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P-annotatePDF-v11
INSTRUCTIONS ON THE ANNOTATION OF PDF FILES
To view, print and annotate your article you will need Adobe Reader version 9 (or higher). This program is freely available for a whole series of platforms that include PC, Mac, and UNIX and can be downloaded from http://get.adobe.com/reader/. The exact system requirements are given at the Adobe site: http://www.adobe.com/products/reader/tech-specs.html.
Note: if you opt to annotate the file with software other than Adobe Reader then please also highlight the appropriate place in the PDF file.
PDF ANNOTATIONS
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Current perspectives
Host-microbial interactions in patients with chronicrhinosinusitisQ1Q2
Q15 Daniel L. Hamilos, MD Boston, Mass
There has been considerable investigation of host-microbialinteractions in patients with chronic rhinosinusitis (CRS) inhopes of elucidating mechanisms of disease and bettertreatment. Most attention has been paid to bacterial infectionand potential underlying defects in innate immunity. Bacterialbiofilm is present in most patients with CRS undergoing surgicalintervention, and its presence is associated with more severedisease and worse surgical outcomes. A role for viral or fungalinfection in patients with CRS is less clear. There is no evidencefor a primary defect in mucociliary clearance in most patientswith CRS. Decreased levels of certain antimicrobial proteins,most notably lactoferrin, have been found in sinus secretions,whereas levels of other antimicrobial proteins have been foundto be normal. No primary defects in Toll-like receptors havebeen found in patients with CRS, although a 50% reducedexpression of Toll-like receptor 9 was reported in patients withrecalcitrant nasal polyps. A polymorphism in a bitter tastereceptor was recently associated with refractory CRS andpersistent Pseudomonas aeruginosa infection. A downregulationof innate immunity by maladaptive TH2 tissue inflammation hasalso been described in patients with recalcitrant nasal polyps,suggesting a link to persistent infection. To date, an effectivemeans of restoring host-microbial balance and mitigatingdisease in patients with CRS remains elusive. (J Allergy ClinImmunol 2013;nnn:nnn-nnn.)
Host-microbial interactions play a critical role in CRS diseaseinitiation and perpetuation. This article aims to summarizeknowledge of host-microbial interactions elucidated in relationto normal sinus physiology and pathology of patients with chronicrhinosinusitis (CRS), including the subsets regarded as chronicrhinosinusitis without nasal polyps (CRSsNP), chronic rhinosi-nusitis with nasal polyposis (CRSwNP), and allergic fungalrhinosinusitis (AFRS).1
Most studies of innate immunity and host-microbial interac-tions in patients with CRS have focused on patients with
‘‘refractory’’ or ‘‘recalcitrant’’ disease. Refractory CRS has beendefined on the basis of failure to stabilize after surgery, antibiotics,saline rinses, and topical steroid treatment.2 Somewhat differ-ently, ‘‘recalcitrant CRS’’ has been defined based on recurrenceof nasal polyps (NPs) after polyp surgery.3 These definitions arenoteworthy because patients with refractory polyposis, forexample, might havemore evidence of infection, whereas patientswith recalcitrant polyposis might have little or no evidence ofinfection but more evidence for maladaptive TH2-biased mucosalinflammation.
MICROBIOLOGY OF CRS Q4
Role of virusesViral upper respiratory tract infections are potentially highly
relevant to CRS. The average healthy adult person experiences 1to 3 common colds per year (http://www.niaid.nih.gov/topics/commoncold/pages/overview.aspx). In healthy subjects the onset
From the Division of Rheumatology, Allergy & Immunology, Massachusetts General
Hospital.
Disclosure of potential conflict of interest: D. L. Hamilos has consultant arrangements
with and has received grants from Merck and received royalties from UpToDate.
Received for publication January 14, 2013; revised June 25, 2013; accepted for publica-
tion June 27, 2013.
Corresponding author: Daniel L. Hamilos, MD, Division of Rheumatology, Allergy &
Immunology, Massachusetts General Hospital, 55 Fruit St, Bulfinch-422, Boston,
and time course of cold symptoms and levels of viral mRNAdetectable in nasal secretions over 21 days have been mappedout after experimental rhinovirus infection.4,5 Patients withasthma or chronic obstructive pulmonary disease manifest asignificantly higher peak rhinovirus 16 viral load and durationof symptoms. Asthmatic patients also manifest a corresponding10-fold decreased induction of type I (b) and type III (l1 andl2/3) interferons.6 Given the similarities between asthma andCRS at the tissue level and the fact that many CRS exacerbationsoccur during the viral season,7 it is plausible that a similar defectexists in patients with CRS. However, experimental rhinovirusinfection has not been studied in patients with CRS. The innateantiviral response to rhinovirus infection involves activation oftype I interferons through interferon-regulatory factor 1 gene acti-vation, an increase in nitric oxide (NO) production, and epithelialproduction of hBD-2Q5 , IL-8, and RANTES.8,9 IL-17Awas found toaugment production of hBD-2 and IL-8 but downregulate produc-tion of RANTES in this model. The chemokine CXCL10 (inter-feron-inducible protein 10) is also induced.10
There has not been a study of the incidence of rhinovirusinfection in patients with CRS. A study by Jang et al11 reportedthat 21% of patients with CRS have detectable rhinovirus infec-tion. This study examined nasal lavage fluid and turbinate epithe-lial cells (collected with a Rhino-probe mucosal curetteQ6 ) from 39patients with CRS and 27 healthy control subjects (HCs). Usingan RT-PCR–based assay, they found that lavage fluid from all pa-tients with CRS and HCs and turbinate epithelial cells from HCswere negative for picornavirus, whereas 8 (21%) of 39 epithelialcell samples from patients with CRSwere positive. Further exam-ination revealed that all 8 patients with CRS with positive resultshad positive results for rhinovirus. It is unclear whether these rep-resented subclinical infections because patients were studied atonly 1 time point.
