symptoms appear
   conductive hearing loss occurs due to middle ear effusion. The impact
   on an individual's quality of life and the disease burden on children
   cannot be exaggerated due to its tenacity and frequent occurrence
   [[92]49]. However, estimating the frequency and prevalence of the
   condition and providing early diagnosis are challenging due to the lack
   of evident symptoms [[93]1]. Pneumatic otoscopy is the major diagnostic
   method currently suggested, and this has high diagnostic accuracy but a
   poor coverage rate due to significant technical difficulties and
   equipment issues, making this method undesirable for mass population
   screening for OME [[94]3, [95]50–[96]52].

   This research project aims to collect adenoid tissue and peripheral
   blood samples from two groups of patients. The isolated exosomes
   obtained from these samples will serve as a valuable material for
   investigating the underlying etiological mechanisms of OME. The
   ultimate objective is to generate novel insights that can contribute to
   developing innovative screening and treatment approaches for OME.

   Considering the potential interference of rhinitis on the research
   results of adenoid hypertrophy and otitis media [[97]53–[98]55], it is
   necessary to exclude the presence of rhinitis when collecting patient
   samples. The following measures are being implemented to achieve this:
   (1) We conduct a detailed clinical assessment of the patients,
   including nasal and sinus examinations, before collecting the samples.
   A preliminary judgment can be made on the presence of rhinitis by
   observing whether there are symptoms of nasal inflammation such as
   nasal congestion, runny nose, itching, etc. (2) We perform nasal
   endoscopy [[99]56] using nasal endoscopes or fiber-optic
   nasopharyngoscopes to observe the whole nasal cavity. This provides a
   clearer view to detect abnormalities in the nasal mucosa such as
   congestion, secretions, etc. (3) If necessary, nasal X-rays, CT scans,
   or MRI examinations can be performed to further evaluate the condition
   of the nasal cavity and sinuses [[100]57]. These examinations can
   reveal nasal inflammation, obstructions, or other abnormalities thereby
   helping to exclude the presence of rhinitis. By applying these methods
   together, it is possible to minimize the interference of rhinitis on
   sample collection and to ensure the collection of valid samples
   directly related to AHOME.

   Exosomes have emerged as promising targets for various research studies
   due to their potential roles in various diseases, and they can be used
   in multiple ways to investigate pathophysiological pathways and to
   elicit inflammatory responses. Additionally, exosomes’ morphological
   and quantitative properties can provide insights into their regulatory
   functions in the cell [[101]27]. Furthermore, the presence of specific
   components within exosomes, such as miRNAs, NADPH oxidase, nitric oxide
   synthases, and protein disulfide isomerase, have been found to
   influence the progression and prognosis of cardiac diseases [[102]58,
   [103]59]. These components are significant in cardiac health and can
   serve as potential molecular targets for therapeutic interventions
   [[104]30].

   Additionally, oncogenic proteins, mRNAs, and miRNAs are associated with
   cancer progression, indicating the involvement of exosomes in cancer
   biology [[105]60]. Moreover, specific proteins like ANXA1 and PSMA7
   have been identified as biomarkers for IBD, and their presence in
   exosomes can provide valuable information for diagnosing and monitoring
   the progression of this disease [[106]32, [107]33]. These examples
   highlight the diverse roles of exosomes in different disease contexts,
   emphasizing their potential as molecular targets and diagnostic
   biomarkers in various research fields.

   Exosomes indeed contain a significant amount of biological information
   that warrants further investigation. Previous studies have demonstrated
   the presence of miRNA-carrying exosomes in middle ear effusion, and the
   abundant miRNAs and proteins in these exosomes appear to be key
   mediators of innate immunity and neutrophilia [[108]41]. In this
   project, high-throughput sequencing will be employed to delve deeper
   into the biological information carried by exosomes. The aim is to
   uncover the downstream pathways of initiation factors that may play a
   role in the development of OME. By identifying these pathways, this
   research will contribute to a more comprehensive understanding of the
   pathophysiology of OME. Moreover, this project seeks to identify and
   establish a novel biomarker for OME. Potential biomarkers specific to
   OME can be identified by analyzing the biological information obtained
   from exosomes using high-throughput sequencing. These biomarkers have
   promise as essential scientific foundations for the development of
   preventive measures, diagnostic techniques, and treatment options for
   OME. The use of high-throughput sequencing technology to investigate
   the biological information in exosomes represents an innovative
   approach that can provide valuable insights into the underlying
   mechanisms of OME.

   This study is a protocol study, and the data will be obtained in the
   future implementation of the trial. The purpose of this paper is to
   present the study design and methodology and to lay the foundation for
   future research. It is anticipated that the outcomes of this project
   will contribute significantly to advancements in the understanding and
   management of OME.

Conclusions

   This study seeks to understand the role of exosomes in children with
   AHOME. The adenoids, which harbor bacterial pathogens and immune system
   molecules, play a significant role in OME. This research aims to
   elucidate the biological functions of exosomes and their involvement in
   the pathophysiological development of OME. We will collect patients’
   peripheral blood and adenoid tissue samples in order to isolate and
   analyze exosomes. The results will shed light on differences in
   exosomes between the two groups before surgery as well as changes in
   microbial diversity in the adenoid tissue. The findings of this study
   might contribute to the development of innovative diagnostic and
   therapeutic approaches for OME.

Acknowledgements