Chronic rhinosinusitis (CRS) is an inflammatory disease of the nasal cavity and paranasal sinuses mucosa that has complex etiopathogenesis and can cause significant physical, emotional and economic burdens for patients and health system [1]. Based on our research at Dr. Saiful Anwar general hospital in Indonesia, CRS cases requiring surgery account for around 38% of all patients admitted to the rhinology division [2]. In recent years, many attempts have been made to describe CRS in terms of endotypes, each defined by different molecular mechanisms and identified by matching biomarkers [1,3].

Based on integrated care pathway European Position Paper on Rhinosinusitis and Nasal Polyps (EPOS) 2020 for primary diffuse CRS, to differentiate between type 2 or non-type 2 endotypes, laboratory examinations are required. Type 2 endotype is indicated by increased immunoglobulin E (IgE) and eosinophilia [1]. Studies have suggested that environmental and genetic factors can also influence inflammatory endotypes in CRS. There is a marked global heterogeneity in inflammatory endotypes among patients with CRS without nasal polyps (CRSsNP) or CRS with nasal polyps (CRSwNP) among the Asian and Caucasian CRS [4], so we need to determine whether the criteria of clinical endotype of CRS listed in EPOS can be applied in our patients. With the development of biologic agents, especially in the cases of “difficult to treat” CRS, the differentiation between endotypes clinically becomes imperative. The implementation of endotype-based therapy is expected to lead to better disease management.

It is known that peripheral blood eosinophils and total serum IgE can be potential markers of type 2 inflammatory endotype CRS, although the results are various [1,4,5]. The eosinophil-neutrophil ratio (ENR), which is the ratio of peripheral blood eosinophil derivatives, is a new biomarker of systemic inflammation that shows a comparison between eosinophil and neutrophil values [6]. ENR values have been widely studied in relation to eosinophilic asthma and allergic rhinitis [7], but their role in CRS is still limited. ENR can show the dominance of inflammation cells without having to determine the exact cut off point in asserting the main inflammation cells in CRS. Thus, the main focus of this study was to analyze the relationship between CRS endotype and peripheral blood eosinophil values, ENR, and total serum IgE in patients diagnosed with CRS.

This was a cross-sectional analytic observational study between December 2021-October 2022 in Otorhinolaryngology Head and Neck Surgery Department. This study was approved by the Saiful Anwar General Hospital ethical committee (No. 400/025/K.3/102.7/2023). The inclusion criteria were patients diagnosed with CRS based on EPOS 2020 with age ≥18-years old without previous history of nasal or sinus surgery. Patients without any other diseases such as eosinophilic granulomatosis with polyangiitis, sinonasal tumor, cystic fibrosis, primary ciliary dyskinesia, autoimmune, and hematologic diseases, CRS which does not only involve the posterior group of paranasal sinuses, does not use systemic or local corticosteroids and/or systemic antibiotics within 4 weeks before Functional Endoscopic Sinus Surgery (FESS), and the patient had laboratories tested for complete blood count, differential count, and total serum IgE 30 days before FESS included in this study. The patients who do not meet the inclusion criteria are being excluded as the subjects of this research. The control groups are adult patients aged ≥18 years, diagnosed with nasal turbinate hypertrophy and septal deviation and eligible for surgical intervention (e.g., turbinoplasty, septoplasty). Patients with allergic rhinitis and patients who used corticosteroids or antibiotics within 4 weeks before specimen collection were excluded from the control group of this study. The control group in this study was only to determine the primary CRS endotype within the study group.

