Immunopathologic characteristics of Chinese pediatric patients with chronic rhinosinusitis

  • Author Footnotes
    † Lijie Jiang, Yinhui Zeng, and Zhaoqi Huang should be considered joint first author.
    Lijie Jiang
    Footnotes
    † Lijie Jiang, Yinhui Zeng, and Zhaoqi Huang should be considered joint first author.
    Affiliations
    Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
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  • Author Footnotes
    † Lijie Jiang, Yinhui Zeng, and Zhaoqi Huang should be considered joint first author.
    Yinhui Zeng
    Footnotes
    † Lijie Jiang, Yinhui Zeng, and Zhaoqi Huang should be considered joint first author.
    Affiliations
    Department of Otolaryngology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China
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  • Author Footnotes
    † Lijie Jiang, Yinhui Zeng, and Zhaoqi Huang should be considered joint first author.
    Zhaoqi Huang
    Footnotes
    † Lijie Jiang, Yinhui Zeng, and Zhaoqi Huang should be considered joint first author.
    Affiliations
    Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
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  • Yiquan Tang
    Affiliations
    Department of Otolaryngology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China
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  • Qingxiang Zeng
    Affiliations
    Department of Otolaryngology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China
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  • Wenlong Liu
    Correspondence
    Corresponding author.
    Affiliations
    Department of Otolaryngology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, China
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  • Jianbo Shi
    Correspondence
    Corresponding author.
    Affiliations
    Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
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  • Author Footnotes
    † Lijie Jiang, Yinhui Zeng, and Zhaoqi Huang should be considered joint first author.
Open AccessPublished:December 05, 2021DOI:https://doi.org/10.1016/j.waojou.2021.100616

      Abstract

      Background

      The histopathology of pediatric chronic rhinosinusitis with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP) is rarely reported due to low prevalence or the unavailability of tissue samples. Hence, we aimed to characterize and compare the histologic features and protein expression of Th1/Th2/Th17-related cytokines in pediatric CRSsNP and CRSwNP.

      Methods

      The histologic characteristics of 15 children with CRSsNP, 52 children with CRSwNP, and 12 control participants were analyzed using hematoxylin and eosin staining. The expression of Th1/Th2/Th17-related cytokines were examined using immunohistochemistry and the enzyme-linked immunosorbent assay.

      Results

      Pediatric subjects with CRSwNP had more intact epithelium and less submucosal mucous glands compared to those with CRSsNP. Tissue eosinophils were more prevalent in the younger CRSwNP group compared to the older CRSwNP or the CRSsNP groups. The protein concentrations of Th2 cytokines were significantly higher in the CRSwNP group than the CRSsNP group or the control group. Moreover, the protein concentrations of Th17 cytokines were significantly higher in the younger CRSwNP group than the older CRSwNP group or the CRSsNP and control groups. The protein concentrations of Th1 and Th17 cytokines were also significantly higher in the CRSsNP group than the control group. Compared with non-eosinophilic CRSwNP, eosinophilic CRSwNP presented with elevated protein concentrations of Th1 and Th17 cytokines.

      Conclusion

      For the first time, we showed that pediatric CRSwNP presents as eosinophilic with Th2/Th17 inflammation, whereas CRSsNP presents as Th1/Th17 inflammation. Our study may provide a theoretical basis for the precise treatment of pediatric CRS in the future.

