Elevation of activated neutrophils in chronic rhinosinusitis with nasal polyps

Abstract

Background: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is well characterized by type 2 (T2) inflammation characterized by eosinophilia in Western countries. However, the presence and roles of neutrophils in T2 CRSwNP are poorly understood.

Objective: We sought to clarify accumulation and inflammatory roles of neutrophils in CRSwNP in a Western population.

Methods: Sinonasal tissues and nasal lavage fluids were obtained from control patients and patients with CRS, and neutrophil markers were determined by ELISA. The presence of neutrophils in tissue was determined by flow cytometry. The gene expression profiles in neutrophils were determined by RNA sequencing.

Results: A neutrophil marker elastase was selectively elevated in nasal polyp (NP) tissue, whereas eosinophilic cationic protein (an eosinophil marker) was elevated in both uncinate and NP tissues of CRSwNP patients. Nasal lavage fluid myeloperoxidase (another neutrophil marker) was also significantly elevated in CRSwNP compared to control patients. Neutrophil markers were more greatly elevated in CRSwNP patients with recurrent disease. Flow cytometric analysis confirmed that neutrophil numbers were significantly elevated in NPs compared to control tissues. RNA sequencing analysis found that 344 genes were >3-fold and significantly elevated in NP neutrophils compared to peripheral blood neutrophils. Gene Ontology analysis suggested that the elevated genes in NP neutrophils were significantly associated with activation. Results suggest that neutrophils are accumulated in T2 NP tissues and that accumulated neutrophils are highly activated and contribute to inflammation in NPs.

Conclusions: Neutrophils may play a heretofore unrecognized meaningful role in the pathogenesis of CRSwNP in Western countries and may be a potentially important therapeutic target in T2 CRSwNP.

Keywords: Neutrophils; chronic rhinosinusitis; endotype; nasal polyps; type 2 inflammation.

Figure 1.. Elevation of a neutrophil marker in nasal polyps.

ECP and NE in uncinate tissues (UT) from control (n=32), CRSsNP (n=42) and CRSwNP (n=38) and NP tissues (n=61) were determined by ELISA. NP samples were further divided into no prior (n=47) and revision (n=14) surgery cases (C). The Spearman rank correlations were assessed by matched NP tissue samples (B, n=61). Results are shown as mean ± SEM. * p<0.05, *** p<0.001, **** p<0.0001 by one-way ANOVA (A) and the Mann-Whitney test (C).

Figure 2.. Elevation of a neutrophil marker in nasal lavage fluids from CRSwNP.

ECP and MPO in nasal lavage fluids from control (n=29) and CRSwNP (n=57) were determined by ELISA. CRSwNP samples were further divided into no prior (n=33) and revision (n=24) surgery cases (C). The Spearman rank correlations were assessed by matched CRSwNP nasal lavage samples (B. n=57). Results are shown as mean ± SEM. * p<0.05, **** p<0.0001 by the Mann-Whitney test (A and C).

Figure 3.. Neutrophils are elevated and activated in NPs.

A representative gating strategy for eosinophils and neutrophils within the live CD45⁺ population is shown in A. We collected inferior turbinate tissues from control patients (Control, n=12) and NP tissues from patients with CRSwNP (NP, n=21) and the numbers of eosinophils and neutrophils in sinus tissues were normalized by mg of tissue (B). The Spearman rank correlation was assessed by matched NP tissue samples (C, n=21). Representative histograms of flow cytometric plots for CD62L, the levels of CD62L on neutrophils by gMFI (geometric mean fluorescence intensity) ratio between CD62L and fluorescence minus one (FMO) control and the frequency of CD62 negative neutrophils in peripheral blood (PB, n=16), control inferior turbinate (Control, n=8) and NPs (n=7) were shown in D. Results are shown as mean ± SEM. ** p<0.01, *** p<0.001, **** p<0.0001 by the Mann-Whitney test (B) and one-way ANOVA (D).

Figure 4.. Bulk RNA-Seq in neutrophils.

A representative cell sorting strategy for neutrophils within the live CD45⁺CD117⁻FcεRI⁻ population is shown in A. The hierarchical clustering was performed using 20 neutrophil specific genes in the RNA-Seq result of human peripheral blood cells from the Human Protein Atlas (http://www.proteinatlas.org) with RNA-Seq result of our neutrophil samples (9 PB and 6 NP) (B). The hierarchical clustering was performed using >2-fold significantly up-regulated (110 genes) or down-regulated (161 genes) genes in CRSwNP PB neutrophils (n=5) compared to control PB neutrophils (n=2) in the bulk RNA-Seq data (C).

Figure 5.. Elevated genes in NP neutrophils are associated with activation.

A Venn diagram identified that 261 and 265 genes were >3-fold significantly elevated in NP neutrophils compared to all peripheral blood (PB) neutrophils (control PB neutrophils + CRSwNP PB neutrophils) and control PB neutrophils, respectively, and hierarchical clustering was performed using these 344 genes in the RNA-Seq data (A). The top 10 GO biological processes associated with elevated genes in NP neutrophils are shown (B). The hierarchical clustering was performed using elevated genes in NP neutrophils associated with a GO term, neutrophil activation (GO:0042119) (C). The expression levels were shown by TPM (transcripts per million) in RNA-Seq data and results are shown as mean ± SEM. * p<0.05, ** p<0.01 by one-way ANOVA (D).

Figure 6.. Elevation of activation markers and innate receptors in NP neutrophils.

The expression of activation markers (A, B) and innate pattern recognition receptors and inflammasome components (C) in control PB neutrophils (Control, n=4), CRSwNP PB neutrophils (CRSwNP, n=5) and NP neutrophils (NP, n=6) was determined by RNA-Seq. We also compared total PB neutrophils (PB, n=9 [4 control PB + 5 CRSwNP PB]) and NP neutrophils in B. The expression levels were shown by TPM (transcripts per million) and results are shown as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 by one-way ANOVA (A, C) and the Mann-Whitney test (B).