Chronic Rhinosinusitis Pathogenesis

Abstract

There are a variety of medical conditions associated with chronic sinonasal inflammation including chronic rhinosinusitis (CRS) and cystic fibrosis. CRS in particular can be divided into two major subgroups based upon whether nasal polyps are present (CRSwNP) or absent (CRSsNP). Unfortunately, clinical treatment strategies for patients with chronic sinonasal inflammation are limited, in part because the underlying mechanisms contributing to disease pathology are heterogeneous and not entirely known. It is hypothesized that alterations in mucociliary clearance, abnormalities in the sinonasal epithelial cell barrier and tissue remodeling all contribute to the chronic inflammatory and tissue deforming processes characteristic of CRS. Additionally, the host innate and adaptive immune responses are also significantly activated and may be involved in pathogenesis. Recent advancements in the understanding of CRS pathogenesis are highlighted in this review with special focus placed on the roles of epithelial cells and the host immune response in cystic fibrosis, CRSsNP and CRSwNP.

Introduction

Chronic rhinosinusitis (CRS) is characterized by chronic inflammation of the sinonasal mucosa and is clinically associated with sinus pressure, nasal congestion, rhinorrhea, and a decreased sense of smell persisting for greater than 12 weeks¹. CRS can be subdivided into 2 major categories based upon whether nasal polyps are present (CRSwNP) or absent (CRSsNP)². While CRS is estimated to affect over 10 million patients in the US and lead to $22 billion in total annual costs^{1, 3}, there are other diseases, such as cystic fibrosis, that also involve chronic sinonasal inflammation and nasal polyp formation that have important clinical implications. In order to advance the current diagnostic and treatment strategies available for affected patients, a better understanding of CRS pathogenesis is needed.

The Sinonasal Microbiome

Much like the gut, the sinonasal cavity has a resident flora that maintains an environment conducive to respiratory health. Substantial effort has recently been made using culture independent techniques, i.e. molecular diagnostics, to attempt to understand and define the microbial community or microbiome of the human sinonasal cavity in the healthy and diseased (CRS) states^4–10. No consistent patterns have emerged in the diseased state to implicate specific organism(s) as causative, but data suggest an imbalance, or dysbiosis, is found in CRS, with a decrease in microbial diversity. Another concept that has emerged is that the correct balance of microbes within the local microbiome may be immunomodulatory and that an imbalance shifts an important regulator of local inflammation¹¹. Puzzles that remain include the existence of similar microbial species in both healthy and CRS patients, albeit in different abundances⁸, the fact that traditional pathogenic microbes e.g. Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Stenotrophomonas maltophilia, and Enterobacter species are also found in healthy cavities, albeit at lower abundances⁸, as well as how innate and adaptive immune mechanisms (described below) can discriminate between bacterial species to help set the nasal microbiome.

Mucociliary Clearance—The Foundation of Sinonasal Innate Immunity

The upper airways play an important role in removing particulates and pathogens from inspired air via mucociliary clearance (MCC)¹², a specialized function unique to the airway epithelium. MCC is the primary physical defense of the respiratory tract, complimenting the physical epithelial barrier (Figure 1). MCC relies upon both mucus production and transport. The airway surface liquid (ASL) lining the respiratory tract consists of two layers. The top is an antimicrobial-rich mucus “gel,” formed by mucins produced by goblet cells and submucosal glands¹³. Mucins are large thread-like glycoproteins¹⁴ with “sticky” carbohydrate side chains¹⁵ that can bind surface adhesins on microorganisms¹⁵, including Mycoplasma pneumonia¹⁶, Haemophilus influenza¹⁷, Moraxella catarrhalis¹⁸, and Pseudomonas aeruginosa¹⁹ and cepacia²⁰. The mucus layer rests on top of a less-viscous fluid periciliary layer (PCL) that surrounds the cilia of airway epithelial cells and allows them to beat rapidly (~8–15 Hz). Membrane-tethered mucins on the apical membrane of ciliated cells may form a “lubricating” brush-like structure that keeps the mucus and PLC layers separate to facilitate MCC²¹. Coordinated and directional ciliary beating (known as the “metachronal wave”²²) facilitates transport of debris-laden mucus through the sinonasal cavity to the oropharynx, where it is swallowed or expectorated.

Figure 1. Overview of sinonasal innate immunity.

