Utilization of Nasal Mucus to Investigate the Pathophysiology of Chronic Rhinosinusitis

Abstract

Background

Nasal mucus is proving to be a useful means by which to study the pathogenesis of chronic rhinosinusitis (CRS). Given the increase in publications examining nasal mucus and the lack of a review on this topic, we will focus on this noninvasive approach to studying CRS. Particular attention will be drawn towards inflammatory cytokines and biomarkers and their influence on disease severity.

Methods

A literature review of papers published in English pertaining to nasal mucus was performed using the PubMed database. The search utilized combinations of the following keywords: sinusitis, polyps, sample collection, nasal mucus, or nasal secretion. Studies solely on acute or bacterial sinusitis, allergic rhinitis, or cystic fibrosis were not included.

Results

A wide variety of materials and methods have been used to collect nasal mucus. Numerous assay types have been performed with the most common being ELISA, cytometric bead array, and proteomics. Most studies have focused on examining the levels of Th1/Th2 cytokines along with chemokines associated with type 2 immunity. Other factors identified include growth factors, senescence-associated proteins, complement, and antimicrobial defenses have also been identified. Nasal mucus cytokines have proven useful in cluster analysis and predicting postoperative improvement in Sino-nasal Outcome Test (SNOT-22) scores. One limitation of the use of nasal mucus is that some studies have suggested that nasal mucus does not always reflect the tissue microenvironment.

Conclusions

Nasal mucus represents a critical tool by which to examine the sinonasal microenvironment in a noninvasive manner. Unlike studies of tissue, it can be utilized in both surgically and medically managed patients and avoids the trauma of biopsies. However, studies are still needed to determine the most effective method for nasal mucus collection. Studies should also take care to confirm that nasal mucus markers do, in fact, reflect the levels of the product studied in the tissue.

Introduction

Much as bronchoalveolar lavage has helped shape our knowledge of the lung microenvironment, nasal mucus is transforming what we know about the pathology of chronic rhinosinusitis (CRS). Analysis of nasal secretions for allergic mediators has been researched since the early 1980s, with initial collection methods predominately being nasal lavage. ¹ Noninvasive methods have since expanded and are categorized as “bulk” surface fluid collection (i.e., nasal aspiration, nasal lavage/washing) or “focal” surface fluid collection (i.e., cotton wool, foam rubber, sponges, absorbent fibrous matrix). ² As research technology has been evolved, the experimental readouts available for use with nasal mucus have expanded greatly. In this review, we will discuss the methods by which nasal mucus from CRS patients is collected, how it has shaped our understanding of inflammatory markers and disease severity, and the strengths and limitations of its use for CRS research.

Literature Review Methodology

A literature review of papers published in English pertaining to nasal mucus was performed using the PubMed database and in accordance with the Preferred Reporting Items for Systemic Reviews and Meta-Analyses (PRISMA) guideline (Figure 1). ³ The search utilized combinations of the following keywords: sinusitis, nasal polyps, sample collection, nasal mucus, or nasal secretion. Studies solely reviewing healthy individuals, acute or bacterial sinusitis, allergic rhinitis, or cystic fibrosis were not included. Studies that utilized nasal mucus for review were included, those that solely utilized other ways (eg, sinonasal tissue biopsy, nasal scraping) were excluded. Included studies spanned from January, 2000 to March, 2021. A total of 747 articles were identified by the searching strategy. No duplicate studies were found within the PubMed database. After screening the title and abstracts and excluding studies without full text, 582 studies were removed. Upon review of 165 full-text articles for inclusion and exclusion criteria, 68 studies were identified. Tables 1 to 5 detail and summarize the mucus collection methods, assay procedures, and study results described in this article.

Figure 1.

PRISMA-compliant literature review diagram.

Table 1.

Nasal Mucus Collection Methods for Chronic Rhinosinusitis (CRS).

Technique	Advantages	Disadvantages	Ref.
Nasal Lavage	Noninvasive Inexpensive	Poor patient tolerance Over dilution High sampling error	4–9
Nasal Aspiration	Noninvasive Fast	Requires presence of mucus at time of suction Risk of mucosal trauma	10–16
Cotton Wool	Noninvasive Reproducible	Limited absorption compared to sponges	17–22
Absorbent Sponge (Polyurethane, Polyvinyl Alcohol, Sinus Pack, Pope Ear Wick)	Decreased sampling error High secretion and protein recovery Reproducible	Risk of mucosal trauma Placement limited by anatomy (eg, septal deviation, sere polyposis)	6,23–44
Leukosorb Paper	Noninvasive Decreased sampling error Highly specific for olfactory binding proteins Reproducible	Proprietary Technology Expensive Limited absorption compared to sponges	45–47

Table 2.