      Keywords

      Introduction

      Chronic rhinosinusitis (CRS) is common in both the adult and child populations.
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      • et al.
      Adult chronic rhinosinusitis.
      ,
      • Rose A.S.
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      • Zanation A.M.
      • Ebert C.S.
      Chronic rhinosinusitis in children.
      CRS is often grouped as chronic rhinosinusitis with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP). In children, nasal polyps (NP) are a rare condition with prevalence of 0.1%.
      • Settipane G.A.
      Epidemiology of nasal polyps.
      The histopathology of pediatric CRSsNP and CRSwNP is rarely reported due to low prevalence or the unavailability of tissue samples. Previous research found that eosinophilic tissue infiltration is more obvious in adults and in older children than in younger children with CRS.
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      • Liu A.H.
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      The study by Coffinet et al found that lymphocytes were more prevalent in young children with CRS, rather than eosinophils.
      • Coffinet L.
      • Chan K.H.
      • Abzug M.J.
      • Simões E.A.F.
      • Cool C.
      • Liu A.H.
      Immunopathology of chronic rhinosinusitis in young children.
      For CRSwNP, Mitroi et al. demonstrated that T cells and eosinophils were the main inflammatory cells in NPs; however, their study included both children and adults who have not been grouped according to age.
      • Mitroi M.
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      • Capitanescu A.
      • et al.
      Differences in the distribution of CD20, CD3, CD34 and CD45RO in nasal mucosa and polyps from patients with chronic rhinosinusitis.
      Moreover, an increasing number of studies had revealed that the immunopathologic features of CRSsNP and CRSwNP are different between various ethnic groups, suggesting that different mechanisms contribute to the pathology of these conditions.
      • Zhang N.
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      • Perez-Novo C.
      • et al.
      Different types of T-effector cells orchestrate mucosal inflammation in chronic sinus disease.
      ,
      • Cao P.-P.
      • Li H.-B.
      • Wang B.-F.
      • et al.
      Distinct immunopathologic characteristics of various types of chronic rhinosinusitis in adult Chinese.
      CRSsNP is believed to be a Th1-dominant inflammation, whereas Th2 and Th17 inflammations are observed in eosinophilic rather than in non-eosinophilic CRSwNP.
      • Zhang N.
      • Van Zele T.
      • Perez-Novo C.
      • et al.
      Different types of T-effector cells orchestrate mucosal inflammation in chronic sinus disease.
      • Cao P.-P.
      • Li H.-B.
      • Wang B.-F.
      • et al.
      Distinct immunopathologic characteristics of various types of chronic rhinosinusitis in adult Chinese.
      • Cho S.H.
      • Hamilos D.L.
      • Han D.H.
      • Laidlaw T.M.
      Phenotypes of chronic rhinosinusitis.
      For the expression of Th cytokines, Cao et al demonstrated that Th1 responses are dominant in Chinese patients with CRSsNP, whereas Th2 responses are found in eosinophilic CRSwNP rather than all CRSwNP types.
      • Cao P.-P.
      • Li H.-B.
      • Wang B.-F.
      • et al.
      Distinct immunopathologic characteristics of various types of chronic rhinosinusitis in adult Chinese.
      But now Delemarre et al reported that CRSsNP also with a moderate type 2 immune response showed a considerable eosinophilic inflammation with clinical impact.
      • Delemarre T.
      • Holtappels G.
      • De Ruyck N.
      • et al.
      Type 2 inflammation in chronic rhinosinusitis without nasal polyps: another relevant endotype.
      In this study, we characterized and compared the histologic features and the protein expression of Th1/Th2/Th17-related cytokines in pediatric CRSsNP and CRSwNP.