In healthy tissue, respiratory epithelial cells are linked by tight junctions to form a protective physical barrier. Inhaled pathogens, such as viruses, bacteria, and fungal spores, are trapped by airway mucus and then removed by mucociliary clearance. Constant beating of cilia drives the pathogen-laden mucus toward the oropharynx, where it is then cleared out of the airway by expectoration or swallowing. Mucociliary clearance is further regulated by secretion of mucus as well as ion and fluid transport, which controls the mucus viscosity. Mucociliary transport is complemented by the generation of reactive oxygen and nitrogen species (ROS and RNS, respectively) and the production of antimicrobial peptides. During more chronic exposure to pathogens, epithelial cells secrete cytokines to activate inflammatory pathways and recruit dedicated immune cells.

Abbreviations: AMP, antimicrobial peptide; LTF, lactotransferrin; IL, interleukin; MCC, mucociliary clearance; MCP-1, monocyte chemotactic protein 1; MIP-1, macrophage inflammatory protein-1; RANTES, Regulated on Activation, Normal T Expressed and Secreted protein; RNS, reactive nitrogen species; ROS, reactive oxygen species, TNFα, tumor necrosis factor alpha;

MCC is regulated by small molecule neurotransmitter (e.g., adenosine trisphosphate, acetylcholine, etc.) and neuropeptide (vasoactive intestinal peptide, substance P, etc.) receptors that regulate mucus and fluid secretion²³ and ciliary beating²⁴, as well as receptors for bacterial products²⁵ and mechanical stresses²⁶. While other mechanisms defend the airway in addition to MCC (described below), the importance of MCC is illustrated by direct links between MCC defects and disease. In cystic fibrosis (CF), defects in the CF transmembrane conductance regulator (CFTR) anion channel result in impaired salt and water secretion²³ as well as possibly enhanced absorption²⁷, creating dehydrated mucus and impaired MCC. CF patients frequently develop severe recurrent sinonasal infections²⁸ that may also seed or exacerbate lung infections. Additionally, the epithelial anion transporter, pendrin, is elevated in nasal polyps compared with uncinate tissue (UT) isolated from healthy controls^{29, 30}. Upregulated pendrin expression in the airway has been linked to IL-4, IL-13, and IL-17A^31–33. However, further studies are needed to investigate how pendrin contributes to MCC, epithelial dysfunction, and CRSwNP pathogenesis. Increased mucus production may also impair MCC. In CRSwNP, Muc5AC was found to be elevated in nasal polyps when compared with UT from CRSsNP or healthy patients²⁹.

Defects involving epithelial cell cilia can also impact MCC and contribute to chronic sinonasal inflammation. For example, in primary ciliary dyskinesia (PCD), abnormal ciliary function and/or structure result in impaired MCC and increased incidences of upper respiratory infections³⁴. More commonly, though, acquired ciliary dysfunction occurs through exposure to environmental or microbial toxins and/or as a secondary consequence of disease through exposure to inflammatory stimuli³⁵. Nonetheless, this likely contributes to pathogenesis of upper respiratory infections. Several pathogens produce compounds that impair ciliary motion and/or coordination, including Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Aspergillus fumagatus, and Pseudomonas aeruginosa^36–39. Hypoxia created by mucostasis or anatomical obstruction may also affect MCC by inhibiting ion transport⁴⁰ or promoting polypogenesis⁴¹. Approaches designed to increase ciliary beating or enhance fluid secretion to thin mucus remain attractive therapeutic strategies for enhancing MCC in CRS with impaired MCC. Additionally, high volume low pressure sinonasal lavage has been demonstrated to be effective in mobilizing the copious secretions associated with CRS⁴².

Epithelial-Derived Antimicrobial Peptides and Radicals

In addition to transporting mucus, sinonasal epithelial cells produce substances that have direct anti-pathogen effects^{43, 44} (Figure 1). These include well-characterized proteins, such as lysozyme, lactoferrin, antitrypsin, defensins, S100 proteins and surfactants. Some are tonically secreted, but the expression of many are up-regulated during infection⁴⁵. Moreover, after epithelial damage, concentrations of these proteins may increase further due to plasma extravasation⁴⁶.

Lysozyme is a small cationic protein secreted submucosal glands⁴⁷. Lysozyme catalyzes the breakdown of the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetyl-D-glucosamine in the outer bacterial cell wall. Additionally, binding of lysozyme to bacterial cell walls facilitates phagocytosis by macrophages⁴⁵. Lysozyme is most effective against gram-positive bacteria, but also has effects against gram-negative bacteria^{48, 49} and fungi⁵⁰. Various studies have examined lysozyme in CRS. However, controversy exists over whether levels are increased⁵¹ or decreased^{52, 53} in CRS. Lysozyme is produced by submucosal glands, which are diminished in nasal polyp tissue, thus lysozyme decrease in polyp tissue may contribute to the variability of reported results⁵⁴.