Nasal Mucus, Inflammatory Markers, and Disease Severity in Chronic Rhinosinusitis (CRS).

Year	Mucus collection method	Assay type used	Study findings	Ref.
2021	Leukosorb paper	LegendPlex Mix, Cytometric Bead Assay (CBA), & ELISA	OC biomarkers can serve as non-invasive measures to categorize CRS into various personalized inflammatory endotypes.	45
	Polyurethane sponge	Proteomics & mass spectrometry	268 uniquely expressed proteins were found in nasal mucus of CRS patients (97 unique to CRSsNP, 132 unique to CRSwNP, 39 common to both). CRS nasal mucus proteome had increased adaptive and innate immunologic pathways, dysfunctional metabolic processes and cell death, and increased tissue remodeling.	31
2020	Leukosorb paper	LegendPlex Mix, CBA, & ELISA	OC biomarkers were disproportionate in CRS versus healthy controls. VEGF-A and EGF had no statistically significant difference across all groups. IL-8 was highest in controls, and lowest in CRSsNP followed by CRSwNP. Both CRSwNP and CRSsNP had elevated biomarkers, but CRSwNP had significantly higher IL5 and IL13. The highest OC opacification scores correlated with increased IL5.	46
	Polyurethane sponge	Transepithelial electrical resistance (TER), ciliary beat frequency, & ELISA	Nasal mucus from healthy control patients demonstrated improved cellular adhesion, mucociliary function, and reduced intercellular permeability when compared to mucus from mixed CRS patients once applied to HNEC	27
	Nasal lavage	Proteomics and Western blotting	Apolipoprotein E and periostin are nasal epithelial cell-derived proteins that can serve as proinflammatory markers for CRS.	4
	Polyvinyl alcohol sponge	Proteomics and Western blotting	Patients with CRSwNP had significant upregulation of nasal mucus exosomal PAPP-A versus control patients.	48
2019	Cotton wool	Bioplex cytokine assay	AERD patients had a predominantly Th2 cytokine profile, which decreased during aspirin desensitization. Six months post aspirin desensitization there was an increase in pro-inflammatory and Th1 cytokines.	21
	Sinus pack	ELISA, Western blot	IL-37 levels are reduced in the nasal secretions of patients with eosinophilic CRSwNP but increased in tissues. TSLP in nasal mucus from eosinophilic CRSwNP is increased.	30
	Control— Pope ear wick CRS—Luken specimen trap	ELISA	IL-25 is elevated in sinonasal tissue and nasal mucus in patients with CRSwNP as compared to controls or CRSsNP.	49
	Polyvinyl alcohol sponge	Proteomic analysis of nasal mucus-derived exosomes	123 significantly different exosomal proteins were detected in the nasal mucus of CRSwNP patients compared to control patients.	29
2018	Polyvinyl alcohol sponge	Proteomics and whole transcriptome sequencing	Exosomal fibrinolysis and coagulation pathway proteomes exhibited strong inverse and significant correlations with the tissue findings.	43
	Polyvinyl alcohol sponge	Proteomic analysis of nasal mucus-derived exosomes	123 significantly different exosomal proteins were detected in the nasal mucus of CRSwNP patients compared to healthy controls.	29
	Polyurethane sponge	CBA	IL-5 and IL-13 were elevated in mixed CRS patients compared to controls. No significant difference was noted in IL-4 among all the groups. IL-5 was most significantly elevated in CRSwNP vs controls and CRSsNP.	28
	Polyurethane sponge	CBA	Nasal mucus type 1 and type 2 cytokines can be used to identify distinct clusters of CRS patients. Of 90 patients undergoing endoscopic sinus surgery, 6 cluster groups were formed.	26
2017	Pope ear wick	ELISA	The antimicrobial peptide dermcidin, previously only thought to be found in human sweat, was discovered in nasal mucus. Concentration based on CRS or control was not investigated.	14
2016	Polyvinyl alcohol sponge	ELISA	In comparison to healthy control patients, CRS patients had significantly elevated P-gp. CRSwNP patients had higher P-gp than CRSsNP.	50
