Microparticles in nasal lavage fluids in chronic rhinosinusitis; potential biomarkers for diagnosis of Aspirin Exacerbated Respiratory Disease

Abstract

Background

Microparticles (MPs) are submicron sized shed membrane vesicles released from activated or injured cells and are detectable by flow cytometry. MP levels have been utilized as biomarkers to evaluate cell injury or activation in patients with pathological conditions.

Objective

To compare MP types and levels in nasal lavage fluids (NLFs) from controls and patients with chronic rhinosinusitis without nasal polyps (CRSsNP), CRS with NP (CRSwNP) and aspirin exacerbated respiratory disease (AERD).

Methods

We collected NLFs from CRSsNP (n=33), CRSwNP (n=45), AERD (n=31), and control (n=24) subjects. Standardized flow cytometry methods were used to characterize the following MP types; endothelial MPs, epithelial MPs (EpCAM(+)MPs, E-cadherin(+)MPs), platelet MPs (CD31(+)CD41(+)MPs), eosinophil MPs (EMR1(+)MPs), mast cell MPs (FcεRI(+)c-kit(+)MPs) and basophil MPs (CD203c(+)c-kit(−)MPs). Basophil activation was evaluated by the MFI of CD203c (CD203cMFI) on basophil MPs.

Results

Activated mast cell MPs (CD137(+)FcεRI(+)c-kit(+)MPs) were significantly increased in NLFs compared to controls in CRSsNP (2.3-fold, p<0.02), CRSwNP (2.3-fold, p<0.03) and in AERD (7.4-fold, p<0.0001). Platelet MPs (3.5-fold, p<0.01) and basophil MPs (2.5-fold, p<0.05) were increased only in AERD. CD203cMFI on MPs was increased in CRSwNP (p<0.002) and AERD (p<0.0001), but not CRSsNP. EpCAM(+)epithelial MPs in CRSwNP were no different from control (p= 0.91) and lower than those in CRSsNP (p<0.02) and AERD (p<0.002).

Conclusion

Based on released MPs, mast cells, platelets, and basophils were more highly activated in AERD than in CRS. Epithelial injury was lower in CRSwNP than in CRSsNP and AERD. MP analysis may help identify phenotypes of CRS, and in distinguishing AERD from CRSwNP.

Introduction

Chronic rhinosinusitis (CRS) is an inflammatory disease of the nose and paranasal sinuses commonly divided into a form with nasal polyps (CRSwNP) and a form without nasal polyps (CRSsNP). There is a particularly severe form of CRSwNP in which subjects have comorbid asthma and sensitivity to aspirin and other COX1 inhibiting drugs referred to as Aspirin Exacerbated Respiratory Disease (AERD). However, there is still an ongoing debate about the underlying pathological mechanisms of this phenotype of disease. Distinguishing AERD subjects from CRSwNP subjects is important because asthma exacerbations secondary to aspirin sensitivity have high morbidity in AERD and because the pathophysiology of these two phenotypes appears to be distinct^{1, 2}. Diagnosis based on patients’ recall of a history of aspirin sensitivity is not always reliable. It was reported that 15–34 % of rhinosinusitis subjects with asthma were unaware of their aspirin intolerance until they developed symptoms during a monitored aspirin challenge, and up to 15% of asthmatic subjects who reported a history of aspirin intolerance had negative aspirin challenge test results ^{1, 3, 4}. However, performing aspirin challenges to diagnose AERD are not routinely performed in a clinical setting given the length of time needed to perform the challenge and the potential risk of inducing a severe reaction. New biomarkers are needed to clarify the diagnosis and pathophysiology of CRSsNP, CRSwNP and AERD, especially to objectively distinguish AERD from CRSwNP.

Microparticles (MPs) are extracellular vesicles ranging in size from 100 to 1000 nm that are released by shedding from the plasma membrane of various cell types, especially when they are activated or undergo apoptosis in response to injury⁵. Growing evidence has suggested that the levels and markers of released MPs are biomarkers of the degree of injury, activation, and apoptosis of cells from which they are derived in various diseases. For example, endothelial MPs are increased in subjects with disorders such as cardiovascular diseases^6–8, renal failure⁹, metabolic diseases¹⁰, and COPD¹¹ in response to endothelial injury. The release of E-selectin endothelial MPs is an indicator of endothelial activation, whereas Annexin V positive PECAM positive endothelial MPs have been used as an indicator of endothelial apoptosis^{12, 13}. MPs are distinct from exosomes that are smaller extracellular vesicles released from cells through exocytosis of multivesicular bodies.⁵ In the case of MPs, the markers expressed on the cell of origin are expressed on the membrane of the MPs since they are formed by blebbing of plasma membrane. Thus, MP analysis can be used to evaluate the type of cells that are injured and the state of activation or apoptosis of injured cells. Assay of MPs has been shown to have significant value in quantifying the degree of cell injury and activation in clinical samples ^{8, 14}.