Lactoferrin (also known as lactotransferrin) chelates and sequesters iron important for bacterial and fungal metabolism⁴⁵. Bacteria may also use iron to catalyze mucin degradation to help break through the protective mucosal barrier, a process likely inhibited by lactoferrin⁵⁵. Lactoferrin also binds certain conserved microbial structures known as pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS) from the gram-negative cell wall and unmethylated bacterial CpG containing DNA. Lactoferrin may act as an anti-inflammatory by inhibiting binding of these molecules to pro-inflammatory receptors⁵⁶. However, the immune regulatory role of lactoferrin is complex, as lactoferrin may also act alone or as a “partner molecule” with PAMPs to promote activation of pattern recognition receptors on immune cells⁵⁶. Lactoferrin binding to LPS can also cause gram-negative bacterial permeabilization⁵⁶. Lactoferrin inhibits entry of RNA and DNA viruses into host cells by binding host viral receptors or the viruses themselves⁵⁷. Lactoferrin levels may be reduced in CRS, especially in those patients with bacterial biofilms⁵⁸.

Collectin (collagen-lectin) proteins, such as surfactant proteins (SP-A and SP-D), C-reactive protein, and mannose-binding lectin, interact with numerous airway bacteria and can activate complement and exhibit antimicrobial properties⁵⁹. Collectins recognize and bind to PAMPs, including LPS, via their calcium-dependent carbohydrate-binding domains, promoting bacterial clearance⁵⁹. LL-37 is produced by the human nasal mucosa^{60, 61} by kallikrein and other proteases from a precursor molecule, cathelicidin. LL-37 activation is regulated by SPINK5 and other protease inhibitors expressed in the epithelium⁶². LL-37 has broad antibacterial properties, and may even have effects against Pseudomonas biofilms in animal models of CRS⁶³. LL-37 may be anti-inflammatory by neutralizing LPS⁶⁴. Of note, the transcription of the LL-37 gene is induced by binding of the bioactive form of vitamin D to its receptor⁶⁵. Sinonasal epithelial cells express the 1-α-hydroxylase enzyme important for synthesis of bioactive vitamin D; when sinonasal epithelial cells are exposed to inactive vitamin D precursors, they synthesize bioactive vitamin D and increase LL-37 production⁶⁶. Vitamin D may thus contribute to airway innate immunity⁶⁷, including in CRS and allergic rhinitis^{68, 69}.

Members of the α-defensin and β-defensin families are also expressed in the sinonasal epithelium^{70, 71}. Defensins are up-regulated in response to bacterial or viral challenge^{72, 73}. Defensins have broad antimicrobial effects against both bacteria and fungi, likely through formation of pores in bacterial and fungal membranes. β-defensins 2 and 3 have been shown to directly bind viruses to inhibit their entry into host cells as well as activate cytokine production to alert immune cells^{74, 75}. Notably, the function of cationic defensins is inhibited under high ionic strength conditions⁷⁶, suggesting that abnormalities in ion transport, as in CF, may reduce defensin function through alteration of ASL electrolyte concentration⁷⁷.

Other antimicrobial peptides may have important roles in CRSwNP. Members of the palate, lung, and nasal epithelial clone (PLUNC) family, including SPLUNC-1, have antimicrobial and surfactant properties but were decreased in nasal polyps compared with healthy sinonasal tissue⁵⁴. In addition to having antimicrobial properties, SPLUNC-1 affects ASL volume by inhibiting activation of the epithelial sodium channel (ENaC)⁷⁸. ENaC mediates sodium and fluid absorption in airway epithelia. Thus, reductions of SPLUNC-1 could have detrimental effects on MCC. Additionally, epithelial defense proteins S100A7 (psoriasin) and S100A8/9 (calprotectin) are reduced in CRSwNP⁷⁹. While antimicrobial protein levels differ between healthy and diseased sinonasal tissue, levels also can vary by location within the sinonasal cavity⁸⁰. For example, lactoferrin is higher in healthy UT compared with healthy inferior turbinate, while S100A7 is higher in inferior turbinate compared with UT⁸⁰. Taken together, regional variability suggests that the sinonasal cavity is not uniform, but rather has complex and unique roles dependent upon specific anatomic location.