2015	Aspiration	ELISA	Nasal mucus from 92% (78/85) of CRS patients was positive for MBP compared to 0% from healthy patients or patients with allergic rhinitis	9
	Polyurethane sponge	CBA	Plasma 25(OH)D3 deficiency is associated with increased sinonasal mucus bFGF and RANTES	25
	Aspiration	ELISA	Tissue factor pathway inhibitor and tissue factor levels were not significantly different in nasal mucus from CRSwNP as compared to controls. Thrombin was elevated in CRSwNP.	51
2014	Nasal lavage	ELISA, CBA, PCR	LPA in nasal mucus was higher in nasal secretions of CRSsNP and CRSwNP as compared to control subjects.	5
	Polyurethane sponge	CBA	Nasal mucus levels of IL-2, IL-4, IL-5, IL-10, and IL-17A, but not TNF-α, correlate with those in sinonasal tissues. CRSsNP had elevated IL-2, 4, 6, 10, and 17A and CRSwNP had elevated L-4, 6, 10, 17A, and TNF-α compared to controls.	24
	Cotton wool	ELISA	ECP levels were most elevated in CRSwNP and tryptase levels were most elevated in AR. However, there was no correlation between ECP and tryptase levels in nasal mucus when compared to tissue eosinophil and mast cell numbers, respectively.	20
	Medtronic sinus secretion collector	ELISA & radioimmunoassay	IL-6, MBP, MPO, and uric acid were significantly elevated during CRS exacerbation.	16
2011	Cotton wool	CBA	IL-5, IL-6, IL-10, and the Th1 cytokine IFN-γ were significantly higher in patients with CRSwNP and asthma compared with non-asthmatic CRSwNP.	19
	Medtronic Juhn TYM-TAP middle ear fluid aspirator	ELISA	Thrombin and TAT complex were significantly increased in the nasal secretion of patients with CRSwNP with asthma compared with the control group.	11
2010	Cotton wool	CBA	IL-5 levels are higher in nasal polyp patients with asthma and are associated with eosinophilic inflammation.	52
	Aspiration	ELISA	Major basic protein was present in nasal secretions of 87% (20/23) of patients with proven CRSwNP compared to 4% (1/21) of control subjects that presented with CRS-like symptoms.	12
2009	Aspiration	Proteomics & multiple reaction monitoring mass spectrometries	Identified 7 out of 10 innate and acquired immunity proteins shared in 6 CRSwNP and 6 control patients. IGLV4-3 deleted brain tumor protein, lysozyme C precursor, mucin B, and Lipocalin were over-expressed in CRSwNP while bactericidal protein and calgranulin were under-expressed in CRSwNP.	36
2007	Aspiration	Proteomics	Identified a total of 35 proteins shared between 4 CRSwNP and 4 control patients.	13
	Aspiration	Proteomics	Identified 83 proteins collected from olfactory nasal mucus of 16 healthy volunteers. Demonstrated that olfactory cleft nasal mucus is a feasible way to obtain biomarkers.	53
	Nasal lavage	Griess assay	Nitric oxide metabolite levels were higher in total CRS patients versus healthy controls. No statistically significant difference in NP between CRSwNP and CRSsNP	54
2005	Aspiration	ELISA	Staphylococcal exotoxins are present in the nasal mucus of patients with CRSwNP at higher rates than controls	15
	Polyurethane sponge	Bioplex cytokine assay	Nasal mucus levels of IL-5 and Ig-E were significantly elevated in CRSwNP versus CRSsNP.	33
2004	Cotton wool	UniCAP	Nasal tryptase was the highest in non-allergic CRSwNP versus controls, AR, NARES, and CRSsNP	18
2003	Sinus packs IVALON 4000 plus	ELISA	Nasal fluid collection via sinus pack represents a noninvasive tool by which to study sinonasal immunity	23
2002	Aspiration	ELISA	CRSsNP had elevated IL-8 versus CRSwNP. Lysozyme and sCD14 levels were the same.	10
2000	Cotton wool	ELISA	Nasal mucus levels of IL-5 and IgE were similar between nonallergic nasal polyposis and AR; soluble ICAM (neutrophil chemotactic) was only increased in chronic, nonallergic sinusitis; no significant correlation between concentrations of cytokines in mucus versus serum.	17