      Methods

       Patients

      This study recruited 15 children with CRSsNP, 52 children with CRSwNP, and 12 children without any sinonasal disease who have undergone optic nerve decompression due to traumatic optic neuropathies (control group) from the medical institution A and B between July 2016 and April 2020. CRSsNP or CRSwNP was determined according to persistent nasal symptoms (>3 months), nasal endoscopic examination, and computed tomographic scans as described by the 2020 European Position Paper on Rhinosinusitis and Nasal Polyps.
      • Fokkens W.J.
      • Lund V.J.
      • Hopkins C.
      • et al.
      European position paper on rhinosinusitis and nasal polyps 2020.
      All enrolled patients did not respond to maxima medical therapy or adenoidectomy.
      • Chandy Z.
      • Ference E.
      • Lee J.T.
      Clinical guidelines on chronic rhinosinusitis in children.
      ,
      • Snidvongs K.
      • Sangubol M.
      • Poachanukoon O.
      Pediatric versus adult chronic rhinosinusitis.
      Asthma or allergic rhinitis were diagnosed according to lung function and through allergen tests.
      • Cheng L.
      • Chen J.
      • Fu Q.
      • et al.
      Chinese society of allergy guidelines for diagnosis and treatment of allergic rhinitis.
      ,
      • Boulet L.-P.
      • Reddel H.K.
      • Bateman E.
      • Pedersen S.
      • FitzGerald J.M.
      • O'Byrne P.M.
      The global initiative for asthma (GINA): 25yearslater.
      All study participants ceased their usage of oral or local corticosteroids and antibiotics 1 month before surgery. Diseased unciform process mucosal samples from patients with CRSsNP, polyp tissues from patients' middle nasal meatus with CRSwNP, and unciform process from control subjects were collected. Patients with prior sinus surgery, immunodeficiency, cystic fibrosis, and Primary ciliary dyskinesia were excluded.
      • Lahiri T.
      • Hempstead S.E.
      • Brady C.
      • et al.
      Clinical practice guidelines from the cystic fibrosis foundation for preschoolers with cystic fibrosis.
      • Pereira R.
      • Barbosa T.
      • Gales L.
      • et al.
      Clinical and genetic analysis of children with kartagener syndrome.
      • Conley M.E.
      • Notarangelo L.D.
      • Etzioni A.
      Diagnostic criteria for primary immunodeficiencies. Representing PAGID (pan-American group for immunodeficiency) and ESID (European society for immunodeficiencies).
      This study was approved by the local ethical committee ([2016]096) and informed consent was obtained from the children's guardians.
      According to“Clinical Consensus Statement: Pediatric Chronic Rhinosinusitis”, the management of children aged 12 years and younger with CRS is distinctly different than management of children aged 13 to 18 years old with CRS,which had raised by Brietzke (Clinical Consensus Statement: Pediatric Chronic Rhinosinusitis). Therefore, we divided the enrolled patients into 6–12 years and 13–18 years groups.

       Morphological study

      Paraffin-embedded sections were stained using hematoxylin and eosin. Tissue eosinophilia was confirmed when the mean eosinophil count from five separate counts was >10% under the high power field (HPF).
      • Conley M.E.
      • Notarangelo L.D.
      • Etzioni A.
      Diagnostic criteria for primary immunodeficiencies. Representing PAGID (pan-American group for immunodeficiency) and ESID (European society for immunodeficiencies).
      • Wang K.
      • Deng J.
      • Yang M.
      • et al.
      Concordant systemic and local eosinophilia relates to poorer disease control in patients with nasal polyps.
      • Lou H.
      • Zhang N.
      • Bachert C.
      • Zhang L.
      Highlights of eosinophilic chronic rhinosinusitis with nasal polyps in definition, prognosis, and advancement.
      The blood cell counts were performed using the LH-785 system (Beckman Coulter, Ireland).
      Epithelial integrity was defined as the proportion of intact epithelium in the entire sample length (400 × ). The epithelial thickness was obtained by measuring 10 random epithelia in each slide (400 × ). The basement membrane thickness was scored as follows (400 × ): 0 = none (<7.5 μm); 1 = mild (7.5 μm–15 μm); 2 = moderate (15 μm–30 μm); or 3 = marked (>30 μm).
      • Dhong H.-J.
      • Kim H.Y.
      • Cho D.-Y.
      Histopathologic characteristics of chronic sinusitis with bronchial asthma.
      • Do T.Q.
      • Barham H.P.
      • Earls P.
      • et al.
      Clinical implications of mucosal remodeling from chronic rhinosinusitis.
      • Kule Z.G.
      • Habesoglu T.E.
      • Somay A.
      • Deveci H.S.
      • Kule M.
      • Gursel A.O.
      Histopathological characteristics of nasal polyps in smokers and non-smokers.
      The submucosa gland hyperplasia in 1 section was scored as follows (100 × ): 0 (<3 glands); 1 (3–10 glands); 2 (11–30 glands); or 3 (>30 cells).
      • Dhong H.-J.
      • Kim H.Y.
      • Cho D.-Y.
      Histopathologic characteristics of chronic sinusitis with bronchial asthma.
      For immunohistochemistry (IHC), the sections were stained overnight at 4 °C using rabbit or mouse antibodies against IFN-γ, IL-4, IL-5, IL-13, IL-17, and IL-23 (Abcam, USA), and then with biotinylated goat anti-mouse/rabbit IgG secondary antibody. Phosphate buffer saline(PBS) was used as the control.
      • Luo X.
      • Xu Z.
      • Zuo K.
      • et al.
      The changes of clinical and histological characteristics of chronic rhinosinusitis in 18 years: Was there an inflammatory pattern shift in southern China?.