We report here the successful use of MP assessment of nasal lavage fluids (NLFs) to evaluate the pathophysiology of CRS using flow cytometry with a standardized MP gating strategy^{15, 16}. Disruption of the epithelial barrier has been speculated to initiate chronic inflammation in CRS^17–19. Eosinophils and mast cells have been reported to be involved in the pathophysiology of CRS^{20, 21}. Elevated basophil counts were reported in polyps of subjects with CRSwNP²². Abnormal platelet activation was reported in AERD^{23, 24}. Our present study demonstrates that measuring MPs in NLFs may be an effective tool to study pathogenesis of CRS, showing consistency with many of these previously published findings. Furthermore, we demonstrate that some MP types have potential use as biomarkers that distinguish AERD subjects from CRSwNP subjects independently of comorbid asthma and ongoing steroid use.

Methods

Patient Population

See Online Repository.

Nasal lavage fluid sampling and processing

See Online Repository.

Measurement of MPs in nasal lavage by flow cytometry

MPs in NLFs were measured by flow cytometry as previously described¹¹. NLF samples were incubated with each specific antibody (Table E1) were analyzed using a BD FACS LSRII flow cytometer and BD FACS DIVA software version 8.0 (BD Biosciences, Erembodegem Belgium). The MP gate was determined using Megamix plus SSC beads (BioCytex, Marseille, France) with a standardized MP gating strategy ^{15, 16}(Fig. E1). The gating strategies of previously unstudied MP types were verified using positive control MPs isolated from culture supernatant of each cell type or plasma (Fig. E8). MP levels were evaluated as absolute MP numbers per one microliter of NLF (/μl NLF) using flowcount beads. Preliminary experiments showed that our MP assay has good repeatability (Fig. E7). Further details are provided in the Online Repository materials.

Characterization of MPs

We quantified MPs from seven different cell types, including some previously unstudied MP types (epithelial MPs, eosinophil MPs, mast cell MPs, and basophil MPs), with activation or apoptosis markers, for a total of 16 different data sets as shown in Table 1. MPs were defined as particles that ranged from 0.1 to 1.0 μm in size and were positively labelled with specific antibodies. Epithelial MPs (E-cadherin(+)MPs: E-cad+EpiMPs, EpCAM(+)MPs: EpCAM+EpiMPs), eosinophil MPs (EMR1(+)MPs: EoMPs), mast cell MPs (FcεRI(+)c-kit(+)MPs: MCMPs) and basophil MPs (CD203c(+)c-kit(−)MPs: BasoMPs) are abbreviated as shown and were defined using cell specific markers as indicated and previously reported^25–30. Activated eosinophil MPs (CD69(+)EoMPs: ActEoMPs) and activated mast cell MPs (CD137(+)MCMPs: ActMCMPs) were defined as previously reported^31–37. Basophil activation was evaluated using CD203c mean fluorescence intensity (CD203cMFI) on basophil MPs. We did not use CD63, another basophil activation marker, because platelets express CD63 and adhere to leukocytes in AERD subjects ³⁸. Apoptotic eosinophil MPs (Annexin V(+)EoMPs: ApoEoMPs), apoptotic basophil MPs (Annexin V(+)BasoMPs: ApoBasoMPs) and apoptotic or degranulated mast cell MPs (Annexin V(+)MCMPs: AV+MCMPs) were defined using Annexin V. Further details are provided in the Online Repository.

Table 1.

Definition of MPs

MP type	Abbreviation	Definition
Endothelial MPs	Endothelial MPs
E-selectin endothelial MPs	ActEndoMPs	E-selectin(+)MPs
PECAM endothelial MPs	EndoMPs	CD31(+)CD41(−)MPs
Epithelial MPs	EpiMPs
EpCAM EpiMPs	EpCAM+EpiMPs	EpCAM(+)MPs
E-cadherin EpiMPs	E-cad+EpiMPs	E-cadherin(+)MPs
Platelet MPs	PltMPs	CD31(+)CD41(+)MPs
Eosinophil MPs	EoMPs	EMR1(+)MPs
Activated EoMPs	ActEoMPs	CD69(+)EMR1(+)MPs
Apoptotic EoMPs	ApoEoMPs	Annexin V(+)EMR1(+)MPs
Mast cell MPs	MCMPs	FceRI(+)c-kit(+)MPs
Activated MCMPs via IgE pathway	ActMCMPs	CD137(+)MCMPs
Apoptotic or degranulated MCMPs	AV+MCMPs	Annexin V(+)MCMPs
Basophil MPs	BasoMPs	CD203c(+)c-kit(−)MPs
Activated basophil	CD203cMFI	CD203c MFI on BasoMPs
Apoptotic BasoMPs	ApoBasoMPs	Annexin V(+)BasoMPs
Annexin V positive MPs	AV+MPs	Annexin V(+)MPs
Erythrocyte MPs	ErythroMPs	CD235a(+)MPs
Membrane dye positive MPs	FM+CFSE+MPs	FM(+)CFSE(+)MPs

MFI; Mean fluorescence intensity, FM; lipid-FM specific dye, CFSE; protein-

Carboxyfluoresceinsuccinimidyl ester (CFSE) specific dye

Statistics

All data were analyzed using non-parametric methods unless stated otherwise. A p-value of less than 0.05 was considered significant. Further details are provided in the Online Repository.