The lipids cholesteryl linoleate and cholesteryl arachidonate may contribute to antimicrobial properties of nasal secretions⁸¹, and their levels may be increased in CRS nasal secretions⁸². The sinonasal mucosa also generates reactive oxygen species (ROS) that can directly damage bacteria. Lactoperoxidase (LPO;⁸³) catalyzes the oxidation of substrates by hydrogen peroxide (H₂O₂). Airway epithelial ciliated cells produce H₂O₂ through the action of NADPH oxidase isoforms, including DUOX1 and DUOX2⁸⁴. A potentially important substrate for the LPO/ H₂O₂ system is thiocyanate. Thiocyanate is oxidized via LPO to hypothiocyanite, a compound with antibacterial, antifungal, and antiviral effects⁸⁵. Both CFTR and pendrin may regulate thiocyanate transport, linking ion transport with host defense⁸⁶. CFTR defects that reduce thiocyanate secretions may impair airway innate defense mechanisms in CF⁸⁷. Pendrin elevation in CRS may also influence thiocyanate transport²⁹.

The generation of nitric oxide (NO) by the sinonasal epithelium is thought to be critical for airway innate immunity, with the major source being the paranasal sinuses⁸⁸. NO is generated by NO synthase (NOS). NOS isoforms vary in their mRNA inducibility as well their sensitivity to intracellular calcium. NOS isoforms are expressed in the cilia and microvilli of epithelial cells⁸⁹, with the maxillary sinus being a site of high expression⁹⁰. NO activates guanylyl cyclase to increase ciliary beating via cyclic-GMP and protein kinase G. NO and reactive derivatives such as peroxynitrite⁹¹ also directly damage bacterial proteins and DNA^{92, 93} and inhibit viral replication⁷⁵. While studies have linked increased NO with host defense in vivo, others have suggested elevated NO is detrimental⁹⁴. The wide range of NO measurement methods and heterogenous study populations have limited the conclusions that can be drawn from studies investigating NO as a diagnostic, prognostic, or efficacy indicator in sinonasal disease⁹⁴.

Regulation of Antimicrobial Compound Production and Secretion

The most well-studied pathway for regulation of antimicrobial compounds by sinonasal epithelial cells is through toll-like receptors (TLRs), which recognize microbial PAMPs⁴⁵ (Figure 2A). Sinonasal cells express ~10 TLRs, with expression changes observed in CRS⁹⁵. TLRs stimulate the direct transcription and/or translation of mucins and AMPs^{45, 96}. TLR responses occur over the course of hours, likely critically important during times of sustained colonization or infection. TLRs activate airway epithelial cells to secrete defense molecules as well as cytokines and chemokines that recruit dedicated immune cells and activate inflammation, which may play an important role in CRS pathogenesis. Epithelial cells from CRS patients produce less IL-8 in response to TLR2 ligands, which may impair immunity. TLRs also play an important role in detection of rhinoviruses, RSV, and influenza, and can stimulate induction of type I interferon⁷⁵.

Figure 2. Role of toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) as well as taste family type 2 receptors (T2Rs) in regulation of sinonasal innate immunity by ciliated epithelial cells.

(A) TLRs located both on the cell surface and in intracellular endosomes of epithelial cells recognize pathogen-associated molecular patterns (PAMPs) and stimulate innate immune responses. PAMPs recognized by specific TLRs are indicated in the figure. TLRs activate downstream intracellular signaling proteins MyD88, TIRAP, TRAM, and TRIF (not shown), which activate transcription factors such as CREB (not shown), NFκB, and interferon response factors (IRFs) that activate transcription of antimicrobial peptides (AMPs), cytokines, and chemokines. The secretion of proinflammatory cytokines and interferons links innate and adaptive immunity. Additionally, the cytoplasmic helicases RIG-1 and MDA5 recognize RNA viruses by detecting intracellular viral double-stranded (ds) RNA, including viral genomic RNA (vRNA) (B) T2R38 expressed in ciliated epithelial cells recognizes bacterial homoserine lactones to stimulate calcium-dependent nitric oxide synthase (NOS) activation and NO production. This NO diffuses into the ASL and has direct antibacterial effects. NO also acts as an intracellular signaling molecule to stimulate ciliary beat frequency through protein kinase G (PKG).