       Enzyme-linked immunosorbent assay (ELISA)

      Tissue homogenates were prepared as previously described.
      • Wang X.
      • Zhang N.
      • Bo M.
      • et al.
      Diversity of T cytokine profiles in patients with chronic rhinosinusitis: a multicenter study in Europe, Asia, and Oceania.
      The IFN-γ, IL-4, IL-5, IL-13, IL-17, and IL-23 protein concentrations were assessed using ELISA kits (R&D, USA) according to the manufacturer's instructions.

       Statistical analyses

      The data were presented as mean ± SD, except for those with additional notes. Despite the sample size between the rhinosinusitis groups and controls was evident, the difference of sample size among groups was acceptable when the CRSwNP group was divided into 6–12 y (n = 24) and 13–18 y (n = 28) CRSwNP subgroup. The Mann-Whitney U test was performed for the comparison of 2 groups, and the Kruskal-Wallis test with Dunn correction was performed in multiple comparisons. Statistical significance was set at P < 0.05.

      Results

       Demographic characteristics of the study population

      Our study included 52 children with CRSwNP, 15 children with CRSsNP, and 12 children in the control group. Their demographic characteristics are presented in Table 1. No statistically significant difference in demographic characteristics was observed between the CRSsNP and CRSwNP groups. Nevertheless, we found that both CRSsNP and CRSwNP were more prevalent in boys than in girls.
      Table 1Demographic characteristics of the study population
      CRSsNPCRSwNPControl
      Numbers155212
      Girls, n (%)2 (13.3%)8 (15.4%)6 (50%)
      Age (y), mean ± SD13.4 ± 3.812.5 ± 3.111.5 ± 6.2
      Allergic rhinitis, n (%)2 (13.3%)6 (11.5%)0 (0%)
      Asthma, n (%)0 (0%)1 (1.9%)0 (0%)
      Abbreviations: CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps.

       Histopathology between pediatric CRSsNP and CRSwNP

      Pediatric subjects in 6–12y or 13-18y CRSwNP group had more intact epithelium and less submucosal mucous glands than those with CRSsNP (P < 0.05). However, the thickness of the epithelium and the basement membranes was not different between the CRSsNP and CRSwNP groups (P > 0.05) (Table 2, Fig. S1). The tissue eosinophil proportion was significantly higher in the 6-12y CRSwNP group (48%) than in the 13-18y CRSwNP group (22%) or the CRSsNP group (Table 3, Fig. S1). However, the blood neutrophil, blood eosinophil, and tissue neutrophil counts were not significantly different among the various subgroups (P > 0.05) (Table 3).
      Table 2Morphologic characteristics of CRSsNP and CRSwNP as per HE staining
      CRSsNPCRSwNP
      6-18 y (n = 15)6-12 y (n = 24)13-18 y (n = 28)
      Epithelial integrity, %55.38 ± 35.553
      Compared with 6–12 y or 13–18 y CRSwNP group, P < 0.05.
      72.174 ± 38.9372.481 ± 35.714
      Mucus glands score1.267 ± 1.335
      Compared with 6–12 y or 13–18 y CRSwNP group, P < 0.05.
      0.957 ± 1.1470.963 ± 1.018
      Epithelium, μm24.357 ± 13.00123.368 ± 15.21424.8 ± 12.768
      Basement membrane, μm10 ± 3.80312.079 ± 4.6211.625 ± 5.878
      The data were expressed as mean ± SD.
      Abbreviations: CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps; HE, hematoxylin and eosin.
      a Compared with 6–12 y or 13–18 y CRSwNP group, P < 0.05.
      Table 3The submucosal inflammatory cell of CRSsNP and CRSwNP by HE staining
      CRSsNPCRSwNP
      6-18 y (n = 15)6-12 y (n = 24)13-18 y (n = 28)
      Absolute valueproportionAbsolute valueproportionAbsolute valueproportion
      Blood NEU4.138 ± 1.220.536 ± 0.1343.707 ± 1.8690.467 ± 0.1294.971 ± 2.170.62 ± 0.121
      Blood EOS0.046 ± 0.050.006 ± 0.0050.041 ± 0.0260.005 ± 0.0030.027 ± 0.0210.004 ± 0.003
      Tissue EOS11.514 ± 15.2790.093 ± 0.13514.93 ± 18.0870.131 ± 0.162
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      12.359 ± 18.4020.095 ± 0.137
      Tissue NEU1.421 ± 2.1920.011 ± 0.0181.373 ± 1.9520.013 ± 0.0181.642 ± 2.3520.012 ± 0.018
      The data were expressed as mean ± SD.
      Abbreviations: NEU, neutrophils; EOS, eosinophils; CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps; HE, hematoxylin and eosin.
      a Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.