Results

Subject demographics

The demographics of the subjects in this study are shown in Table 2. There were no significant differences in age, gender, ethnicity, atopy or past smoking status among the groups. In addition, there were no significant differences in the distribution of comorbid atopy among CRSsNP, CRSwNP, and AERD subjects. The proportion of comorbid asthma was significantly higher in CRSwNP than in CRSsNP (p<0.03), and was significantly higher in AERD than in CRSsNP (p<0.0001) or CRSwNP (p<0.0001). Furthermore, when compared with CRSsNP, the proportion of oral steroid use was significantly higher in AERD than in CRSsNP (p<0.002) or CRSwNP (p<0.05), but there was no significant difference in the prevalence of intranasal steroid use.

Table 2.

Demographics of the 133 subjects

	Control (n=24)	CRSsNP (n=33)	CRSwNP (n=45)	AERD (n=31)
Gender, male/female	16/8	17/16	30/15	14/17
Ethnicity, White/non-White	19/5	29/4	34/11	22/9
Age (years), mean ± standard deviation	44.5±14.2	42.1±15.0	46.9±11.3	46.7±11.7
Smoking status, never/ex-smokers 1)	21/3	25/8	34/11	23/8
Comorbidity
Asthma (%)	-	8 (24%)	22 (49%)*	31 (100%)*,#
Atopy (%)	5 (21%)2)	16 (49%)	28 (62%)	20 (65%)
Steroid use
Oral (%)	-	0 (0%)	4 (9%)	8 (25%)*#
Dose (prednisone mg/day), median [Range]	-	-	7.5 [5–10]	7.5 [5–30]
Intranasal (%)	-	6 (18%)	9 (20%)	8 (26%)
Fluticasone propionate	-	6	6	5
Mometasone furoate	-	0	3	2
Dexamethasone	-	0	0	1

¹⁾Current smokers were excluded and all subjects stopped smoking more than 6 months before enrollment.

²⁾None were on medication for atopy at the time of enrollment.

^*p< 0.05, vs. CRSsNP,

^#p< 0.05, vs. CRSwNP

Comparisons of endothelial MP (EndoMP), and epithelial MP (EpiMP) levels in NLFs

EndoMPs (PECAM endothelial MPs) were measured as a marker for endothelial injury^9,8. ActEndoMPs (E-selectin endothelial MPs) were measured as a marker for endothelial activation^{7, 11, 12, 39, 40}. Compared with control, ActEndoMPs were significantly increased in CRSsNP (1.8-fold vs. control median, p<0.02), CRSwNP (1.7-fold, p<0.03), and AERD (3.2-fold, p<0.0001) (Fig. 1.A). EndoMPs were also significantly increased in CRSsNP (1.7-fold, p<0.002), CRSwNP (1.4-fold, p<0.03) and AERD (1.6-fold, p<0.0005) (Fig. 1.B). EpCAM is located on the basolateral side of epithelial cells, whereas E-cadherin is localized in adherens junctions⁴¹. Loss of intercellular junctions and cellular detachment from basement membranes are observed during epithelial injury^{42, 43}. We measured both EpCAM+EpiMPs and E-cad+EpiMPs to evaluate degrees and conditions of epithelial injury. Compared with control, EpCAM+EpiMPs were significantly increased in CRSsNP (1.6-fold, p<0.05) and AERD (2.4 fold, p<0.001) (Fig. 1.C) but were not increased in CRSwNP (p= 0.91). Levels in CRSwNP were lower than in CRSsNP (p<0.02) and AERD (p<0.0003). E-cad+EpiMPs were significantly increased in CRSsNP (2.9-fold, p<0.0003), CRSwNP (1.5-fold, p<0.05) and AERD (2.3-fold, p<0.0001) (Fig. 1.D). Similarly, E-cad+EpiMPs in CRSwNP were significantly lower than in CRSsNP (p<0.004) and AERD (p<0.002).

Fig. 1. Comparison of MPs among CRSsNP, CRSwNP, AERD, and control subjects.