Table 3.

Nasal Mucus and Quality of Life Assessment in Chronic Rhinosinusitis (CRS).

Year	Mucus collection method	Assay type used	Study findings	Ref.
2020	Polyurethane sponge	CBA	Preoperative nasal mucus cytokines correlate with postoperative SNOT-22 scores; higher IL-5 predicted lower (improved) scores whereas higher IL-2 predicted higher (worse) scores.	34
2018	Polyurethane sponge	Proteomics	Proteomic and pathway analysis of nasal mucus from control subjects and CRSwNP patients found the top 2 identified pathways were the initial triggering of complement and the complement cascade. Other altered pathways included glycolysis and gluconeogenesis, pathogenic Escherichia coli infection, carbon fixation, pentose phosphate pathway, and cell communication. Nasal mucus C3 correlated with a more severe SNOT22 score.	37
	Polyurethane sponge	CBA	IL-4 correlated significantly with rhinologic symptom domains (SNOT-22) in mixed CRS patients, IL-6 correlated with worse emotional scores and general health (SF-8) and IL-21 had significant correlations with extranasal rhinologic symptom domains (SNOT-22) in CRSsNP.	35
	Polyurethane sponge	CBA	High IL-13 CRS patients correlated with significantly worse preoperative SNOT-22. No significant SNOT-22 correlations with low/high IL-5 or low IL-13 CRS patients.	28
	Polyvinyl alcohol sponge	ELISA	Higher P-gp secretions demonstrated significantly higher SNOT-22 scores and higher Lund-Mackay scores	50
2016	Nasal lavage	ELISA	IL-8 and HSP70 levels in nasal lavage fluid positively correlated with Lund-Mackay scores.	6
2015	Nasal lavage	ELISA	HMGB1 levels in nasal lavage fluid positively correlated with Lund-Mackay scores.	7
2014	Polyurethane sponge	CBA	IL-17A levels were reduced after endoscopic sinus surgery when using hydrodebrider sinus irrigation; no significant change was seen in TNF-A, IL-6, IL-10 nor symptom visual analog scale scores.	55
2009	Nasal lavage	Griess Assay	There was a positive correlation between NO nasal mucus levels and symptomatic visual analog scoring in CRS patients post FESS.	8

Table 4.

Nasal Mucus and Olfactory Dysfunction in Chronic Rhinosinusitis (CRS).

Year	Mucus collection method	Assay type used	Study findings	Ref.
2020	Polyurethane sponge	CBA	Worse TDI was associated with higher expression of type 1 and type 2 cytokines in CRS. Specifically, CRSwNP was associated with higher IL-4 and IL-5 and CRSsNP with higher TNF-a and IL-10.	40
	Leukosorb paper	LegendPlex Mix, CBA, & ELISA	OC biomarkers (CCL2, CCL3, IgE, IL5, IL6, IL9, IL10, and IL13) have a correlation with worse olfactory function. This was seen in CRSsNP and CRSwNP, indicating that localized tissue inflammation without OC obstruction from polyposis can contribute to olfactory dysfunction.	46
2019	Leukosorb paper	CBA	In CRSwNP patients, CCL2, IL-5, IL-6, IL-13, IL 10, IL-9, TNF-α, CCL5, and CCL11 were significantly correlated with olfaction. IL-6, IL 10, VEGF-A, and IgE correlated with OC opacification for CRSsNP, only CXCL5 correlated with olfaction and OC opacification.	47
2018	Polyurethane sponge	CBA	Biomarkers from the olfactory cleft resemble middle meatus. Elevations in IL-2, IL-5, IL-6, IL-10, and IL-13 are associated with reduced olfactory function in CRS patients.	41
2016	Polyurethane sponge	Cytometric bead array	IL-5 levels inversely correlated with TDI in patients with CRSwNP and CRSsNP; IL-6, IL-7, and VEGF-A positively correlated with TDI in CRSwNP patients.	38

Table 5.

Nasal Mucus and Treatment Studies in Chronic Rhinosinusitis (CRS).

Year	Mucus collection method	Assay type used	Study findings	Ref.
2020	Luken specimen trap	Mass spectrometry	Doxycycline and Roxithromycin antibiotic concentrations were significantly lower in nasal mucus in comparison to sinonasal tissue and systemic circulation in patients with CRS.	42
2019	Sinus packs IVALON 4000 plus	ELISA	Charcot-Leyden crystal concentration in nasal mucus reflects tissue eosinophil levels and can predict clinical response to glucocorticosteroids.	32
	Polyvinyl alcohol sponge	ELISA	Periostin, serpinF2, PAPP-A, and CST2 levels decreased after FESS and gradually increased despite medical treatment in CRSwNP patients. Patients requiring revision surgery had a higher increase in Periostin, PAPP-A, and CST2.	44
2018	Cotton wool	ELISA	Compared to controls, RANTES and Eotaxin levels were higher in the nasal secretions of CRSwNP, regardless of atopic status. Fluticasone furoate treatment reduces levels of both mediators Fluticasone furoate treatment reduce levels of both mediators.	22
	Polyurethane sponge	CBA	Subgroups of CRS patients with elevated IL-5 and IL-13 had higher rates of revision sinus surgery.	28
2015	Pope ear wick	Glucose colorimetric assay	CRS patients have independently elevated nasal mucus glucose levels that were not altered by topical or systemic corticosteroid usage.	56
	Polyurethane sponge	ELISA	Isotonic saline irrigation decreases innate sinonasal antimicrobial activity more significantly than “low-salt” or hypertonic saline.	57
2014	Polyurethane sponge	CBA	ESS with intraoperative hydrodebrider sinus irrigation lowered postoperative levels of IL-17A but did not alter IL-6, IL-10, or TNF-a. No clinical significance was noted in findings.	55
2012	Cotton wool	Flow Cytomix, UniCAPTM100	An eight-week course of 500 mg clarithromycin statistically reduced ECP levels in nonatopic CRSwNP, and IL-6 levels in atopic CRSwNP patients. Mean polyp size scores reduced in both groups posttreatment, regardless of atopic status.	58
2009	Nasal lavage	Griess Assay	Nitric oxide metabolite levels were decreased in CRS patients and increased to normal levels after FESS.	8
2002	Sinus packs	ELISA	Elevated TGF-B1, TGF-B2, EGF, and PDGF were noted in post-op-FESS patients for mixed CRS, whereas concentrations of EGF decreased	39
2000	Nasal lavage	ELISA	Following an 8-to-12-week course of clarithromycin, polyp shrinkage correlated with a significant reduction in IL-8 scores in patients with CRSwNP.	59