       The expression of Th1/Th2/Th17-related cytokines in pediatric CRSsNP and CRSwNP

      The IFN-γ positive cell counts were not significantly different among the various subgroups (P > 0.05) (Table 4, Fig. S2). By contrast, the IL-5, IL-13, IL-17, and IL-23 positive cell counts in the 6-12y CRSwNP group were significantly higher compared with the 13-18y CRSwNP or the CRSsNP groups (P < 0.05) (Table 4, Fig. S2). Moreover, the IL-4 positive cell count was significantly higher in the 6-12y and 13-18y CRSwNP groups compared to the CRSsNP group (P < 0.05) (Table 4, Fig. S2).
      Table 4The expression of Th1/Th2/Th17-related cytokines between pediatric CRSsNP and CRSwNP as per immunochemistry
      CRSsNPCRSwNP
      6-18 y (n = 15)6-12 y (n = 24)13-18 y (n = 28)
      IFN-γ0.522 ± 0.2940.56 ± 0.3310.504 ± 0.307
      IL-40.023 ± 0.0110.068 ± 0.104
      Compared with CRSsNP, P < 0.05.
      0.044 ± 0.039
      Compared with CRSsNP, P < 0.05.
      IL-50.165 ± 0.1040.228 ± 0.195
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      0.131 ± 0.083
      IL-130.265 ± 0.2170.433 ± 0.215
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      0.321 ± 0.209
      IL-170.182 ± 0.150.251 ± 0.166
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      0.116 ± 0.079
      IL-230.138 ± 0.0740.203 ± 0.107
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      0.15 ± 0.08
      The data were expressed as mean ± SD.
      Abbreviations: CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps.
      a Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      b Compared with CRSsNP, P < 0.05.