(A) ActEndoMPs, (B) EndoMPs, (C) PltMPs, (D) EpCAM+EpiMPs, (E) E-cad+EpiMPs, (F) EoMPs, (G) ActEoMPs, (H) ApoEoMPs (I) MCMPs, (J) ActMCMPs, (K) AV+MCMPs, (L) BasoMPs, (M) ApoBasoMPs *; p< 0.05, **; p< 0.01, ***; p< 0.001

Comparisons of platelet MP (PltMP) levels in NLFs

Platelet MPs are released from activated platelets, associated with phosphatidylserine externalization, and regarded as markers for platelet activation^44–46. Compared with control, PltMPs were significantly increased only in AERD (3.5-fold, p<0.0003) (Fig. 1.E).

Comparisons of eosinophil MP (EoMP), mast cell MP (MCMP), and basophil MP (BasoMP) levels in NLFs

When compared with control, EoMPs were significantly increased in CRSsNP (1.8-fold, p<0.0001), CRSwNP (1.6-fold, p<0.0001) and AERD (1.9-fold, p<0.0001) (Fig. 1.F). ActEoMPs were also significantly increased in CRSsNP (2.2-fold, p<0.01), CRSwNP (2.3-fold, p<0.03) and AERD (3.1-fold, p<0.001) (Fig. 1.G). ApoEoMPs were significantly increased in CRSsNP (1.6-fold, p<0.0001), CRSwNP (2.2-fold, p<0.0001) and AERD (2.4-fold, p<0.0001) (Fig. 1.H). Compared with control, MCMPs were significantly increased only in AERD (4.3-fold, p<0.002) and were not increased in CRSsNP or CRSwNP (Fig. 1.I). In contrast, ActMCMPs were significantly increased in CRSsNP (2.4-fold, p<0.03), CRSwNP (2.4-fold, p<0.02), and AERD (7.4-fold, p<0.0002) (Fig. 1.J). AV+MCMPs were significantly increased only in AERD (4.9-fold, p<0.0003) (Fig. 1.K). When compared with control, BasoMPs were significantly increased only in AERD (2.5-fold, p<0.05) (Fig. 1.L). However, CD203cMFI (the mean fluorescence intensity of CD203c on BasoMPs) was increased in both CRSwNP (p<0.002) and AERD (p<0.0001) (Fig. 2). ApoBasoMPs were increased only in AERD (2.3-fold, p<0.006) and were not increased in either CRSsNP or CRSwNP (Fig. 1.M).

Fig. 2. Comparison of CD203c mean fluorescence intensity (MFI) on BasoMPs among CRSsNP, CRSwNP, AERD, and control subjects.

*; p< 0.05, **; p< 0.01, ***; p< 0.001

Comparisons of Annexin V positive MP (AV+MP), erythrocyte MP (ErythroMP), and membrane dye positive MP (FM+CFSE+MP) levels in NLFs

When compared with control, the total level of Annexin V(+)MPs (AV+MPs), regardless of cell type of origin, was significantly increased in CRSwNP (1.4-fold, p<0.02) and AERD (1.4-fold, p<0.02) but not in CRSsNP (Fig. E2.A). When compared with control, ErythroMPs were not increased in CRSsNP, CRSwNP or AERD subjects (Fig. E2.B). To observe the trends of total MP levels in NLFs, we measured membrane dye positive MPs (FM(+)CFSE(+)MPs: FM+CFSE+MPs) as previously described⁴⁷. When compared with control, FM+CFSE+MP levels were significantly increased in CRSsNP (1.6-fold, p<0.0007), CRSwNP (1.3-fold, p<0.003) and AERD (2.0-fold, p<0.0001). In addition, FM+CFSE+MP levels were higher in AERD than in CRSsNP (p<0.005) and CRSwNP (p<0.0003) (Fig. E2.C).

Receiver Operating Characteristic (ROC) curve analysis of MP levels for distinguishing AERD from CRSwNP

We assessed the feasibility of using levels of MPs as biomarkers to distinguish subjects with AERD from subjects with CRSwNP (45 CRSwNP and 31 AERD subjects) using area under the curve (AUC) analysis. There were significant differences in levels of ActEndoMPs, PltMPs, EpCAM+EpiMPs, E-cad+EpiMPs, MCMPs, ActMCMPs, AV+MCMPs, BasoMPs and ApoBasoMPs between CRSwNP and AERD. The results of this analysis are summarized in Table 4. There were significant differences in distribution of oral corticosteroid use and asthma between the two groups. The multivariate logistic analysis showed that the following MP types had cut-off values that were significantly associated with AERD, independent of asthma and oral steroid use (Table E2): cut-off values of PltMPs, 206/μL (AUC 0.77, sensitivity 68%, specificity, 84%) (Fig. 3.A); EpCAM+EpiMPs, 3687/μL (AUC 0.75, sensitivity 52%, specificity 91%) (Fig. 3.B); E-cad+EpiMPs, 1027/μL (AUC 0.72, sensitivity 74%, specificity 64%) (Fig. 3.C); MCMPs, 854/μL (AUC 0.70, sensitivity 74%, specificity 64%); ActMCMPs, 424/μL (AUC 0.71, sensitivity 58%, specificity 80%); AV+MCMPs, 192/μL (AUC 0.72, sensitivity 81%, specificity 60%) (Fig. 3.D); and ApoBasoMPs, 0.69/μL (AUC 0.69, sensitivity 61%, specificity 73%).