In an in vitro experiment Wang et al12 infected NPs and nasalturbinate epithelial cells from 16 patients with CRSwNP andsphenoid sinus and turbinate epithelial cells from 19 HCs withrhinovirus (rhinovirus 16). No significant differences in rates ofinfection or induction of IL-6 or IL-8 were found.12 Our groupfound that cultured airway epithelial cells from patients withCRSsNP had an exaggerated response to stimulation with thecombination of double-stranded RNA (a Toll-like receptor[TLR] 3 agonist and surrogate for viral infection) plus cigarettesmoke extract, with exaggerated production of RANTES and hu-man b-defensin (hBD) 2.13
Finally, although studies are quite limited, there is a lack ofevidence for persistence of viral infection in patients with CRS.Again using PCR methodology, Wood et al14 found no evidencefor common respiratory tract viruses, including parainfluenza 1,2, and 3; respiratory syncytial virus; human metapneumovirus;adenovirus; rhinovirus; coronavirus; bocavirus; cytomegalovirus;or influenza A or B virus in sinus mucosal samples from 13 pa-tients with CRS.14
Whether upper respiratory tract viruses could contributecausally to the inception of CRS analogous to their hypothesizedrole in asthma pathogenesis remains unexplored.15
Bacterial involvement in patients with CRSBacteriology of CRS determined by using conven-
tional culture techniques. Studies with conventional culturetechniques in children with CRS cultured in the absence of
antibiotic treatment reported positive cultures in roughly 60% ofcases, with the most common pathogens being Haemophilus in-fluenzae, Streptococcus pneumoniae, and Moraxella catarrhalis(reviewed byMeltzer et al1). Studies by Brook et al16,17 using spe-cial techniques to optimize recovery of anaerobic bacteria identi-fied these bacteria in roughly 80% of children with CRS.
Prospective studies in adults identified a positive bacterialculture in a variable percentage of patients with CRS (reviewed byMeltzer et al1). Coagulase-negative Staphylococcus species wasthe most common aerobic isolate in several studies, often accom-panied by Staphylococcus aureus and viridians streptococci.Organisms associated with acute bacterial rhinosinusitis werecultured in some cases. In several studies gram-negative entericrods, including Pseudomonas aeruginosa, Klebsiella pneumo-niae, Proteus mirabilis, Enterobacter species, and Escherichiacoli were also isolated. These organisms are rarely found in mid-dle meatus cultures from healthy subjects. More recent studies ofintraoperative sinus cultures with simultaneous analysis of cul-tures and biofilm reported positive cultures in 72.6% to 80% ofcases, with a predominance of S aureus and P aeruginosa in theisolates.18-20
The frequency with which anaerobic organisms have beenrecovered from adults with CRS has varied widely, withanaerobes found mainly by investigators using special techniquesto optimize their recovery.21,22 Several species, including pig-mented Prevotella, Fusobacterium, Bacteroides, and Peptostrep-tococcus species, were isolated. In support of a role for anaerobicbacteria in chronic maxillary sinusitis, Finegold et al22 foundrecurrence of signs and symptoms to be twice as frequent whencultures yielding anaerobic bacterial counts of greater than 103cfu/mL. Further supportive evidence came from the detection ofIgG antibodies to anaerobic organisms commonly recoveredfrom sinus aspirates, namely Fusobacterium nucleatum and Pre-votella intermedia. Antibody levels to these organisms decreasedin the patients who responded to therapy but did not decrease inthose in whom therapy failed.23 Recent studies with moleculartechniques have shed new light on the potential role of anaerobesin patients with CRS (see below).
Role of atypical bacterial infection in patients with
CRS. Studies with conventional culture techniques or moleculartechniques to overcome issues of detection of nonculturablebacteria suggest that atypical mycobacterial infection is rare inpatients with CRS but should be sought in patients with refractoryCRS (see additional text in this article’s Online Repository atwww.jacionline.org).