Abbreviations: AERD, aspirin-exacerbated respiratory disease; bFGF, basic fibroblast growth factor; CBA, cytometric bead array; CCL, C-C motif ligand; CCL2, chemokine ligand 2; CCL20, chemokine ligand 20; CDKN2A/p16INK4a, cyclin-dependent kinase inhibitor 2A; CRS, chronic rhinosinusitis; CRSsNP, CRS without nasal polyps; CRSwNP, CRS with nasal polyps; CXCL, C-X-C ligand; ECP, eosinophil cationic protein; ELISA, enzyme linked immunosorbent assay; ESS, endoscopic sinus surgery; GM-CSF, granulocyte macrophage colony-stimulating factor; HSP70, heat shock protein 70; sCD14, soluble CD-14; ICAM, intercellular adhesion molecules; IgE, immunoglobulin E; IL, interleukin; LPA, lysophosphatidic acid; MBP, major basic protein; MMP, matrix metallopeptidases; MPO, myeloperoxidase; OBP, odorant binding protein; QOL, quality of life; RANTES, regulated on activation, normal T cell expressed and secreted; SCF, stem cell factor; SNOT22, sinonasal outcomes test-22 questions; TAT, thrombin–antithrombin complex; TDI, threshold, discrimination, and identification; Th1/Th2, T-helper 1/T-helper 2; TNFa, tumor necrosis factor alpha; TIMP, tissue inhibitor of metalloproteinases; VEGF-A, vascular endothelial growth factor-A; PAPP-A, pappalysin-A; CST2, cystatin 2; P-gp, P-glycoprotein.

Nasal Mucus Collection Methods

Currently, no method is accepted as the “gold standard” by which nasal mucus should be collected. Most reports have used either cotton wool, absorbent sponges, or aspiration. Table 1 summarizes some of their reported advantages and disadvantages. Since it is easily accessible and generally nontraumatic, placement of cotton wool was among the first collection methods for CRS-related research and reproduced several times since.^{17–22,52,56} Though not as common due to expenses from proprietary technology, Leukosorb (Pall Scientific, Port Washington, NY) is another noninvasive filter paper with an excellent ability to limit dilution of soluble biomarkers, particularly from the olfactory cleft (OC), and withstand degradation in freezing or room temperature storage for at least 24 h.^2,45,60

Absorbent sponges (ie, polyvinyl alcohol sponge, polyurethane sponge) have also served as reliable collection methods.^{10,23–32,61} A 2004 study by Watelet et al, ²³ showed that absorbent sinus packs (IVALON 4000Plus, comprised of polyvinyl alcohol) yielded excellent mucus volume and protein recovery in vitro and in subsequent human models (control vs CRSwNP). In addition, there was a minimal day-to-day variation of growth factor concentrations in nasal mucus over a three-day sampling period of eight patients. Polyurethane sponges have also been found to have eight times the amount of key protein (e.g., tryptase, eosinophilic cationic protein) recovery and nonsignificant differences with sample evaporation at 1, 2, and 4 weeks when stored at various temperatures (4 °C, − 20 °C, and − 80 °C). ⁶² Although this was discovered in a review of allergic rhinitis patients, the findings have influenced polyurethane sponge usage in various CRS-related studies. To date, no study has compared side by side these sponges’ absorbency, protein, or volume retention properties.