Table 4.

Receiver operating characteristic (ROC) curve analysis of MP levels for distinguishing AERD from CRSwNP

	Cut-off value	AUC [95% CI]	Sensitivity	Specificity
ActEndoMPs	2079	0.68 [0.54–0.79]	52	80
PltMPs	206	0.77 [0.64–0.86]	68	84
EpCAM+EpiMPs	3687	0.75 [0.63–0.85]	52	91
E-cad+EpiMPs	1027	0.72 [0.59–0.82]	74	64
MCMPs	854	0.70 [0.57–0.81]	74	64
ActMCMPs	424	0.71 [0.57–0.82]	58	80
AV+MCMPs	192	0.72 [0.59–0.82]	81	60
BasoMPs	6869	0.64 [0.50–0.76]	32	91
ApoBasoMPs	338	0.69 [0.56–0.81]	61	73

Fig. 3. Receiver operating characteristic (ROC) curve of PltMP, EpCAM+EpiMP, E-cad+EpiMP, AV+MCMP levels for distinguishing AERD subjects from CRSwNP subjects.

(A) PltMPs, (B) EpCAM+EpiMPs, (C) E-cad+EpiMPs, (D) AV+MCMPs

Comparisons of MP levels between CRSwNP subjects without asthma and CRSwNP subjects with aspirin tolerant asthma, and comparison between CRSwNP subjects with aspirin tolerant asthma and AERD subjects

Although the proportion of females was significantly higher in CRSwNP without asthma than CRSwNP with aspirin tolerant asthma (ATA) (p<0.03), there was no significant difference in the other patient demographic endpoints between these two groups (Table E5). There was no significant difference in levels of any MP types between CRSwNP without asthma and CRSwNP with ATA (Table E6). There was no difference in the patient demographic endpoints between CRSwNP with ATA and AERD. Even when compared with the more narrowly defined group of CRSwNP with ATA, levels of PltMPs (p<0.0003), EpCAM+EpiMPs (p<0.0005), E-cad+EpiMPs (p<0.02), MCMPs (p<0.02), ActMCMPs (p<0.02), AV+MCMPs (p<0.02) and ApoBasoMPs (p<0.006) were still higher in AERD. There was no significant difference in levels of the other MP types between these two groups (ActEndoMPs: p=0.061, EndoMPs: p=0.588, EoMPs: p=0.386, ActEoMPs: p=0.241, ApoEoMPs: p=0.698, BasoMPs: p=0.165 and CD203cMFI on BasoMPs: p=0.116).

Discussion

This is the first study to evaluate the pathophysiology of CRS and AERD by analyzing released MP levels and types in NLFs. The findings are summarized in Table 3. MPs were detected in NLFs of both control and CRS subjects, and there were similarities as well as significant differences in MP types and levels comparing NLFs from subjects with CRSsNP, CRSwNP, and AERD. MPs contain various bioactive effectors originating from the parent cells that can actively modulate immune responses under both normal healthy and pathological conditions^{5, 48, 49}. Our results indicate that MPs were released from activated immune cells and injured structural cells into the nasal cavity under pathological and healthy normal conditions and may play roles in cell to cell communication in the nose and paranasal sinuses. Furthermore, we demonstrate that several MP types, have potential use as biomarkers to distinguish AERD from CRSwNP subjects independently of asthma and steroid intake, especially PltMPs (AUC 0.77, sensitivity 68%, specificity, 84%), EpCAM+EpiMPs (AUC 0.75, sensitivity 52%, specificity 91%), and AV+MCMPs (AUC 0.72, sensitivity 81%, specificity 60%). Current estimates of AERD prevalence among CRS patients and among the general population are variable due to variations in diagnostic methods and definitions¹. In this study, aspirin intolerance was diagnosed based on clinical history (reports from patients and/or in patient charts). However, clinical history alone may not be sufficient to diagnose all AERD subjects⁵⁰. As such, oral aspirin challenges remain the gold standard for confirming this disease ^{1, 3, 4}. Future analysis of MPs in a large cohort of subjects with challenge-confirmed AERD is essential to establish the usefulness of MP levels in NLFs as biomarkers. If validated in such a study, the use of MP may help to better estimate AERD prevalence and help standardize the diagnosis of AERD.

Table 3.