Bacterial biofilm in patients with CRS. Biofilm forma-tion is an important survival mechanism for microorganismsthrough attachment to surfaces.24 Formation of biofilm is a com-plex process controlled by different genetic pathways dependingon growth conditions and exposure to membrane-targeting antibi-otics.25 Furthermore, biofilm-associated bacteria are known tohave enhanced resistance to antimicrobial agents relative tofloating (planktonic) bacteria.26 Biofilm formation on sinonasalmucosal surfaces was first described in 200427 and later in severalother studies.28-33
Multiple techniques for biofilm detection have been describedand are discussed in the additional text in this article’s OnlineRepository. Table E1 in this article’s Online Repository at www.jacionline.org summarizes the results of several studies of biofilm(including fungal biofilm) in patients with CRS, including thetechniques used for biofilm identification. Most studies have not
subcategorized patients as having CRSsNP, CRSwNP, AFRS, oreosinophilic mucin chronic rhinosinusitis (EMCRS). One study34
found biofilm to be present in only 2 of 12 patients with NPs, alower prevalence than reported in most other studies of CRS.When the studies using scanning electron microscopy (SEM),transmission electron microscopy (TEM), or confocal scanninglaser microscopy (CSLM) are taken in total (excluding the studyconfined to patients with NPs), the prevalence of biofilm in theCRS case series summarized in Table E1 was 56.3%.
Is the presence of biofilm of prognostic value?. Thepresence of bacterial biofilm, as determined by using CSLM, wasfound to be associated with more severe sinus disease preoper-atively (worse radiologic and symptom scoring) and worse sinussymptom and nasal endoscopy scores 16 months after sur-gery.30,35 The presence of bacterial biofilm was found to be asso-ciated with more severe preoperative disease radiology and nasalendoscopy scoresQ7 .30 Single-organism H influenzae biofilm wasassociated with mild clinical and radiographic disease andnormalization of sinus mucosa a short time after surgery. Incontrast, polymicrobial biofilm or biofilm containing S aureuswas associated with more severe disease and a poorer postopera-tive course.36 The presence of bacterial biofilmwas strongly asso-ciated with persistent mucosal inflammation after endoscopicsinus surgery (53% of biofilm-positive patients vs 0% ofbiofilm-negative patients).31 One study showed that the likeli-hood of detecting bacteria with biofilm-forming capacity in-creases in relation to prior endoscopic sinus surgery, possiblyreflecting the severity of their disease.37 Similarly, Zhang et al38
found an association of biofilm forming-capacity in vitro fromclinical samples with prior sinus surgeries and nasal steroid usein the month before sample collection but no association withthe presence of NPs, allergy, or Samter triad and an associationwith positive culture results at least suggesting that biofilm mightbe of greater relevance in patients with nonpolypoid CRS. In sum-mary, these studies suggest that that mucosal biofilm is a markerof more severemucosal disease and a predictor of poorer outcomeafter sinus surgery. On the basis of animal models of rhinosinusi-tis, biofilm formationmight reflect the severity, chronicity, or bothof sinus infection.39
Intracellular bacteria in patients with CRS. Intracel-lular intraepithelial Staphylococcus aureus (IESA) was first re-ported in sinus epithelium by Corriveau et al40 using a peptidenucleic acid fluorescence in situ hybridization (FISH) assay. Inthis study IESA was found in the epithelium of patients withCRS but was also seen in some HCs, raising the question of itspathologic significance. In a recent study using a similar S aureusFISH probewith propidium iodide counterstain and CSLM, IESAwas detected in 56% of patients with CRS undergoing sinus sur-gery but none of 8 HCs.41 Simultaneous analysis revealed thepresence of S aureus biofilm in 100% of the IESA-positive tissuesand 50% of the tissue from IESA-negative patients with CRS. TheCSLM-FISH/propidium iodide technique has the advantage ofbeing able to detect IESA and S aureus biofilm in the same tissuesample.41,42
Presently, it is unclear whether the presence of IESA has anyprognostic or pathologic significance beyond that afforded by thepresence of biofilm, and there are no studies linking the presenceof IESA to any specific defects in epithelial innate immunity.
Bacteriology of patients with CRS using molecular
techniques. The evolving view of bacterial involvement inpatients with CRS has expanded beyond that of infection with
individual pathogens to considerations of increased bacterialburden, biofilm formation and alterations in the microbialcommunity (ie, the microbiome).
The microbiome is the universe of culturable and nonculturablemicroorganisms present in a specific ecologic niche, such as thegastrointestinal tract or the sinus mucosa. Analysis of the micro-biome has gained much recent attention as a novel means ofstudying host-microbial relations in various organs, including therespiratory tract and sinuses. This has been made possible by theadvent of broad-platform molecular techniques, including micro-arrays and sequencing methods, allowing for identification of thefull microbiome based on microbial RNA. The most commonlyused technique is pyrosequencing to identify bacterial speciesbased on the conserved 16S eubacterial 16S ribosomal gene.43
Techniques to study themicrobial community are rapidly emergingbut include methods to quantify bacterial diversity, representationof specific bacterial species, and bacterial ‘‘load’’ or ‘‘burden.’’