Abbreviations: AMP, antimicrobial peptide, CCL20, Chemokine (C-C motif) ligand 20; CXCR3, Chemokine (C-X-C Motif) Receptor 3; ER, endoplasmic reticulum, GM-CSF, Granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; IP₃, inositol trisphosphate; IP₃R, inositol trisphosphate receptor; IRF, interferon regulatory factor; MDA-5, Melanoma Differentiation-Associated protein 5; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; NO, nitric oxide; NOS, nitric oxide synthase; PLCβ2, phospholipase C isoform β2; RIG-1, retinoic acid-inducible gene 1; Th1, T helper cell 1; TLR, toll-like receptor;

Human ciliated epithelial cells also express T2R bitter taste receptors, originally identified on the tongue. At least one T2R, T2R38, is expressed in ciliated epithelial cells, detects bitter acyl-homoserine lactone quorum sensing molecules secreted by gram-negative bacteria and stimulates an increase in mucociliary clearance and production of bactericidal levels of nitric oxide²⁵ (Figure 2B). T2R38 function has also been linked to antibacterial immune responses in vitro²⁵ as well as gram-negative upper respiratory infection²⁵ and chronic rhinosinusitis susceptibility in vivo^97–101. Bitter taste receptors are also expressed in specialized epithelial cells termed solitary chemosensory cells (SCCs), a specialized cell type that stimulates the rapid release of stored AMPs from surrounding cells^{96, 102}. Because of the rapid innate immune responses observed during stimulation of bitter taste receptors in the airway, it appears that they constitute an “early-warning” arm of the sinonasal innate immune system. T2Rs SCCs are also regulated by T1R sweet taste receptors (reviewed in^{103, 104}), a mechanism which may be important in diabetics and CRS patients, both of which have elevated glucose in their ASL^105–107. Taste receptors are emerging as a “front line” defense mechanism against microbial invaders, and there are exceedingly common functional polymorphisms found in all the taste receptor genes¹⁰⁸. Thus, each individual’s “bitterome,” or collection of T2R polymorphisms, may define the permitted and excluded microbes, thus shaping each person’s baseline microbiota.

Epithelial Cell Barrier in Host Defense

In addition to secreting anti-microbial products, epithelial cells lining the sinonasal mucosa are involved in other aspects of host defense. Epithelial cells form a physical barrier involving tight junctions, adherens junctions, and desmosomes that protect the underlying sinonasal tissue from damage caused by inhaled pathogens, allergens, and other irritants. However, in CRS, studies have suggested that this barrier is compromised. Reductions in tight junction proteins, occludin-1 and zonula occludens 1, were observed in CRSwNP compared to healthy controls and this was associated with decreased epithelial electrical resistance¹⁰⁹. Other markers of epithelial cell dysfunction commonly observed in CRS include abnormal ion transport as mentioned previously, goblet cell hyperplasia, basal cell proliferation, acanthosis and acantholysis.

There remains some debate as to whether epithelial cells in CRS are inherently dysfunctional or if exposures to external or internal factors induce this dysregulation. In support of the former argument, ex-vivo cultures of epithelial cells taken from patients with CRSwNP showed reduced electrical resistance when compared to healthy sinonasal tissue¹⁰⁹. This is in contrast to another recent study that found no difference in electrical resistance in epithelial cells cultured from patients with CRSwNP, CRSsNP, or healthy controls¹¹⁰. Interestingly, in this latter study, Oncostatin M, a member of the IL-6 family of cytokines, was elevated in nasal polyps compared to healthy controls and could induce tissue permeability, disrupt tight junctions, and decrease electrical resistance in cultured human epithelial cells¹¹⁰. It will be important to resolve whether there are epithelial cell intrinsic defects in barrier and whether elevated levels of Oncostatin M and other inducers of the epithelial mesenchymal transition (EMT) in CRSwNP contribute to epithelial barrier dysfunction. It should be noted that loss of epithelial barrier has been reported in asthma and atopic dermatitis as well, and both epithelial cell intrinsic and extrinsic mechanisms have been discovered (e.g. filaggrin mutations and type-2 cytokine mediated changes).

Finally, pathogens are another possible external factor that could directly impact the sinonasal epithelial cell barrier. Pseudomonas aeruginosa was shown to transiently disrupt the tight junction proteins occludin and claudin-1 in cultured human nasal epithelial cells¹¹¹. Additionally, Staphylococcus aureus as well as various fungi have been found to secrete products that could also disrupt zona occludens-1 in human nasal epithelial cells in vitro¹¹². In some cases, the microbes produce proteases that can either cleave the junctional proteins or activate epithelial changes through protease activated receptors such as PAR2^{113, 114}. It is possible that the pathogen-induced disruption of the sinonasal epithelium could promote further bacterial infection, colonization, or even biofilm formation and in turn further potentiate the overall chronic disease process.