Comparisons to controls of MPs in nasal lavage among CRSsNP, CRSwNP and AERD

	Control	CRSsNP	CRSwNP	AERD
Endothelial MPs (EndoMPs)
ActEndoMPs	-	1.8-fold	1.7-fold	3.2-fold
	653 [243–3301]	1198 [283–4823]	1103 [223–7180]	2078 [401–10231]
EndoMPs	-	1.7-fold	1.4-fold	1.6-fold
	2206 [869–4657]	3634 [1495–6869]	3055 [1146–7924]	3588 [1990–8840]
Epithelial MPs (EpiMPs)
EpCAM+EpiMPs	-	1.6-fold	NS	2.4-fold
	1534 [266–3301]	2383 [403–9769]	1606 [343–6680]	3687 [463–8995]
E-cad+EpiMPs	-	2.9-fold	1.5-fold	2.3-fold
	586 [150–3182]	1669 [249–8202]	886 [146–3252]	1326 [404–8542]
Platelet MPs (PltMPs)	-	NS	NS	3.5-fold
	77 [0–535]	112 [0–450]	65 [0–874]	267 [12–2462]
Eosinophil MPs (EoMPs)	-	1.8-fold	1.6-fold	1.9-fold
	1867 [486–3017]	3342 [1136–7651]	2938 [1105–8100]	3577 [832–8154]
ActEoMPs	-	2.2-fold	2.3-fold	3.1-fold
	142 [0–1050]	312 [14–3306]	326 [20–1787]	443 [52–2373]
ApoEoMPs	-	1.6-fold	2.2-fold	2.4-fold
	589 [99–1005]	961 [276–2757]	1284 [260–3527]	1401 [60–3762]
Mast cell MPs (MCMPs)	-	NS	NS	4.3-fold
	461 [59–3912]	536 [35–4003]	557 [73–5275]	1989 [58–6176]
ActMCMPs	-	2.4-fold	2.4-fold	7.4-fold
	59 [0–1211]	143 [0–1558]	140 [0–1619]	437 [0–2407]
AV+MCMPs	-	NS	NS	4.9-fold
	138 [0–682]	145 [0–2175]	152 [0–1446]	675 [29–3521]
Basopil MPs (BasoMPs)	-	NS	NS	2.5-fold
	2786 [227–5274]	2825 [305–6953]	2519 [873–9578]	3497 [1289–18841]
CD203cMFI	-	NS	Increased	Increased
	1.35 [1.02–2.58] X103	1.30 [0.70–3.50] X103	1.91 [0.93–8.73] X103	2.56 [1.09–6.81] X103
ApoBasoMPs	-	NS	NS	2.3-fold
	162 [2–924]	161 [33–934]	195 [0–1708]	375 [0–3308]
Annexin V positive MPs (AV+MPs)	-	NS	1.4-fold	1.4-fold
	4358 [1684–8147]	5044 [2015–8316]	6242 [2437–15629]	6293 [2784–22001]
Erythrocyte MPs (ErythroMPs)	-	NS	NS	NS
	2154 [475–4556]	1547 [538–4405]	1732 [336–4198]	2186 [514–4181]
Membrane dye positive MPs (FM+CFSE+MPs)	-	1.6-fold	1.3-fold	2.0-fold
	20794 [13242–37119]	33913 [16302–53933]	28587 [12000–68764]	43271 [19218–77955]

All data of MP levels are presented as median [range].

NS; not significant vs. control

##-fold; significantly higher by ##-fold vs. control

Increased; significantly higher in MFI vs. control

Convincing evidence implicates epithelial barrier disruption in the pathogenesis of asthma, eczema and CRS^{19, 51}. However, there have been no reports evaluating the degree of epithelial injury in CRS and comparing CRSsNP, CRSwNP and AERD. In this study, both EpCAM+EpiMPs and E-cad+EpiMPs were significantly increased in CRSsNP and AERD when compared with control subjects (Fig. 1.C,D). E-cadherin is localized only in adherens junctions⁴¹. Thus, the increased E-cad+EpiMPs in the nasal cavity may reflect a loss of intercellular adherens junctions in CRSsNP and AERD as observed during epithelial injury⁴³. In contrast, EpCAM is located on the basolateral side⁴¹. The increased EpCAM+EpiMPs may reflect detachment of epithelial cells from basement membranes in CRSsNP and AERD as observed during epithelial injury⁴². Our results indicate that epithelial barrier disruptions are induced by loss of both intercellular junctions and cellular attachment to the basement membrane in CRSsNP and AERD. On the other hand, EpCAM+EpiMPs and E-cad+EpiMPs were both significantly lower in CRSwNP when compared with CRSsNP or AERD, indicating a lower degree of epithelial injury in CRSwNP than in CRSsNP or AERD. Interestingly, although E-cad+EpiMPs were significantly increased, EpCAM+EpiMPs were not increased in CRSwNP. Epithelial barrier disruption may be induced mainly by loss of intercellular junctions in CRSwNP. Variable epithelial barrier disruption may reflect heterogeneity of inflammatory mechanisms and histological changes among CRSwNP, CRSsNP and AERD.