Stephenson et al20 used pyrosequencing with modified 16S eu-bacterial primers to amplify the 600-bp region of 16S rRNAgenes. Eighteen patients with CRS (83% with NPs) werecompared with 9 control subjects. Conventional cultures identi-fied bacterial growth in 82% of patients with CRS (the percentagepositive in control subjects was not mentioned), with an averageof 1.4 isolates per sample. In contrast, the results of pyrosequenc-ing were positive in 100%, with a mean of 10 and a range of 1 to20 isolates per sample. The most prevalent organisms in patientswith CRS were anaerobic bacteria, Diaphorobacter and Peptoni-philus species, whereas coagulase-negative Staphylococcusspecies were identified in 50% of samples. S aureus and Coryne-bacterium and Propionibacterium species were also identified incontrol subjects.
Feazel et al44 obtained middle meatus swabs and comparedbacterial culture and pyrosequencing results in 15 patients withCRS (including 2 with NPs) versus 5 HCs. Standard bacteriologiccultures were positive for all 15 patients with CRS and 5 HCs,with an average of 2.8 isolates reported per subject (range, 1-5isolates). No statistically significant differences were found be-tween the number of isolates obtained from patients with CRSand control subjects. The most commonly cultured organismswere coagulase-negative staphylococci (75%), S aureus (50%),and Propionibacterium acnes (30%). By using pyrosequencing,the most prevalent DNA sequence types detected werecoagulase-negative staphylococci (100%), Corynebacterium spe-cies (85.7%), Pacnes (76.2%), and S aureus (66.7%). In general,cultured organisms comprised less than 15% of the organismsidentified bymeans of sequencing, but bacteria identified by usingculturewere identified bymeans of pyrosequencing inmost cases.Some of the nonspecific CRS culture results, such as ‘‘mixedgram-negative rods,’’ were associated with pyrosequencing detec-tion of moderate mixed anaerobes representing 75% of the totalsequences identified. This confirms a long-held contention ofsome investigators that studies reporting a low prevalence of an-aerobes in CRS samples might have had methodologic flawsrelated to sample collection, bacterial culture, or both.
In the study by Feazel et al,44 S aureuswas selectively enrichedin patients with CRS by means of culture (10/16 patients withCRS vs 0/5 HCs). DNA sequences representative of S aureuswere detected in a similar percentage of patients with CRS(11/16 [69%]) and HCs (3/5 [60%]), but a greater abundance(bacterial burden) of S aureus sequences was found in patientswith CRS. In this study both antibiotic exposure and asthma
were associated with reduced microbial diversity and increased Saureus abundance.
ROLE OF FUNGAL COLONIZATION AND FUNGAL
BIOFILM IN PATIENTS WITH CRSUsing a special technique of ‘‘mucus preservation’’ (sample
collection on saline-moistened nonstick gauze to prevent adsorp-tion of mucus into gauze and prompt transport of samples to thelaboratory for staining), Ponikau et al45 reported that 93% of pa-tients with CRS had detectable fungal hyphae in mucus obtainedfrom diseased sinuses. Virtually all patients with CRS and HCsalso had a positive fungal culture of nasal secretions, with a broadarray of fungi isolated. The latter finding was confirmed by an in-dependent group of investigators who found a positive fungalstain or culture in 87% of patients with CRS and 91.3% ofHCs.46 Gosepath et al47 analyzed tissue specimens from patientswith CRS using a highly sensitive PCR assay and detected fungalDNA in all 27 patients with CRS with both a universal fungalprimer and a primer specific for Alternaria species. In contrast,although the universal primer detected fungal DNA in 10 of 15HCs, none of these subjects had positive results with the Alterna-ria species–specific primer.
Fungal biofilm has also been described in patients with CRS.Healy et al48 used specific bacterial FISH DNA probes and a pan-fungal probe to detect biofilm in mucosal samples from 11 pa-tients with EMCRS and AFRS or CRS. Three mucosal samplesfrom control subjects were also collected. Samples were alsoanalyzed by using epifluorescent microscopy. Bacterial biofilmwas demonstrated in 9 of 11 samples and 2 of 3 control specimens,and fungal elements were detected in association with bacterialbiofilm in the majority of cases.
Foreman et al49 analyzed 50 patients with CRS and 10 controlsubjects for the simultaneous presence of bacterial and fungal bio-film by using bacteria-specific FISH probes and a universal probefor fungus. They identified bacterial biofilm in 72% and fungalbiofilm in 22% of patients with CRS. In this study there weretoo few patients to compare the prevalence of fungal biofilm in pa-tients with EMCRS, AFRS, and CRS.
BARRIER FUNCTION IN SINUS EPITHELIUM AND
ITS RELEVANCE TO CRSEpithelial barrier function is important for maintaining
mucosal hydration and preventing penetration of foreign parti-cles, including microbes, into the subepithelial layer. Defectiveepithelial barrier function has been found to be a key risk factorfor development of atopic dermatitis, leading to increased trans-epidermal water loss and possibly contributing directly toincreased susceptibility to S aureus skin infection.50,51
Tight junction proteins are essential to normal epithelial barrierfunction. They are composed of a branching network of sealingstrands, with each strand acting independently. The key tightjunction proteins are the claudins and the occludins. In theintestine tight junction proteins efficiently restrict most microbesfrom penetrating into the subepithelial tissue.52 Acute reversiblechanges in barrier function have been associated with intestinalviral or bacterial infections, including rotavirus and enteropatho-genic E coli.53 Whether primary defects in epithelial barrier func-tion increase susceptibility to inflammatory bowel disease (IBD)is not presently established.53
No primary defects in epithelial tight junction proteins havebeen described in patients with CRS or NPs. Soyka at al54 foundthat NPs have decreased transtissue resistance and an irregular,patchy decreased expression of the tight junction proteins occlu-din and zonula occludens 1. These effects are likely secondary totissue inflammation because it was shown that in vitro culture ofHCs or NP epithelial cells in the presence of IL-4 resulted indownregulation of occludin and zonula occludens 1 expression,whereas culturing with IFN-g caused an upregulation of theseproteins.