The Host Immune Response: Inflammatory Mediators

The host immune system is also thought to play a prominent role in CRS pathogenesis. The effector functions of innate and adaptive immune cells as well as the various mediators they produce can all contribute to the chronic inflammatory environment characteristic of CRS (Figure 3). To this end, the type of inflammatory cytokines and chemokines identified within the sinonasal mucosa has historically been used to help define particular subsets of CRS. For example, cystic fibrosis has classically been characterized by a type-1 inflammatory response with elevated levels of interferon (IFN)-γ in the sinonasal tissue compared to healthy controls^{115, 116}.

Figure 3. Role of the Host Immune Response in CRSwNP.

The dysregulated epithelial barrier in CRS can lead to enhanced exposures to various inhaled allergens, bacteria, fungi, and viruses. Additionally, colonization with Staphylococcus aureus may also occur. In CRSwNP nasal polyps, epithelial cells can release various inflammatory mediators, most notably thymic stromal lymphopoietin (TSLP), which in turn promote the development of a type-2 immune response. Innate immune cells including innate type-2 lymphoid cells (ILC2), mast cells, and eosinophils are all elevated in nasal polyps. These cells can release type-2 cytokines that further perpetuate the ongoing inflammatory response as well as specific granule proteins that can contribute to tissue injury. Adaptive immune cells including both naive B cells and activated plasma cells are also elevated in nasal polyps and contribute to increased local production of antibodies within the sinonasal tissue. Finally, type-2 cytokines are also thought to contribute to decreased t-PA and increased FXIIIa which, in the setting of increased vascular leak, lead to increased fibrin deposition and cross-linking within nasal polyps.

In contrast, CRSwNP, one of the most extensively studied CRS subtypes, is typically regarded as having a type-2 inflammatory environment, at least in the US and Europe (see below). Type-2 inflammation refers to a response typically driven by the type-2 cytokines IL-4, IL-5 and IL-13. Such inflammation typically is characterized by infiltration of large numbers of eosinophils, basophils and mast cells. Thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine that plays an important role in promoting type-2 responses, is elevated and has enhanced activity in nasal polyps of CRSwNP when compared to healthy sinonasal tissue¹¹⁷. There remains some debate as to whether IL-33 and IL-25, other epithelial derived cytokines that promote type-2 inflammation, are elevated in nasal polyps^118–121. Other classic type-2 inflammatory mediators including IL-5, IL-13, Eotaxin-1 (CCL11), Eotaxin-2 (CCL24) and Eotaxin 3 (CCL26) are often the products of epithelium and have been reported to be elevated in nasal polyps compared to healthy control tissues^122–127.

It is important to note, however, that most early studies examining CRSwNP evaluated patients of European descent. More recently, studies have reported that nasal polyps from Asian patients living in Asia or second generation Asians residing in the United States have an enhanced type-1 inflammatory environment with elevated levels of IFN-γ and reduced levels of IL-5^{128, 129}. It remains unclear why Asian patients are more likely to have a type-1 inflammatory profile in nasal polyps compared to patients from Western countries but a yet-to-be-identified genetic factor may play a role¹²⁹.

Finally, unique characteristics that distinguish CRSsNP from other CRS subsets remain under debate. Historically, CRSsNP was thought to have a predominant type-1 inflammatory environment with more IFN-γ and less IL-5 expression than in nasal polyps from CRSwNP^{124, 125}. More recently, however, this classification has been under review with several studies reporting similar levels of IFNγ expression in CRSwNP nasal polyps when compared to CRSsNP UT, control UT, or control IT tissues^{117, 128}. In a more comprehensive analysis, no differences in gene expression levels of several interferon family members (IFNγ, IFNα2, IFNα8, and IFNβ1) were found between CRSwNP nasal polyps, CRSsNP UT, and control UT¹²⁶. Furthermore, no significant difference in IFNγ protein levels was observed among all the sinonasal tissues examined¹²⁶. Taken together, these findings suggest CRSsNP may not necessarily be more “type-1” than CRSwNP. However, regional variations in protein expression levels within the sinonasal cavity could possibly explain the discrepancies in IFNγ observed among the nasal polyp, ethmoid mucosa, inferior turbinate, middle turbinate and uncinate process sinonasal tissues⁸⁰. Given these findings, further work is needed to more extensively characterize the cytokine environments in CRSsNP as well as CRSwNP.