The study found that CRS was found to be affected patients in their productive age with the age range between 18 to 65 years old [1]. Our research found a similar mean age of patients affected with CRS for 36.08±3.0 years old. With the increase in age, the production of the S100 family protein was decreased which causing the cell proliferation, repair, and epithelial defense to be impaired and lead to the increased risk of abnormal microbial colonization following the chronic inflammation [9]. In this study, we found that the prevalence of CRS was more dominant in female (57.7% vs. 42.3%). CRS prevalence according to gender as a whole is still a debate, but epidemiology studies reported female tend to experience CRS two times higher rather than males [1,2,10], meanwhile other studies found higher prevalence on males [11]. From the available data, heterogeneity of sex prevalence in CRS cases was obtained which was influenced by several factors such as environmental, hormonal, and geographical [10,11]. According to EPOS 2020 [1], the prevalence of cardinal symptoms in CRS patients with and without polyps who are going to undergo surgery, the most common complaints are nasal blockage/congestion and the lowest complaints of facial pain are in CRSwNP [1]. In this study, similar results were obtained, complaints of nasal blockage/congestion were more dominant in non-type 2 endotypes, while headaches were lowest in type 2 endotypes. The SNOT-22 is now regarded as the most appropriate instrument in the evaluation of health-related quality-of-life impairment in CRS patients [1]. The SNOT-22 can be stratified into ‘mild’ being defined on the SNOT-22 score as 8-20, ‘moderate’ as >20-50 and ‘severe’ as >50 [1,12]. The overall severity rating of symptoms is obviously highly dependent upon the population being studied [1,12]. Patients in secondary care awaiting surgery report mean symptom severity scores in the moderate to severe range [1,12], similar results were obtained in this study with the mean total score 32.35±8.3, which is included in the moderate severity level. In this study we obtained nasal polyp more prevalent on type 2 CRS endotype. However, it should be emphasize that not all of nasal polyps are related or equals with type 2 inflammation and vice versa.

In determining the endotype, this study used the same biomarkers as those used in the study by Stevens, et al. [8]. Type 1 was characterized by the detection of IFN-γ, type 2 CLC, and/or ECP, while type 3 IL-17A. Based on their study, there was a higher percentage of type 2 than non-type 2 [8]. In contrast, our study finds that there was a higher percentage of non-type 2 than type 2. These suggested that there was a difference in environmental factors and genetic influences between countries that are thought to be the basis for variations in the types of mucosal inflammation in CRS [4,8,13,14].

This study determining the endotype without using the typical definition of CRS based on the eosinophilic count. The classification of type 2 CRS is characterized by eosinophilic inflammation, whereas non-type 2 is neutrophilic [15]. However, the definition of CRS based on the eosinophilic count might be prone to bias and not reflecting the actual activation of eosinophils in CRS. Moreover, the protein biomarkers such as ECP, major basic protein and eotaxin were considered to be more accurate in determining the degranulated or eosinophil infiltration compared to eosinophilic count [16]. This is also supported by other study conducted by Maharani, et al. [17] showing that there was no significant association between ECP endotype biomarker and the type of tissue inflammation based on predetermined cut-offs. The ECP hold a potential as a biomarker for type 2 for its association with eosinophilic type of inflammation. We propose using ECP as the main biomarker to identify type 2 CRS. The CRS endotyping based on the tissue inflammation response, which may be influenced by various factors, should be replaced with more specified biomarkers [17].

The gold standard for establishing a CRS endotype is by analyzing the gene or protein expression in the patient’s sinonasal tissues. Several genes and proteins have been studied which are the best markers for identifying the type of mucosal inflammation of CRS [4,8,13]. The use of these biomarkers is very limited due to invasive sampling, biomarker measurements that are not included in routine investigations in hospitals, and high costs that will be incurred by patients before surgery. Therefore, establishing the endotype using other practical parameters is currently the focus of CRS research, such as this study.

In this study, there was no statistically significant difference in peripheral blood eosinophil count (p>0.05) between endotypes, but there was a tendency for the percentage of eosinophils in the peripheral blood to be higher in type 2 endotypes. Unlike the result of our research, others have evidence that suggests that peripheral blood eosinophils are a relevant and reliable biomarker for eCRS with or without nasal polyps. However, it’s use as a replacement marker for tissue eosinophilia remains limited [4,5,18]. Gitomer, et al. [19] found no correlation between blood eosinophils and tissue eosinophils, because local eosinophil actions at the tissue level such as activation and migration often occur without an increase in blood eosinophils [19]. Peripheral blood eosinophil counts may also be artificially increased by parasitic infections, comorbid allergies, autoimmune disorders, or drug side effects [20]. In addition, because the nose is a small organ with minimal impact on the blood compartment, there is a high risk of an undiagnosed type 2 immune reaction on the basis of haematological parameters alone [20].