       Protein expression of Th1/Th2/Th17-related cytokines by tissue

      The IFN-γ protein concentrations were significantly elevated in patients with CRSsNP, as well as among the 6-12y or 13-18y CRSwNP groups, compared to the 6-12y or 13-18y control group (P < 0.05) (Table 5). The protein concentrations of Th2 cytokines (IL-4, IL-5, IL-13) were significantly higher in both the 6-12y or 13-18y CRSwNP groups compared to the CRSsNP and the 6-12y or 13-18y control groups (P < 0.05) (Table 5). The protein concentrations of Th17 cytokines (IL-17, IL-23) were significantly higher in the 6-12y CRSwNP group compared to the13-18y CRSwNP and CRSsNP groups and the 6-12y or 13-18y control group (P < 0.05) (Table 5). The protein concentrations of Th1 and Th17 cytokines were significantly higher in the CRSsNP group compared to the 6-12y or 13-18y control group (P < 0.05) (Table 5).
      Table 5The tissue protein expression of Th1/Th2/Th17-related cytokines as per ELISA
      CRSsNPCRSwNPControls
      6-18 y (n = 15)6-12y (n = 24)13-18 y (n = 28)6-12 y (n = 5)13-18 y (n = 7)
      IFN-γ39.5 ± 21.7
      Compared with control, P < 0.05.
      46.8 ± 31.2
      Compared with control, P < 0.05.
      42.3 ± 22.6
      Compared with control, P < 0.05.
      19.4 ± 15.323.6 ± 17.8
      IL-459.7 ± 18.554.6 ± 12.8
      IL-518.6 ± 9.6183.2 ± 65.6
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      Compared with control, P < 0.05.
      133.9 ± 58.5
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      Compared with control, P < 0.05.
      12.5 ± 6.614.8 ± 9.2
      IL-13168.9 ± 41.5278.6 ± 76.3
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      213.4 ± 48.7
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      IL-17239.5 ± 173.6
      Compared with control, P < 0.05.
      573.2 ± 288.1
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      Compared with control, P < 0.05.
      146.8 ± 101.9
      Compared with control, P < 0.05.
      41.5 ± 13.749.8 ± 16.3
      IL-23173.6 ± 99.5378.2 ± 119.6
      Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      211.3 ± 108.5
      The data were expressed as mean ± SD.
      The protein concentrations are in pg/mL.
      Abbreviations: CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps; ELISA, enzyme-linked immunosorbent assay.
      a Compared with CRSsNP or 13–18 y CRSwNP, P < 0.05.
      b Compared with control, P < 0.05.

       Comparison of cytokine expression between eosinophilic and non-eosinophilic CRSwNP and CRSsNP

      Eosinophilic and non-eosinophilic CRSsNP account for 40% and 60% of the CRSsNP group, respectively. By contrast, eosinophilic and non-eosinophilic CRSwNP account for 36.5% and 63.5% of the CRSwNP, respectively.
      Compared to non-eosinophilic CRSsNP, eosinophilic CRSsNP was found to have increased protein concentrations of IFN-γ, IL-5, IL-13, and IL-17 (P < 0.05) (Table 6). Similarly, compared to non-eosinophilic CRSwNP, eosinophilic CRSwNP had increased protein concentrations of IFN-γ, IL-5, IL-17, and IL-23 (P < 0.05) (Table 6).
      Table 6The expression of Th1/Th2/Th17-related cytokines between pediatric CRSsNP and CRSwNP as per immunochemistry
      CytokinesCRSsNPCRSwNP
      EOS(n = 6)Non-EOS(n = 9)EOS(n = 18)Non-EOS (n = 34)
      EOS0.223 ± 0.125
      Compared with Non-EOS, P < 0.05.
      0.012 ± 0.0150.278 ± 0.16
      Compared with Non-EOS, P < 0.05.
      0.032 ± 0.026
      IFN-γ0.694 ± 0.179
      Compared with Non-EOS, P < 0.05.
      0.427 ± 0.310.675 ± 0.325
      Compared with Non-EOS, P < 0.05.
      0.483 ± 0.287
      IL-40.021 ± 0.0130.025 ± 0.010.051 ± 0.0530.059 ± 0.089
      IL-50.235 ± 0.096
      Compared with Non-EOS, P < 0.05.
      0.119 ± 0.0850.256 ± 0.199
      Compared with Non-EOS, P < 0.05.
      0.132 ± 0.086
      IL-130.45 ± 0.218
      Compared with Non-EOS, P < 0.05.
      0.141 ± 0.1010.41 ± 0.2160.369 ± 0.221
      IL-170.261 ± 0.186
      Compared with Non-EOS, P < 0.05.
      0.122 ± 0.0880.242 ± 0.186
      Compared with Non-EOS, P < 0.05.
      0.151 ± 0.11
      IL-230.162 ± 0.0850.121 ± 0.0660.212 ± 0.102
      Compared with Non-EOS, P < 0.05.
      0.154 ± 0.089
      The data were expressed as mean ± SD.
      Abbreviations: EOS, eosinophils; CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps.
      a Compared with Non-EOS, P < 0.05.