The Innate Immune Response

There have been numerous studies examining the role of innate immune cells in the development of CRS pathology. In CRSwNP, type-2 innate lymphoid cells (ILC2) are thought to be early contributors to the type-2 inflammatory response. These specialized innate effector cells are elevated in nasal polyps and are activated by epithelial derived-cytokines such as TSLP and IL-33 to secrete IL-5 and IL-13^{130, 131}. Interleukin-5 is a potent activator and survival factor for eosinophils and studies have shown that ILC2 numbers are doubled in eosinophilic compared to non-eosinophilic nasal polyps¹³².

While eosinophils are one of the major hallmarks of Western nasal polyps, mast cells and basophils, other type-2 innate inflammatory cells, are also elevated in CRSwNP compared to healthy controls^{133, 134}. These innate effector cells can release a variety of inflammatory mediators and toxic granules that can perpetuate the chronic type-2 inflammatory response and induce sinonasal mucosal damage. Interestingly, a unique subset of mast cells was found in nasal polyp glandular epithelial cells that produced tryptase, carboxypeptidase A3, and chymase¹³⁴. Since chymase is a known inducer of mucus, it is hypothesized that these specialized mast cells may play a role in the over-production of mucus commonly seen in CRSwNP¹³⁴. Furthermore, a recent small study in CRSwNP reported that mast cells may be a reservoir for S. aureus and thus contribute to the chronicity of this infection in certain patients¹³⁵.

In contrast to Western nasal polyps, Asian nasal polyps are characterized by reduced numbers of eosinophils as well as decreased levels of IL-5, Eotaxin, and eosinophil cationic protein, a protein found in eosinophil granules^{128, 129}. In cystic fibrosis, neutrophils and macrophages are commonly detected^{115, 116}. Unfortunately, no single defining innate effector cell has been identified in CRSsNP to date but this CRS subtype is associated with a lack of type-2 inflammation as previously discussed.

The Adaptive Immune Response

Along with the innate immune response, the adaptive immune system also contributes to the chronic inflammation seen in CRS. T cells represent a major component of adaptive immunity and, despite conflicting reports in CRSsNP, CD3⁺ T cells have been shown to be elevated in nasal polyps compared to healthy sinonasal tissue^{124, 127}. Upon further analysis of CD3⁺ T cell populations by flow cytometry, no differences were reported in the number of either CD4⁺ T cells or CD8⁺ T cells in nasal polyps of CRSwNP versus inferior turbinates of patients with CRSsNP¹²⁷. However, in this study, the ratio of CD4⁺ to CD8⁺ T cells was significantly higher in CRSwNP than CRSsNP¹²⁷. Finally, there have been several conflicting reports regarding the importance of regulatory T cells in CRS pathogenesis. Van Bruaene and colleagues initially identified a decrease in Foxp3 expression as well as the regulatory cytokine TGF-beta¹²⁵. A later study found that suppressor of cytokine signaling 3 (SOCS3) could negatively regulate Foxp3 expression in human airway mucosa and that SOCS3 protein levels were elevated in inflammatory cells in the airway mucosa¹³⁶. These findings suggested that T regulatory cells were impaired in CRSwNP thus leading to an imbalance in pro- and anti-inflammatory responses. However, a later study using flow cytometric analyses reported increased numbers of T regulatory cells in CRSwNP¹³⁷. Taken together, further studies examining T regulatory cells are needed to elucidate their role in CRSwNP and to determine if they are important in CRSsNP pathology.

In addition to T cells, B cells may also significantly contribute to the ongoing sinonasal inflammation observed in CRS¹³⁸. In CRSwNP, naive B cells as well as activated plasma cells were found to be elevated in nasal polyps when compared to CRSsNP or healthy control tissue^139–141. This influx in B cells may be secondary to the elevated levels of B cell chemotactic factors CXCL13 (BCA-1) and CXCL12 (SDF-1a, Stromal cell-derived factor-1) observed in nasal polyps¹⁴². BAFF and IL-6, both mediators important in B cell activation and proliferation, are increased in nasal polyps compared to controls with levels of BAFF strongly correlating with expression of CD20, another B cell marker^{140, 143}.

Interestingly, B cells in nasal polyps also appear to have local effector functions. Levels of IgG1, IgG2, IgG4, IgA, IgE and IgM were all elevated in nasal polyps versus healthy sinonasal tissue¹⁴¹. Importantly, there was no concomitant elevation in these antibodies in the peripheral blood of the same CRSwNP patients suggesting that B cell antibody production in CRSwNP is not a systemic response but rather driven by a stimulus within the local sinonasal inflammatory environment¹⁴¹.