The accumulation of eosinophils in polyps has been known for over a century^{22, 52, 53}. In our studies, ECP levels in NLFs were significantly increased in CRSwNP and AERD when compared with CRSsNP (Fig. E5), indicating that a high degree of eosinophil degranulation occurs in CRSwNP and AERD, but not CRSsNP. However, we were surprised to find that there was no significant difference in EoMP and ActEoMP levels among CRSsNP, CRSwNP and AERD (Fig. 1.F,G). Because eosinophils accumulate in most polyps, our results may indicate that the number of released EoMPs and ActEoMPs per eosinophil was higher in CRSsNP, suggesting that the degree of activation per eosinophil was higher in non-polypoid diseases than in polypoid disease. Eosinophils may be also activated in non-polypoid diseases without eosinophil accumulation and degranulation. Although correlations between ActEoMPs and ECP levels were weak (CRSwNP: r_s= 0.358, p<0.04, AERD: r_s= 0.111, p= 0.63), one recent article reported relationships between exosome release and eosinophil degranulation⁵⁴. Exosomes in eosinophils contain EPO, MBP, and ECP, and are secreted after induction of degranulation in vitro⁵⁴. In contrast with MP release, exosome secretion can occur both spontaneously and constitutively, and is controlled in different cell types by various environmental influences^{5, 54}. Eosinophil degranulation may be a specific feature of eosinophil activation under the microenvironment of polyps. Analysis of extracellular vesicles may improve our understandings and quantification of eosinophil activation in CRS.

Tryptase positive mast cells are increased in CRS, along with increased IgE levels in many cases, and have been implicated in pathogenesis^{21, 55}. ActMCMPs were increased 2-fold in CRSsNP and CRSwNP, and increased an impressive 7.4-fold in AERD when compared with control (Fig. 1.J). In addition, ActMCMPs showed strong correlations with ActEoMPs (Fig. E4). These results indicate that mast cells are profoundly activated in AERD subjects, more so than in CRSsNP and CRSwNP. Furthermore, the correlations of ActMCMPs with ActEoMPs suggest that the activation pathways for these two cells may be similar, or related. Interestingly, AV+MCMPs were significantly increased (4.9-fold) only in AERD and were not increased in CRSsNP or CRSwNP. Because phosphatidylserine externalization occurs in the process of mast cell degranulation⁵⁶, our results may indicate that mast cell activation, rather than apoptosis, is what we are detecting with this MP analysis. There was no significant difference in mast cell MP levels between AERD subjects with and without atopy (data not shown). Non-IgE dependent mast cell activation was also reported to play an important role in the pathophysiology of AERD⁵⁷. However, there are no established mast cell activation cell surface markers specific for IgE independent pathways. Discovery of markers unique to non-IgE dependent mast cell activation and evaluation of non-IgE pathway activation using MP assays could be useful in future studies.

In this study, MP findings indicate that basophils are not activated in CRSsNP, but are activated in CRSwNP and AERD (Fig. 2). This may be in concordance with the Type 2 cytokine pattern in CRSwNP and AERD. In addition, ApoBasoMPs were significantly increased only in AERD but not in any other subject group (Fig. 1.M), indicating that apoptosis is induced in basophils only in AERD. Furthermore, BasoMP levels in AERD were higher than in CRSwNP (Fig. 1.L), indicating that the degree of basophil activation is likely higher in AERD than CRSwNP. In a recent study, we reported lower basophil counts in polyps in AERD compared with CRSwNP and speculated that the basophils are likely migrating to AERD polyps and then become undetectable due to either degranulation or apoptosis²². The finding in the present study suggests that the highest level of activation and apoptosis in basophils occurs in AERD polyps, supporting this previous speculation.

PltMPs were significantly increased only in AERD, and not in the other phenotypes of CRS in this study (Fig. 1.E). In addition, PltMPs were strongly correlated with ActEoMPs in AERD (Table E3) (Fig. E3). Platelet MPs have previously been reported to be shed from activated platelets and are regarded as both effectors and biomarkers for platelet activation ^{44, 45}. Our results provide further support to the concept that platelets are activated in AERD subjects, even without treatment with COX1 inhibitor drugs. Laidlaw and Boyce reported that platelets adhere to eosinophils and neutrophils and can be found within nasal polyp tissue in AERD subjects to a greater extent than in CRSwNP³⁸. The present studies support their conclusion, and suggest that the high degree of platelet activation is unique to AERD among the types of CRS that we studied here. The use of MPs to assess the level of activation of platelets within NLF may provide an important new tool to study disease prevalence, progression and remission in AERD.