Many studies have been carried out on the ratio of blood parameters by various researchers [6,21,22]. The ENR, which is the ratio of peripheral blood eosinophil derivatives, is a new biomarker of systemic inflammation that shows a comparison of eosinophil and neutrophil values [6]. ENR values have been widely studied related to eosinophilic asthma [7] and allergic rhinitis [23,24], but their role in CRS is still limited. Study on ENR in CRS with using nasal polyp (NP) tissue samples was already conducted by Golebski, et al. [25] and Shaghayegh, et al. [26] Previous studies has found that NP on CRS patients are characterized by an eosinophilic inflammation with high tissue eosinophilia, high IL-5 and IgE, Th2-polarized responses, and an enrichment of ILC2s [25]. Golebski, et al. found a positive correlation between ILC2s and the ENR in NP, suggesting that ILCs are involved in the recruitment of eosinophils and neutrophils and that ILC2s in CRSwNP activate eosinophils. Other study by Shaghayegh, et al. [26] found that NP or mucosal samples had eosinophils to neutrophils ratio that significantly higher in CRSwNP patients compared to CRSsNP (p<0.0001) and controls (p=0.0009). This finding, indicates the presence of an ongoing immune response dominated by type-2 inflammation [26]. However, study on ENR in CRS using blood samples was first studied by Bayer, et al. [6] studied whether hematological indices of the peripheral blood are associated with revision surgery in patients with CRS undergoing endoscopic sinus surgery (ESS) and found by univariate analysis that increasing ENR values were associated with high ESS revision procedure (p<0.001) [6].

Based on previously reported studies, we are interested in examining the blood ratio of eosinophils and neutrophils in CRS and see its relationship with type 2 and non-type 2 CRS endotypes based on the inflammatory biomarkers CLC, ECP, IFN-γ, and IL-17A. The present study of ours is the first one to report the possible relationship between CRS endotypes based on EPOS 2020 (type 2 and non-type 2) using biomarkers of type 2 inflammation (CLC and ECP) and non-type 2 (IFN-γ, IL-17) with the blood eosinophils-neutrophils ratio. Significant results on the ENR value in this study, can also be explained that the variable measurement level with a ratio is a better measurement value than relative numeric value. The ratio can show how big or small a quantity is when compared to the others, in this case the proportion of eosinophil values increases when compared to the neutrophil values in patients with type 2 endotype CRS. It can be concluded that the ENR value can be an indicator/index of peripheral blood of eosinophil derivatives beneficial in type 2 endotype CRS.

In this study, there was no statistically significant mean difference (p>0.05) between total serum IgE and CRS endotypes. It is widely understood that locally produced IgE is a more potent pathophysiological cause of disease than systemic IgE in CRS [27-29]. It is hypothesized that this is due to local class switching to IgE in the pathogenesis of CRS and not from the systemic circulation. Total serum IgE, positive skin prick test, or previous immunoassay for serum specific IgE did not show a significant association with elevated tissue IgE [29]. Local class switching to IgE and local IgE production have also been shown to be associated with Staphylococcus aureus which plays an important role as a disease modifier in CRSwNP and asthma, by releasing enterotoxins. Staphylococcus aureus enterotoxin (SE) has been shown to act as a superantigen that activates T-cells polyclonally, releases Th2 cytokines and amplifies eosinophilic inflammation, and B-cells can induce polyclonal IgE and immunoglobulin G (IgG)/IgG4 production. Other effects of SE on local inflammation are inhibition of Treg cells, decreased eosinophil apoptosis, and induction of chemokines from epithelial cells [30]. Research by Zhang, et al. [30] found that tissue mast cell reactivity in CRS patients with nasal polyps when exposed to allergens and the presence of specific IgE against inhalant allergens or Staphylococcal enterotoxin B (SEB) was associated with tissue, but not with serum. Polyclonal IgE induced by the SEB superantigen contributes to persistent inflammation by continuously activating mast cells [30].

The difference in the results of this study with other studies can also be related to differences in inflammatory endotypes in Indonesia and around the world [4,8,13,14]. The environmental and geographical influences shown by the diversity of inflammatory endotypes in various countries play a role in increasing the complexity of diagnosis and implementation of endotype-based CRS therapy. How environmental, genetic factors, or a combination of both may affect the development of type 1, type 2, or type 3 inflammatory endotypes, will ultimately influence the role of endotype association with blood eosinophils, ENR, and total serum IgE.

The limitation of this study is our samples are CRS cases with more severe inflammation who had failed to respond to medication. We don’t have the data in those who had less severe inflammation and does not require surgery. As a result, the conclusions from this study are based on a patient population with moderate to severe disease. For the direction we want to conduct similar research that includes outpatient CRS with a larger number of samples.

In conclusion, there’s a relationship between ENR values and CRS endotypes, meanwhile no relationship was found between values of peripheral blood eosinophil and total serum IgE with CRS endotypes. ENR value ≥0.06 can be used in screening and diagnosing clinically of endotype type 2 of CRS.