Unfortunately, the specificity of the majority of antibodies detected locally in nasal polyps in CRSwNP remains unclear. A certain subset of patients with CRSwNP who are also colonized with Staphylococcus aureus (S. aureus) can develop specific IgE antibodies directed against S. aureus enterotoxins within nasal polyp tissue¹⁴⁴. Additionally, other studies have reported elevated levels of IgG and IgA autoantibodies, particularly against nuclear antigens, locally produced in nasal polyps¹⁴⁵. However, it remains unclear how these autoantibodies may contribute to disease pathology in CRSwNP.

Finally, even less is known regarding the role that B cells may play, if any, in CRSsNP. Naive B cells and plasma cells were not increased in UT from patients with CRSsNP compared to controls¹⁴¹. Additionally, there were no significant elevations in the immunoglobulin subtypes IgG, IgA, IgE, or IgM¹⁴¹, IgA or IgG autoantibodies¹⁴⁵ or specific IgE to S. aureus¹⁴⁴ locally in CRSsNP sinonasal tissue versus healthy controls. Interestingly, however, patients with CRSsNP are unique in that IgD is locally elevated within the sinonasal tissue in this population unlike in healthy controls or patients with CRSwNP¹⁴⁶.

Tissue Remodeling

As in other chronic inflammatory diseases, tissue remodeling also occurs in CRS. However, the histological characteristics as well as mechanisms contributing to this upper airway remodeling are hypothesized to differ between CRSsNP and CRSwNP. Traditionally, CRSsNP is characterized by fibrosis, basement membrane thickening, and goblet cell hyperplasia. Transforming growth factor beta (TGF-β), a key mediator in promoting fibrosis and airway remodeling, was found to be elevated in CRSsNP compared to CRSwNP and to healthy controls¹⁴⁷. Likewise, expression of the TGF-β receptor as well as mediators critical in TGF-β signaling was also enhanced in CRSsNP sinonasal tissue¹⁴⁷. Interestingly, there appears to be some regional variation in TGF-β protein expression throughout the sinonasal cavity with the inferior and middle turbinates having the lowest level of expression in CRSsNP¹⁴⁸.

In contrast to CRSsNP, CRSwNP is histologically characterized by a significant inflammatory cell infiltrate, the formation of pseudocysts, and stromal tissue edema. Collagen levels are reduced in nasal polyps¹⁴⁹ but there have been conflicting reports regarding TGF-β. One study suggests that TGF-β is reduced in CRSwNP¹²⁵ while another study reported elevated levels when compared to healthy controls and/or CRSsNP¹⁵⁰. More recently, TGF-β expression was found to be lower in epithelial cells but elevated in stromal cells in nasal polyps compared to the same cell subtypes in healthy sinonasal tissue¹⁵¹. These regional differences may also explain recent conflicting reports regarding the expression levels of Activin A, a member of the TGF-β superfamily hypothesized to play a role in remodeling in nasal polyps^{152, 153}.

Another aspect of CRSwNP pathogenesis relates to the growth of nasal polyps themselves. In general, plasma proteins are enriched in affected sinus and polyp tissue due to vascular leak, and can traverse the dysfunctional epithelial barrier into the lumen in CRS. In nasal polyps, studies have shown that fibrin deposition is increased which, in turn, can form a scaffold, trapping plasma proteins and enhancing tissue edema¹⁵⁴. This fibrin mesh is further stabilized by Factor XIIIa, which is also elevated in CRSwNP and thought to be another signature of a type-2 inflammatory environment¹⁵⁵. Additionally, nasal polyps also have reduced levels of tissue plasminogen activator, an enzyme critical for the breakdown of fibrin mesh, as well as d-dimer, a product of fibrin degradation¹⁵⁴. Taken together, these studies suggest that an imbalance in fibrin formation (elevated fibrin and factor XIIA) and degradation (reduced t-PA and d-dimer) may contribute to the polyp growth observed in CRSwNP (Figure 3).

Conclusions

In conclusion, significant advances have been made in the understanding of CRS pathogenesis. Mechanisms involving mucocilliary clearance, epithelial barrier dysfunction, the host immune response, and tissue remodeling all are thought to work in concert and contribute to the chronic inflammation characteristic of CRS. It is the goal of all working in this field that laboratory and clinical findings will continue to build the foundation upon which more developmental studies may be performed that advance diagnostic and therapeutic strategies for the benefit of affected patients.