Immunopathologic characteristics of Chinese pediatric patients with chronic rhinosinusitis

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.


INTRODUCTION
Chronic rhinosinusitis (CRS) is common in both the adult and child populations. 1,2 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%. 3 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. 4 The study by Coffinet et al found that lymphocytes were more prevalent in young children with CRS, rather than eosinophils. 5 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. 6 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. 7,8 CRSsNP is believed to be a Th1dominant inflammation, whereas Th2 and Th17 inflammations are observed in eosinophilic rather than in non-eosinophilic CRSwNP. [7][8][9] 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. 8 But now Delemarre et al reported that CRSsNP also with a moderate type 2 immune response showed a considerable eosinophilic inflammation with clinical impact. 10 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.

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. 11 All enrolled patients did not respond to maxima medical therapy or adenoidectomy. 12,13 Asthma or allergic rhinitis were diagnosed according to lung function and through allergen tests. 14,15 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. [16][17][18] 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). [18][19][20] The blood cell counts were performed using the LH-785 system (Beckman Coulter, Ireland).

Statistical analyses
The data were presented as mean AE 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 ¼ 24) and 13-18 y (n ¼ 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.

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.

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).

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 noneosinophilic CRSwNP account for 36.5% and 63.5% of the CRSwNP, respectively.

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. 4 Eosinophilic and neutrophilic inflammation are the two main CRS inflammatory types in both children and adults. 26 opposite from the inflammation pattern observed among Caucasian children. 4 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. 8 However, the neutrophilic inflammation in all types of pediatric CRS is very minor compared to that in adult CRS. 13 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 noneosinophilic 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. 25 Interestingly, Baba et al. found elevated Th2 cytokine levels in eosinophilic CRS without Th17 cytokines in the Japanese population. 30 Our previous study also found that an eosinophilic shift of diffuse rhinosinusitis inflammatory pattern in southern China over the last 18 years. 24 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.

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.