Unlike tight junctions, less is known of the role of adherensjunction proteins and desmosomal proteins in epithelial barrierfunction. Decreased expression of the epithelial adherens junctionprotein E-cadherin was described in NPs and linked toother manifestations of epithelial-mesenchymal transition.55
Decreased expression of the desmosomal junction proteins des-moglein 2 and desmoglein 3 was reported.56 In the latter studyculture of human bronchial epithelial cells with TNF-a or IL-13downregulated desmoglein 2 expression, suggesting that thedefect in desmoglein expression in NPs might be secondary tothe underlying inflammatory process.56
MUCOCILIARY TRANSPORT AND ITS RELEVANCE
TO CRSMucociliary clearance is an essential process in normal sinus
function. Impairments in mucociliary clearance lead to muco-stasis, bacterial colonization, biofilm formation, and CRS.Genetically engineered mice that lack normal cilia spontaneouslyhave bacterial sinusitis.57,58
The classic example of abnormal ciliary function is primaryciliary dyskinesia (PCD; also known as immotile cilia syndrome).PCD is a rare, usually autosomal recessive disorder characterizedby sinopulmonary disease, laterality defects (eg, situs inversus),and male infertility. PCD has traditionally been diagnosed basedon ultrastructural defects in cilia, which are identified in about90% of patients with PCD and involve the outer dynein arms,inner dynein arms, or both.59 Q8Mutational analyses have discov-ered that 38% of patients with PCD carry mutations of the dyneingenes DNAI1 and DNAH5.59 Because it is estimated that normalciliary function involves more than 1000 gene products, addi-tional mutations accounting for the remaining 62% of patientswith PCD are likely to be uncovered. Recently, screening of146 unrelated PCD families identified patients in 6 familieswith reduced outer dynein arms carrying mutations inCCDC103.60 The CCDC103 gene product might function as atightly bound, axoneme-associated protein. A somewhat similarclinical syndrome known as Young syndrome is characterizedby obstructive azoospermia with normal sperm structure andfunction and recurrent sinopulmonary infections.61 Unlike pa-tients with PCD, no mutations in ciliary structural genes havebeen identified in patients with Young syndrome, and the problemhas been presumed to be caused by an abnormality in mucus.62
Several studies have established that CRS is associated with anacquired reduction in mucociliary clearance.63-67 Mucociliaryclearance typically normalizes after clearance of infection andrestoration of normal sinus drainage.63,65,66 This has beenconfirmed by in vitro studies showing that sinonasal mucosal ex-plants from patients with CRS show blunted ciliary beat fre-quency responses to cholinergic and adrenergic stimulation butnormal responses within 36 hours of culturing.68 Lai et al69
reported increased expression of the centrosomal protein 110(Cp110) in inflamed sinus tissue from patients with CRS.Cp110 is known to prevent the terminal step in ciliary maturation.They further showed that Cp110 could be induced in vitro byproinflammatory cytokines in cultured sinus epithelial cellswith concomitant inhibition of ciliogenesis. In contrast, there isno evidence for a primary defect in mucociliary clearance to ac-count for CRS. To date, genetic association studies of patientswith refractory CRS have not uncovered any gene polymorphismsthat would suggest a primary defect in mucociliary clearance.70
HOST DEFECTS IN THE INNATE IMMUNE
RESPONSE ASSOCIATED WITH CRS
Key antimicrobial proteins and peptides in host
innate immunityComposition of sinus mucosal secretions. Fig 1[F1-4/C] 13,71-84
illustrates the general structure of the sinus epithelium, submuco-sal mixed seromucinous glands, and stroma and summarizes themany proteins and peptides produced by sinus mucosal cells.
Submucosal seromucinous mixed glands comprise a series ofducts with interconnecting serous and mucous tubules thatterminate in acini.85 Alcian blue can be used to stain acidic muco-glycoproteins within epithelial goblet cells and submucosalglands, whereas serous glands are not stained.86 Serous glandsare reported to be the major site of production of antimicrobialpeptides, although many of the antimicrobial peptides illustratedare produced by both surface epithelial cells and submucosalglands. Protease-activated receptor 2 has been demonstrated onsubmucosal glands, and a heightened glandular secretory
response to protease-activated receptor 2 agonists, includinghouse dust mite extract, has been described in patients withCRS.87
Lysozyme. Lysozyme catalyzes the breakdown of bacterialcell wall peptidoglycan by cleaving b[1-4]glycosidic linkagesbetween N-acetylmuramic acid and N-acetylglucosamine (http://www.ncbi.nlm.nih.gov/gene/4069). Lysozyme localizes primar-ily to serous cells of submucosal mixed glands and neutrophils,with weak staining of the mucus cells of submucosal mixedglands and goblet cells.71,88 Decreased immunostaining for lyso-zyme was reported in longstanding cases of CRS89; however,more recent studies reported increased rather than decreased im-munostaining in patients with CRS.71,88,90 In the study by Woodset al,90 increased immunoreactivity for lysozyme was found inmucosal biopsy specimens of both patients with CRSsNP andthose with CRSwNP, including low-level immunostaining in theepithelium.