Though not as routinely used anymore, aspiration and/or lavage methods have been utilized to collect nasal mucus. Initially, they were reported to optimize sinonasal fluid acquisition while being noninvasive to the patient, and reproducible for the examiner. ²³ However, patient compliance to nasal lavage is highly variable and lacks specific anatomical localization. ¹⁷ Furthermore, nasal collection methods from Klimek and Rasp has demonstrated wide variability in dilutions of nasal lavage specimens leading to the inability to determine an appropriate dilution factor. ⁶³

Nasal Mucus, Inflammatory Markers & Disease Severity

Some of the most studied aspects of nasal mucus have been inflammatory cytokines and their variations based on disease state; Table 2 summarizes all these findings. Nasal mucus in CRS patients was first evaluated by Kramer et al among allergy screen-tested patients with allergic rhinitis (AR), nonallergic (NA) CRSsNP, and NA CRSwNP. Within NA CRS patients, significantly elevated interleukin-5 (IL-5) and immunoglobulin-E (Ig-E) levels were only seen in NA CRSwNP. ¹⁷ Similar findings of upregulated T-cell type 2 (TH2) mediators (IL-4, IL-6, IL-10, IL-13, and IL-25) within CRSwNP have been replicated numerous times since, albeit only a few stratified by atopic status.^{18,19,24,28,33,47,49,52} This reinforces the early theory that Th2 pathway with increased cytokine concentrations may be key in the pathogenesis of CRSwNP, and potentially irrespective of underlying allergies.

While much has been described Th2 and type 2 cytokines association with CRSwNP, T-cell type 1 (TH1) cytokines have had a less defined role in CRSsNP within nasal mucus review studies. A variety of findings have noted both elevated, decreased, or similar levels of interferon-y (IFN-y), tumor necrosis factor (TNF), and IL-2 within CRSsNP in comparison to healthy subjects or CRSwNP.^33,38,46,64 Subsequently, has reinforced the increasing theory of heterogeneity within the inflammatory profile of CRSsNP patients.

Several chemokines and products of granulocytes in the nasal secretions have also been studied. A study in 2017 used ELISAs to reveal that the nasal mucus levels of RANTES and eotaxin were elevated in patients with CRSwNP, and treatment with fluticasone furoate decreased the levels of these chemokines. ²² The high RANTES levels in this study came in support of previous results published by our group, which showed that RANTES levels are elevated in the nasal mucus of patients with low plasma vitamin D, which in turn is associated with an increased risk of CRS. ²⁵ Thymic stromal lymphopoietin (TSLP) and lysophosphatidic acid (LPA) have also been demonstrated to be higher in patients with eosinophilic CRSwNP and within CRSwNP and CRSsNP patients compared to control patients, respectively.^5,30 Mediator major basic protein (MBP), which can trigger the release of histamine, was found in one study to be elevated in 87% of CRSwNP patients versus only 4% of controls. ¹² This observation was confirmed in another study that added that MBP was absent in the nasal mucus of patients with AR. ⁹ MBP was also found to be elevated in the nasal mucus during CRS exacerbations. ¹⁶ Early studies of eosinophilic cationic protein (ECP), another eosinophil-derived product in nasal mucus, described that it was elevated in AR, but not CRSsNP or nonatopic CRSwNP. ¹⁷ However, more recent studies have shown that the nasal discharge levels of ECP are significantly elevated in CRSwNP patients when compared to allergic rhinitis or controls. ²⁰ Furthermore, the same study showed that the mast cell marker tryptase was elevated in allergic rhinitis when compared to patients with CRS or control patients. Neither of these markers correlated with the number of eosinophils or mast cells in tissue biopsies, however. ²⁰ Despite that, this study poses that these granulocyte markers in nasal mucus may be utilized to distinguish between different upper airway diseases.

While the inflammatory factors discussed above are the ones most reviewed in the literature, several studies have analyzed the influence of separate proteins and metabolites within the nasal mucus of CRS patients. Proteins involved in cellular proliferation such as pappalysin-A (PAPP-A) and periostin (PST) have repeatedly been shown to be significantly higher among CRS patients versus controls.^29,44,48 P-glycoprotein, a transmembrane efflux pump that is upregulated in TH2 cells has additionally shown overexpression within CRS patients compared to control subjects.^29,50

Molecules involved in the coagulation cascade were also studied in the nasal mucus by ELISAs in two different studies.^11,51 While the levels of tissue factor and the tissue factor pathway inhibitor in the nasal mucus were comparable between CRSwNP patients and control patients, ⁵¹ thrombin and thrombin-anti-thrombin complexes were found in higher abundance in the nasal mucus of patients with CRSwNP with asthma when compared to control patients. ¹¹ Thrombin is thought to promote the inflammatory response to the upper airway diseases via its action on protease-activated receptors. ¹¹ Adding to the list of pro-inflammatory molecules, the high-mobility group box 1 (HMGB1) chromatin protein, which has been described as a player in CRS, has also been identified in nasal mucus ⁶⁵ ; in yet another study, this protein was shown to be correlated with the severity of inflammation in CRS. ⁷ Together, all these studies highlight the broad range of factors and their influences on disease severity that can be identified from nasal mucus.