      Discussion

      In the current study, we investigated and compared the pathological patterns and concentrations of Th-related cytokines in CRSsNP and CRSwNP among Chinese children. To the best of our knowledge, this is the first time that the immunologic endotypes of CRS was explored among Chinese children.
      We found that pediatric subjects with CRSwNP had more intact epithelium and less submucosal mucous glands compared to those with CRSsNP. Similarly, Chan et al. reported that pediatric CRS is characterized by thinner epithelium and basement membranes, and less submucosal mucous glands.
      • Chan K.H.
      • Abzug M.J.
      • Coffinet L.
      • Simoes E.A.F.
      • Cool C.
      • Liu A.H.
      Chronic rhinosinusitis in young children differs from adults: a histopathology study.
      Eosinophilic and neutrophilic inflammation are the two main CRS inflammatory types in both children and adults.
      • Bachert C.
      • Akdis C.A.
      Phenotypes and emerging endotypes of chronic rhinosinusitis.
      ,
      • Heath J.
      • Hartzell L.
      • Putt C.
      • Kennedy J.L.
      Chronic rhinosinusitis in children: pathophysiology, evaluation, and medical management.
      Berger et al and Coffinet et al showed that tissue eosinophilia is significantly greater in adult CRS compared to pediatric CRS.
      • Coffinet L.
      • Chan K.H.
      • Abzug M.J.
      • Simões E.A.F.
      • Cool C.
      • Liu A.H.
      Immunopathology of chronic rhinosinusitis in young children.
      ,
      • Berger G.
      • Kogan T.
      • Paker M.
      • Berger-Achituv S.
      • Ebner Y.
      Pediatric chronic rhinosinusitis histopathology: differences and similarities with the adult form.
      However, Baroody et al found that eosinophilic inflammation was prevalent in children with refractory CRS.
      • Baroody F.M.
      • Hughes C.A.
      • McDowell P.
      • Hruban R.
      • Zinreich S.J.
      • Naclerio R.M.
      Eosinophilia in chronic childhood sinusitis.
      Our present study suggests that the CRS pathological type in Chinese children is significantly different from those of Caucasian children. Moreover, eosinophilic inflammation is more prevalent in the young CRSwNP group (48%) than the old CRSwNP group (22%) in our study, which is opposite from the inflammation pattern observed among Caucasian children.
      • Chan K.H.
      • Abzug M.J.
      • Coffinet L.
      • Simoes E.A.F.
      • Cool C.
      • Liu A.H.
      Chronic rhinosinusitis in young children differs from adults: a histopathology study.
      Moreover, we found that non-eosinophilic infiltration accounts for most inflammation patterns (>60%) among children with CRSwNP or CRSsNP. This is consistent with the results of a study by Cao et al, which involved Chinese adults with CRS.
      • Cao P.-P.
      • Li H.-B.
      • Wang B.-F.
      • et al.
      Distinct immunopathologic characteristics of various types of chronic rhinosinusitis in adult Chinese.
      However, the neutrophilic inflammation in all types of pediatric CRS is very minor compared to that in adult CRS.
      • Snidvongs K.
      • Sangubol M.
      • Poachanukoon O.
      Pediatric versus adult chronic rhinosinusitis.
      To exclude the problem of antibodies, we tested 2 types of antibodies from different suppliers (RD BAF3174 and abcam9535) a, and arrived at similar results. These differences between Chinese adult and pediatric populations with CRS may be attributed to genetics and environmental factors.
      An increased Th2/Th17 response was found in eosinophilic CRSwNP rather than in non-eosinophilic CRSwNP. Wang et al. found that adult CRSwNP in Beijing presented mixed patterns of Th1/Th2/Th17 expression, whereas subjects from Chengdu present with a lower Th2 expression.
      • Wang X.
      • Zhang N.
      • Bo M.
      • et al.
      Diversity of T cytokine profiles in patients with chronic rhinosinusitis: a multicenter study in Europe, Asia, and Oceania.
      Interestingly, Baba et al. found elevated Th2 cytokine levels in eosinophilic CRS without Th17 cytokines in the Japanese population.
      • Baba S.
      • Kagoya R.
      • Kondo K.
      • Suzukawa M.
      • Ohta K.
      • Yamasoba T.
      T-cell phenotypes in chronic rhinosinusitis with nasal polyps in Japanese patients.
      Our previous study also found that an eosinophilic shift of diffuse rhinosinusitis inflammatory pattern in southern China over the last 18 years.
      • Luo X.
      • Xu Z.
      • Zuo K.
      • et al.
      The changes of clinical and histological characteristics of chronic rhinosinusitis in 18 years: Was there an inflammatory pattern shift in southern China?.
      These pathological differences among similar populations could possibly be attributed to environmental factors.
      Our qualitative (IHC) and quantitative (ELISA) analyses suggest that pediatric CRSwNP (regardless of age) presented as Th1/Th2 inflammation, whereas CRSsNP presented as Th1/Th17 inflammation. Moreover, Th1/Th2/Th17 inflammation was more prevalent in the young CRSwNP group than the old CRSwNP group, which may be explained by the mutual enhancement of Th2 and Th17 inflammation. Moreover, the high Th2 and Th17 cytokine levels in the young CRSwNP group may be also explained by the higher eosinophilic inflammation in this group. Consistently, when the subjects were grouped according to the proportion of eosinophils, we also found that both eosinophilic CRS and CRSwNP presented as Th2/Th17 inflammation.
      There are several limitations to this study. First, the small sample size used in this study remains as a limitation towards conducting further analysis. Further multi-center studies and larger sample that compare CRS are therefore needed. Second, we did not perform IHC in the control group due to the limited availability of samples. Third, the effect of Th cytokines on dispersed nasal polyp cells was not explored in this study.
      In conclusion, this study showed for the first time that pediatric CRSwNP presents as eosinophilic with Th2/Th17 inflammation, whereas CRSsNP presents as Th1/Th17 inflammation. With age, an increasing number of pediatric CRSwNP cases present as non-eosinophilic inflammation, which is similar to the inflammation pattern in adult CRS. Our study may provide a theoretical basis for the precise treatment of pediatric CRS in the future.