It should be noted that there are some interesting patterns of baseline MP levels in NLFs of control subjects. Although platelet MPs and endothelial MPs are the major populations of MPs in the plasma ^{58, 59}, baseline PltMP levels were very low in NLFs (about 4% of EndoMP). In addition, although basophils are rare populations in both the nasal cavity and blood, baseline BasoMP levels were the highest (as high as EndoMPs) among the MP types in NLFs. We also measured baseline BasoMPs and EndoMPs in the plasma (n=9), and found that baseline BasoMP levels were around 40% of the level of EndoMPs (Table E4). MP levels in NLFs and plasma reflect the balance between MPs that are released from the cells of origin and MPs that are taken up by target cells. We speculate that this balance may be different between the nasal cavity and the circulation, or among MP types. Differences in the rate of turnover among the cell types may also influence baseline levels of each MP type. Mechanisms of MP release and uptake in in vivo models have not been clarified yet and should require further study.

There are a number of limitations of this study. Although there was no difference in the prevalence of nasal steroid use among CRSsNP, CRSwNP and AERD, the number of subjects using oral corticosteroids in AERD was significantly higher than in the other forms of CRS. Glucocorticoids can induce apoptosis in immune cells such as eosinophils⁶⁰, and basophils⁶¹. However, there was no significant difference in levels of any MP types between AERD subjects with and without oral steroid use in this study (data not shown). The increased ApoEoMPs, and ApoBasoMPs may be specific features of AERD. In addition, platelet MPs in plasma were reported to be significantly increased in subjects with asthma, which is one of the triad of conditions defining AERD⁶². Although growing evidence has suggested that abnormal platelet activation is uniquely associated with the pathophysiology of AERD independently of asthma^{23, 24} further studies are necessary to clarify this point. However, PltMP levels in NLFs were higher in AERD than in CRSwNP with aspirin tolerant asthma, and the multivariate logistic analysis showed our cut-off values of platelet MPs, epithelial MPs, and mast cell MPs were significantly associated with AERD independently of asthma in this study. Furthermore, MP assay in NLFs is representative of inflammation in the entire nasal cavity and NLFs have not yet been isolated from specific anatomic structures within the nose and sinuses. Thus, it is impossible to clarify the parts of the nasal cavity from which MPs in NLF are originating. To address this weakness, we are now developing a new assay using a sponge based approach⁶³ to collect MPs released from specific anatomic structures in nasal cavity.

In conclusion, we found evidence for activation of endothelium, epithelium, mast cells, basophils, eosinophils and platelets in the various forms of CRS and also found clear differences among the phenotypes of disease. Furthermore, we found that some MP types, especially EpCAM+EpiMPs, PltMPs, and AV+MCMPs, are potential biomarkers for distinguishing AERD from CRSwNP in the clinic. Although platelet activation markers were reported to be potential diagnostic biomarkers for AERD in blood²³, MP analysis in NLF may make diagnosis of AERD more robust by evaluating both EpiMPs and AV+MCMPs in addition to PltMPs. Recently, type 2 innate lymphoid cells have been detected in nasal polyp tissues and interest has focused on their role in the pathophysiology of CRS^{64, 65}. At present, definitively detecting ILC2 requires a cocktail of several cell surface markers, making detection of ILC2-derived MP challenging. Developing reliable MP analysis of these cells is of great interest to us. We measured NLF samples collected from patients taken in the operating room in this study. However, in results not shown, we have recently found that MPs were also detectable in NLFs collected from patients during a routine clinical office visit. We believe that MPs in NLFs may become an invaluable tool in the study of CRS and AERD in the laboratory and provide new biomarkers for diagnosis and treatment of CRS in the clinic.

Key messages.

• Microparticles may be useful to assess the degree of cell injury, activation, and apoptosis relevant to the pathophysiology of CRS and AERD.

• Analysis of MP subsets suggests that mast cells, platelets, basophils and endothelial cells are all more highly activated in AERD than in CRS.

• Platelet MPs, epithelial MPs and mast cell MPs may have utility as biomarkers to distinguish AERD from CRSwNP in both clinical and research settings.

Abbreviations

CRS

Chronic rhinosinusitis

CRSsNP

CRS without nasal polyps

CRSwNP

CRS with nasal polyps

NP

Nasal polyps

AERD

Aspirin Exacerbated Respiratory Disease

MPs

microparticles

FcεRI

high-affinity IgE receptor

EpCAM

Epithelial cell adhesion molecule

EMR1

EGF-like module-containing mucin-like hormone receptor-like 1

MFI

mean fluorescence intensity

NLFs

Nasal lavage fluids

ActMPs

activated MPs

ApoMPs

apoptotic MPs

EndoMPs

endothelial MPs

EpiMPs

epithelial MPs

EoMPs

eosinophil MPs

MCMPs

mast cell MPs

BasoMPs

basophil

MPs FM

lipid-FM specific dye

CFSE

protein-Carboxyfluoresceinsuccinimidyl ester (CFSE) specific dye

ATA

Aspirin tolerant asthma