Nasal Mucus & Quality of Life Studies

Correlations between inflammatory markers and validated quality of life (QOL) metrics is another area of research utilizing nasal mucus. Table 3 summarizes the findings currently published. In a prospective review utilizing the 22-item Sino-Nasal Outcome Test (SNOT-22) and 8-item short-form health survey (SF-8), Chowdhury et al noted interesting findings: IL-4 correlated significantly with rhinologic symptom domains (SNOT-22) in mixed CRS patients, IL-6 correlated with worse emotional scores and general health (SF-8) among CRSwNP, and IL-21 had significant correlations with extranasal rhinologic symptom domains (SNOT-22) in CRSsNP. ³⁵ In a separate study by the same group, higher preoperative IL-5 was associated with significant postoperative SNOT-22 improvement whereas higher IL-2 predicted postoperative worsening, among all CRS patients. ³⁴ While awareness of inflammatory markers associated with OD or QOL metrics is growing, several limitations (ie, larger randomized control trials, diurnal, and seasonal variations) must be addressed in future studies before inference of causality can be made.

Use of Nasal Mucus to Study Olfactory Loss

Nasal mucus has also proven to be useful in identifying factors related to olfactory dysfunction (OD) within CRS. Table 4 summarizes the most pertinent findings that have been published. Previous work by our group has shown that secreted chemokines and cytokines from the OC correlate with olfactory function testing, thus serving as clinically useful biomarkers for OD. ³⁸ In 2016, we noted a significant inverse correlation between IL-5 levels and objective olfactory function among all patients with CRS. Interestingly, IL-6, IL-7, and vascular endothelial growth factor A (VEGF-A) were positively correlated in patients with CRSwNP, such that increased levels were associated with improved olfaction. ³⁸ In a follow-up multiinstitutional study in 2019, we only noted a positive correlation between CRSsNP with VEGF-A. Meanwhile, there was a significant inverse correlation with C-C motif ligand 2 (CCL2), IL-5, IL-6, IL-13, IL-10, IL-9, TNF-α, CCL5, and CCL11 with olfactory scores in CRSwNP. ⁴⁷ These findings were supported in separate reviews, where elevations in IL-2, IL-5, IL-6, IL-10, and IL-13 by Wu et al, ⁴¹ and IL-4 and IL-5 by Han et al ⁴⁰ were associated with worse OD in CRSwNP. Although, Han et al ⁴⁰ noted worse OD in CRSsNP with elevated IL-10 and tumor necrosis factor-α (TNF-α). Of note, both studies measured inflammatory markers from the middle meatus, and Wu et al ⁴¹ observed that inflammatory markers in the OC directly correlated with levels obtained from the middle meatus. This suggests that the OC may not represent an exclusive inflammatory microenvironment as previously thought and may be more reflective of inflammatory changes within the whole sinonasal cavity.

Nasal Mucus and Treatment Studies

The analysis of biomarkers in nasal mucus has provided essential data in monitoring and tailoring treatment responses in CRS. With its objective measures, it can serve as an additional tool for assessing the actual impact of various therapeutic modalities. Table 5 summarizes the most pertinent medical and surgical findings available. Repeated large-volume, saline-based nasal irrigations are most often considered first-line treatment for CRS due to enhancement of mucociliary clearance. Interestingly, Woods et al noted that this may subsequently remove innate sinonasal immune defenses (eg, lysozyme, lactoferrin) and theoretically increase the risk for microbial colonization. However, these findings were only seen in isotonic and lactated ringers’ solutions. Meanwhile, hypertonic irrigations had no decrease in antimicrobial activity, and “low-salt” irrigations only had transient decreases with a return to baseline within 1 to 6 h. ⁵⁷