      Abbreviations

      EOS, Eosinophils; NEU, Neutrophils; CRSsNP, Chronic rhinosinusitis without nasal polyps; CRSwNP, Chronic rhinosinusitis with nasal polyps; ELISA, Enzyme-linked immunosorbent assay; HE, Hematoxylin and eosin.

      Declaration

      All the authors consent to the publication of this manuscript.

      Ethical statement

      The study approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University ([2016]096) and informed consent was obtained from the children's guardians.

      Consent for publication

      Not applicable.

      Data availability statement

      All data generated or analyzed during this study are included in this published article and its supplementary information files.

      Funding

      This study was funded by grants from the National Natural Science Grant of China (No. 81970861 ), the Guangdong Province Natural Science Grant (No. 2021A1515010940 ), and the Science and Technology Program of Guangzhou (No. 202102020079 ).

      Authors' contributions

      Study design: Wenlong Liu, Jianbo Shi; experiment: Lijie Jiang, Yinhui Zeng, Zhaoqi Huang; data collected and analysis: Lijie Jiang, Yinhui Zeng, Zhaoqi Huang, Qingxiang Zeng; manuscirpt drafting: Wenlong Liu and Lijie Jiang.

      Declaration of competing interest

      The authors declare that they have no relevant conflicts of interest.

      Acknowledgments

      Not applicable.

      Appendix A. Supplementary data

      Fig. S1
      Fig. S1Representative graphs of HE-stained sinus mucosal tissue (original magnification: 400 × ) from pediatric CRSsNP or CRSwNP. (A) CRSsNP, incomplete epithelium and more submucosal mucous glands. (B) CRSsNP, intact epithelium and less submucosal mucous glands. (C-E). The eosinophils in the CRSsNP (C), young CRSwNP (D) and old CRSwNP (E) groups. Abbreviations: CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps; HE, hematoxylin and eosin
      Fig. S2
      Fig. S2Representative graphs of immunohistochemistry for Th1/Th2/Th17 cytokine staining (original magnification: 200 × ) in the CRSsNP, young CRSwNP, and old CRSwNP groups. Abbreviations: CRSsNP, chronic rhinosinusitis without nasal polyps; CRSwNP, chronic rhinosinusitis with nasal polyps

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