Allergy Asthma Immunol Res.  2020 Jan;12(1):42-55. 10.4168/aair.2020.12.1.42.

Elastase-Positive Neutrophils Are Associated With Refractoriness of Chronic Rhinosinusitis With Nasal Polyps in an Asian Population

• Kim DK
• Kim JY
• Han YE
• Kim JK
• Lim HS
• Eun KM
• Yang SK
• Kim DW

• KMID: 2462571
• DOI: http://doi.org/10.4168/aair.2020.12.1.42

Abstract

PURPOSE
Various immune cells, including eosinophils and neutrophils, are known to contribute to the development of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the current understanding of the role of neutrophils in the development of CRSwNP still remains unclear. Therefore, we investigated risk factors for refractoriness of CRSwNP in an Asian population.
METHODS
Protein levels of 17 neutrophil-related mediators in nasal polyps (NPs) were determined by multiplex immunoassay, and exploratory factor analysis using principal component analysis was performed. Immunofluorescence analysis was conducted to detect human neutrophil elastase (HNE) or myeloperoxidase (MPO)-positive cells. Tissue eosinophilic nasal polyp (ENP) and tissue neutrophilia (Neu(high)) were defined as greater than 70 eosinophils and 20 HNE-positive cells, otherwise was classified into non-eosinophilic nasal polyp (NENP) and absence of tissue neutrophilia (Neu(low)).
RESULTS
In terms of disease control status, NENP-Neu(low) patients showed the higher rate of disease control than NENP-Neu(high) and ENP-Neu(high) patients. Linear by linear association demonstrated the trend in refractoriness from NENP-Neu(low) to NENP-Neu(high) or ENP-Neu(low) to ENP-Neu(high). When multiple logistic regression was performed, tissue neutrophilia (hazard ratio, 4.38; 95% confidence interval, 1.76-10.85) was found as the strongest risk factor for CRSwNP refractoriness. Additionally, exploratory factor analysis revealed that interleukin (IL)-18, interferon-γ, IL-1Ra, tumor necrosis factor-α, oncostatin M, and MPO were associated with good disease control status, whereas IL-36α and IL-1α were associated with refractory disease control status. In subgroup analysis, HNE-positive cells and IL-36α were significantly upregulated in the refractory group (P = 0.0132 and P = 0.0395, respectively), whereas MPO and IL-18 showed higher expression in the controlled group (P = 0.0002 and P = 0.0009, respectively). Moreover, immunofluorescence analysis revealed that IL-36R⁺HNE⁺-double positive cells were significantly increased in the refractory group compared to the control group. We also found that the ratio of HNE-positive cells to α1 anti-trypsin was increased in the refractory group.
CONCLUSIONS
Tissue neutrophilia had an influence on treatment outcomes in the Asian CRSwNP patients. HNE-positive cells and IL-36α may be biomarkers for predicting refractoriness in Asians with CRSwNP. Additionally, imbalances in HNE and α1 anti-trypsin may be associated with pathophysiology of neutrophilic chronic rhinosinusitis.

Keyword

Neutrophils; leukocyte elastase; interleukin 36; risk factors; rhinitis; sinusitis; nasal polyps; Asians

MeSH Terms

Asian Continental Ancestry Group*
Biomarkers
Eosinophils
Fluorescent Antibody Technique
Humans
Immunoassay
Interleukin 1 Receptor Antagonist Protein
Interleukin-18
Interleukins
Leukocyte Elastase
Logistic Models
Nasal Polyps*
Necrosis
Neutrophils*
Oncostatin M
Peroxidase
Principal Component Analysis
Rhinitis
Risk Factors
Sinusitis
Biomarkers
Interleukin 1 Receptor Antagonist Protein
Interleukin-18
Interleukins
Leukocyte Elastase
Oncostatin M
Peroxidase

Figure

• 

  Fig. 1 Treatment outcomes according to tissue eosinophilia and neutrophilia in CRSwNP. (A) Distribution of NP tissues according to the tissue eosinophilia and tissue neutrophilia (B) Comparison of disease control status according to the endotype of NP tissues based on tissue eosinophilia and neutrophilia. NP, nasal polyp; ENP, eosinophilic nasal polyp; NENP, non-eosinophilic nasal polyp; Neuhigh, tissue neutrophilia; Neulow, without tissue neutrophilia.

• 

  Fig. 2 Biplots for neutrophils-associated markers according to disease control status. Arrows are displayed for neutrophils-associated markers against the first 4 principal components. The dots are displayed for individual patients, and they are divided into three colors based on their disease control status; the refractory group consists of the partly controlled and uncontrolled patients. (A) Neutrophils-associated markers against the first 2 components. (B) Individual patients categorized by their disease control status against the first 2 components. (C) Neutrophils-associated markers against the third and fourth 2 components. (D) Individual patients categorized by their disease control status against the third and fourth 2 components. FAMD, factor analysis of mixed data; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; MPO, myeloperoxidase; MMP, matrix metallopeptidase; CXCL, chemokine (C-X-C motif) ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; OSM, oncostatin M; DCS, disease control status.

• 

  Fig. 3 Phenotypes of neutrophils in the refractory NPs. (A) Representative images of HNE-positive cells, MPO-positive cells, and IL-36R-positive cells (B) Ratio of double positive (HNE and IL-36R) cells/HNE-positive cells. Cell nuclei were counterstained with DAPI (blue). NP, nasal polyp; HNE, human neutrophil elastase; MPO, myeloperoxidase; IL, interleukin; DAPI, 4′,6-Diamidino-2-phenylindole dihydrochloride.

• 

  Fig. 4 Imbalance of neutrophil elastase and α1 anti-trypsin in the refractory nasal polyps. (A) Concentration of α1 anti-trypsin between the controlled and the refractory groups. (B) The ratio of human elastase-positive cells/α1 anti-trypsin between the controlled and the refractory groups.

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