Lactoferrin. Similar to lysozyme, lactoferrin is detectedprimarily in serous cells of submucosal glands.88 Although Zhanget al88 reported an increase in lactoferrin immunostaining in pa-tients with CRS, Psaltis et al91 reported a decrease in lactoferrinat both the mRNA and protein levels in patients with CRS andan even further decrease in expression in the presence of bacterialbiofilm.92
Secretory leukocyte proteinase inhibitor. Secretoryleukocyte proteinase inhibitor (SLPI) protects epithelial tissuesfrom serine proteases, including trypsin, leukocyte elastase, andcathepsin G (http://www.ncbi.nlm.nih.gov/gene/6590). It is acationic protein with a very high affinity for leukocyte elastase.93
It also has antibacterial and other immunomodulatory properties
FIG 1. Structure of the sinus epithelium, submucosal mixed seromucinous glands, and stroma and
summary of proteins and peptides produced by sinus mucosal cells of potential relevance to innate or
adaptive immune responses. Seromucinous glands are stained positively for CXCL1 (GRO-a). Adapted from
references 13 and 71 to 83. *The NGAL protein was observed in the epithelium, infiltrating inflammatory
cells, and submucosal gland of NPs but was rarely detected in normal inferior turbinate tissue.84
MBL-associated serine protease (MASP) 1 and 2 and MBL are soluble proteins that can arise in sinus
and is the third most abundant antimicrobial protein in the upperairways.94 The antibacterial properties of SLPI are thought toreside in the N-terminal domain of the protein, where its activitycould be mediated by its cationic charge.94 SLPI has antibacterialactivity againstPaeruginosa, S aureus, Staphylococcus epidermi-dis, and Candida albicans.94
The role of SLPI in innate immunity and control of inflamma-tion is complex. Furthermore, it has been shown that thedecreased levels of SLPI found in Pseudomonas species–infectedlungs of patients with cystic fibrosis is due to degradation byneutrophil elastase.95 SLPI has been recovered from purulentmaxillary sinus secretions in patients with maxillary sinusitis,96
but there have been no studies linking abnormalities of SLPI toCRS.
Antimicrobial peptides defensins, cathelicidins,
DMBT-1, and S-100 proteins. Defensins and cathelicidinsare the main families of antimicrobial peptides present in airwaysecretions and expressed by the airway epithelium.97 They repre-sent small (3-5 kDa) cationic peptides that are either producedconstitutively or induced by microbial products (including TLRligands), cytokines, or growth factors.
The a-defensins are produced mainly by neutrophils andintestinal Paneth cells, whereas hBD-1, hBD-2, hBD-3 andhBD-4 are primarily produced by epithelial cells.97 In epithelialcells hBD-1 production is constitutive, whereas hBD-2, hBD-3,and hBD-4 production is inducible. Negligible expression ofhBD-2 and hBD-3 was found in normal nasal mucosa.98 Reducedexpression of hBD-2 was found in epithelial cells isolated fromNPs.3
In human subjects only 1 cathelicidin (hCAP-18) is expressed.In neutrophils the cathelicidin proform hCAP-18 is proteolyti-cally processed by proteinase 3, resulting in the release ofLL-37.99 In skin serine proteinases control cleavage of hCAP-18 to LL-37.100 LL-37 was detected in epithelial cells, submuco-sal glands, and inflammatory cells in nasal tissue explants andinduced by culture with fungal allergens from Aspergillus andAlternaria species.101,102 No clear pattern of deficiency in the pro-duction of LL-37 in sinus tissue has been demonstrated in patientswith CRS.102
Bactericidal/permeability-increasing protein and
Plunc family proteins. A family of proteins, includingbactericidal/permeability-increasing protein (BPI) and LPS-binding protein (LBP), has antimicrobial effects against gram-negative bacteria. Both bind to bacterial LPS, causing growtharrest and inhibition of LPS-induced activation of inflammatoryresponses.103,104 Although BPI and LBP are found in airway se-cretions, there has been little study of their levels in nasal or sinussecretions.
Plunc proteins, members of the palate lung and nasal epithe-lium clone (PLUNC) family, are structurally related to LBP andBPI. Recent studies suggest physiologically relevant concentra-tions of PLUNC-inhibitedPaeruginosa biofilm formation in vitrothat do not act directly as a bactericide.105,106Q9 This finding sug-gests that PLUNC protein might have relevance to patients withrefractory CRS.