Inhaled corticosteroids also have a well-established role in treatment due to their impact on sinonasal mucosal inflammation. Spadijer Mirkovic et al ²² specifically noted significant decreases in RANTES and Eotaxin-2 levels in patients with CRSwNP who were solely treated with fluticasone furoate for 2 weeks. Glucocorticoid responsiveness can still be variable among all CRS patients, and a proportion exhibit steroid insensitivity. Wu et al ³² found that increased levels of Charcot Leyden crystals (CLCs) measured in nasal mucus had predictive significance in identifying glucocorticoid response in patients with CRSwNP. Apart from steroids, several studies have also identified decreasing IL-6, IL-8, and ECP levels correlated with a reduction in nasal polyp size following extended clarithromycin oral administration.^58,59

Following the failure of medical management, functional endoscopic sinus surgery (FESS) is the most accepted therapeutic option available. Watelet et al were among the first to demonstrate increases in various growth factors (eg, TGF-B1, TGF-B2, EGF, and PDGF) within nasal mucus of CRS patients versus controls in the acute postoperative period. ³⁹ Turner et al noted that CRS patients stratified with higher IL-5 and IL-13 levels had a greater number of revision surgeries. Additionally, an earlier pilot study from our group found decreased postoperative levels of IL-17A when an intraoperative hydrodebrider sinus irrigation system was used in CRS mixed patients. ⁵⁵ These findings combined show the impact nasal mucus may have in deciding aggressive upfront surgery as well as acute versus chronic postoperative management.

Strengths & Limitations to the Use of Nasal Mucus

Several questions remain about the ideal handling of nasal mucus including stability of sample storage and specificity. Limited research has been published on the duration of sample storage and temperature influences specific to CRS nasal mucus. ⁶² Nasal mucus is known to be a heterogeneous collection of proteins and cytokines (signal) admixed with high levels of proteases leading to subsequently degraded protein fragments (noise). ⁶⁶ While the addition of protease inhibitors, prompt transport of samples on ice, centrifugation in a refrigerated centrifuge, and storage at < −70 °C can reduce this degradation, some protein loss and decreased specificity may occur. In lieu of this, degradation-resistant exosomes containing proteomic and genomic sequences from sinonasal tissue have gained interest in CRS research. Utilizing polyvinyl acetate sponge mucus collection methods and ultracentrifugation with SOMAscan^tm proteomic array, Mueller et al ⁴⁸ have shown an improved signal-to-noise ratio over whole nasal mucus. In turn, exosomes may provide more resilient and specific biomarkers within nasal mucus for serial and prospective research.

Specific sites (eg, olfactory cleft, middle meatus) of nasal mucus collection and obtaining mucus reflective of sinonasal tissue should also be considered with their relationship to outcomes. Differences in nasal versus sinus mucins have been reported and as such, nasal lavage samples that contain both types of mucus may not accurately reflect the microenvironment of CRS. ⁶⁷ Furthermore, a recent study by our group has shown that levels of OBP, which is produced only by olfactory epithelium, are increased in nasal mucus collected from the OC versus that from the middle meatus. ⁶⁸ While recent studies have shown that Charcot-Leyden crystal concentration in nasal mucus reflects tissue eosinophil levels, ³² not everything found in nasal mucus reflects nasal tissue. For example, our group previously reported that while numerous type 1 and type 2 cytokines correlate between tissue and nasal mucus, TNF-α did not. ²⁴ Mueller et al ²⁹ also noted a poor correlation between nasal mucus and tissue proteomes. Of note, exosomes isolated from the nasal mucus were found to have significant overlap with tissue proteomes. Similarly, studies examining IL-37 found that while it was elevated in nasal tissue of CRSwNP patients, there was no difference in the amounts found in nasal mucus between control subjects and those with CRSwNP. ³⁰ As mentioned previously, a study using ECP as a biomarker for eosinophils and tryptase for mast cells, found no correlation with either of these cell types in tissue biopsy or cytobrush scrapings. ²⁰ Overall, these studies highlight that as new biomarkers are explored in nasal mucus, it will be important to confirm they in fact represent the sinonasal tissue microenvironment

Conclusions

Rhinology research is in an exciting time of tremendous growth and advancement. One tool helping to expand our knowledge in the field is the utilization of nasal mucus. To date, nasal mucus studies have already served as a useful tool in patient cluster analysis and identified new therapeutic targets from proteomic analyses. The future of allergy testing may also involve local sampling instead of systemic (serum) assays. For example, Immunocap© assay and allergen microarray have been shown to be successfully used for direct sampling of nasal mucus.^69,70 While the use of nasal mucus for biomarker research expands, the development of new nasal mucus biomarkers will need to be rigorously tested to confirm that the analytes in mucus markers truly reflect the tissue environment. With an ever-increasing number of biological medications available for the possible treatment of CRS, the nasal mucus may serve as a critical, noninvasive step in the development of personalized medicine.