SPLUNC1 and LPLUNC2 were found to be differentiallyexpressed in serous andmucous cells, respectively, of submucosalglands in uncinate tissue. Decreased SPLUNC1 and LPLUNC2levels were found in NPs relative to those seen in healthy controluncinate tissues, likely reflecting the decreased number ofsubmucosal glands in NPs because their levels correlated with
decreased lactoferrin levels.107 Similarly, Wu et al108 foundPLUNC protein levels to be reduced in NPs and somewhatincreased in patients with CRS relative to HCs, which is consis-tent with other studies showing an increased number of submuco-sal glands in patients with CRSsNP relative to HCs.109
DMBT1 (gp-340). The deleted in malignant brain tumor 1(DMBT1) gene encodes alternatively spliced proteins referred toas gp-340 (DMBT1gp340) and salivary agglutinin (DMBT1SAG).The former is secreted in bronchoalveolar lining fluid, whereasthe latter is present in saliva, but the 2 molecules are identical.DMBT1 interacts with and agglutinates several gram-negativeand gram-positive bacteria, including Streptococcus mutans, abacterium responsible for dental caries. DMBT1 interacts withsurfactant protein A (SP-A) and surfactant protein D (SP-D)and a variety of other host proteins involved in innate immunityor wound healing. A precise role for DMBT1 in human diseasehas yet to be elucidated. DMBT-1 is overexpressed in NPs mainlyin submucosal glands.110
Ficolins and collectins (SP-A, SP-D, conglutinin, and
mannose-binding lectin). Ficolins and collectins (‘‘collage-nous lectins’’) are soluble innate pattern recognition receptors thathave an innate function resembling that of antibodies in adaptiveimmunity.111 Subunits of ficolins and collectins recognize carbo-hydrate arrays of their microbial targets through globular trimericcarbohydrate-recognition domains. SP-A, ficolins, and mannose-binding lectin (MBL) also share structural similarity with C1q,and ficolins and MBL (but not SP-A) activate complement.Whether collectins interact with immune cells through receptorsis unclear.111
There are no studies of ficolin in the upper airway. SP-Aexpression was reported to be increased in sinus mucosal biopsyspecimens in patients with CRSsNP112 and also present in NPs.113
However, Woodworth et al72 found comparable expression of SP-A and SP-D in HCs and patients with CRSwNP. In these studiesSP-A and SP-D immunoreactivity was found in epithelial cellsand submucosal seromucinous glands. One other study reportedreduced immunostaining for SP-D in nasal submucosal glandsand a lack of induction of SP-D byAspergillus andAlternaria spe-cies allergens in cultured nasal explants from patients with nonal-lergic fungal eosinophilic sinusitis.114 In contrast, these allergensinduced SP-D production in HCs and patients with CRS. Rama-nathan et al3 showed that culturing human SNECs Q10in the presenceof the TH2 cytokines IL-4 or IL-13 for 36 hours reduced expres-sion of antimicrobial innate immune genes by using real-timePCR, ELISA, and flow cytometry, including TLR9, hBD-2, andSP-A. In these experiments IL-4 and IL-13 reduced expressionof these innate factors in both HCs and patients with CRS. How-ever, given that mucosal SP-A expression is increased in patientswith CRSwNP, the significance of in vitro suppression of SP-A byIL-4/IL-13 is unclear.
MBL, also referred to as mannose-binding protein, is acalcium-dependent serum protein that binds carbohydrate de-terminants on the surfaces of a wide range of pathogens (viruses,bacteria, fungi, and protozoa), thereby activating the complementcascade or acting directly as an opsonin.115 MBL is a member ofthe collectin family of proteins and has structural similarity tocomplement component C1. MBL deficiency is associated withan increased incidence of upper respiratory tract infections inchildren, presumably because their adaptive immune systemsare still immature. However, a study of MBL deficiency (definedby expression of an MBL deficiency allele) in 9245 Danish adults
found no significant differences in infectious disease prevalenceor mortality in MBL-deficient subjects versus control subjects.116
However, there has been a paucity of studies of MBL levels or ge-notype in a population of patients with CRS. One study foundincreased serum levels of both C3 and MBL in patients withCRSsNP and those with CRSwNP.117
Complement components. The complement componentC3 and serum amyloid A protein are produced by sinonasalepithelial cells.73 Serum amyloid A functions as an acute-phaseopsonin against gram-negative bacteria, including E coli and Paeruginosa, through an outer membrane protein A family mem-ber.118 No differences were found in expression of C3 or serumamyloid A in patients with CRS compared with that seen inHCs.73 Increased expression of factor B, C3, and C5 mRNAwas found in tissues from patients with AFRS and those withCRSwNP compared with that seen in HCs,119 but the functionalsignificance of this is unclear.
NO. NO is constitutively produced at high levels in sinusepithelium by virtue of high constitutive levels of inducible nitricoxide synthase (or NOS2A).120 The NO concentration in ahealthy maxillary sinus (9.1 6 3.8 ppm) exceeds that necessaryfor antibacterial effects in vitro121 and is vastly higher than thatproduced in the nose or lungs (normal exhaled NO value, <50ppb). The antimicrobial effects of NO in the sinuses might alsorelate to stimulation of increased ciliary beat frequency and com-plex reactivities between NO radical superoxide, metals, andthiols.122 NO production is also downstream of innate signalingthrough bitter taste receptors (see below).
Recent experiments have demonstrated an NO-responsivequorum-sensing mechanism in Vibrio harveyi123 and Shewanellaoneidensis124 that regulates biofilm formation. In these bacteriaNO stimulates biofilm formation by controlling levels of the bac-terial cyclic diguanosine monophosphate.124 Whether a similarmechanism exists in other pathogens remains to be explored,but it has been reported that low NO levels (0.9-2.0mmol/L) stim-ulate biofilm formation in S aureus.125
Innate signaling mechanisms through epithelial
cellsMultiple innate pattern recognition receptor pathways are
either known or potentially relevant to sinus epithelium, includingtransmembrane TLRs and intracellular nucleotide-binding olig-omerization domain (NOD) receptors,126,127 dectin receptors, andbitter taste receptors. Table E2 in this article’s Online Repositoryat www.jacionline.org summarizes the pattern recognition recep-tors involved in microbial recognition by airway epithelial cells,their microbial ligands, and abnormalities described in patientswith CRS. Fig 2[F2-4/C] 18,29,128 summarizes some of the salient featuresof host-microbial interactions involved in triggering innate im-mune responses in patients with CRS.
Microbial recognition through TLR receptorsSinonasal epithelial cells express TLRs 1 through 10.74,129
TLR2, TLR3, TLR4, and TLR9 signaling has been demonstratedin sinonasal epithelial cells.13,130 TLR ligation in airway epithe-lial cells results in activation of specific intracellular signalingpathways (reviewed by Bals and Hiemstra131), leading to produc-tion of (1) innate antimicrobial peptides and (2) cytokines andchemokines that amplify innate responses (eg, neutrophil
infiltration) and activate adaptive immune responses. The TLR2receptor has the greatest diversity of ligands and recognizes awide array of gram-positive and gram-negative bacteria, as wellas fungi, in part because of formation of heterodimers withTLR1 or TLR6.132 The TLR4 pathway is extremely importantin host responses to gram-negative bacterial infection in theairway, and polymorphisms in TLR4 have been associated withmore gram-negative bacterial infections in patients in an intensivecare unit.133 The response of human tracheobronchial epitheliumto LPS requires the TLR4 ‘‘coreceptor’’ CD14.134
Studies of TLR signaling pathways have focused on patientswith ‘‘refractory’’ CRS unresponsive to medical management orthose with ‘‘recalcitrant’’ NPs. Increased expression of TLR2 wasfound in patients with recalcitrant CRS.135 An exaggeratedresponse to TLR3 plus cigarette smoke extract was found incultured epithelial cells from patients with CRSsNP, with excessproduction of RANTES and hBD-2.13 Reduced baseline expres-sion of TLR9 was found in cultured epithelial cells from patientswith refractory NPs,130 and this might be linked to the effects oflocally produced TH2 cytokines.3
Microbial recognition through dectin receptorsDectin-1 is a type II transmembrane protein with a C-type
lectin–like carbohydrate recognition domain, a transmembraneregion, and a cytoplasmic tail containing an immunoreceptortyrosinase activation motif.136 Dectin-1 binds specifically tob-1,3 glucans and induces intracellular signaling. Dectin-1 andTLR2/TLR6 signaling combine to enhance the responses trig-gered by each receptor.137,138 Dectin-1 deficiency has been asso-ciated with mucocutaneous candidiasis.139 Although dectinreceptors are regarded as receptors on myeloid cells, Sunet al140 recently showed that dectin-1 receptors could be inducedon airway epithelial cells by Alternaria species in a TLR2-dependent manner with induction of TNF-a, GM-CSF, IL-8,hBD-2, and hBD-9. Although fungal colonization and mucosalresponses to fungi have received much attention, dectin-1 hasnot yet been studied in patients with CRS.
Microbial recognition through NOD-like receptorsNOD2 is a member of the NOD-like receptor protein family
that initiate inflammatory responses when exposed to ligandsderived from bacterial components intracellularly.141,142 Poly-morphisms in NOD2 (CARD15) lead to impaired NOD2 functionand increased susceptibility to Crohn disease (CD), a conditionmarked by excessive inflammatory responses to normal bacterialflora. The ligand for NOD2 is muramyl dipeptide, the ‘‘minimalbioactive peptidoglycan motif common to all bacteria.’’143
NOD2 ordinarily downregulates responses to TLR stimulation,141
and it has been suggested that NOD2 polymorphisms in patientswith CD result in a decrease in negative regulation of TLR re-sponses and a pathologic increase in responses to normal gut flora.Alternatively, it has been suggested that impaired activation of theNOD2 pathway might facilitate invasion of the intestinal epithe-lial cells through commensal or pathogenic bacteria.142
NOD2 defects in patients with IBD are expressed widely inbodily tissues, raising the question as to whether there is anyassociation between IBD and CRS. One study found a prevalenceof chronic ‘‘sinonasal disease’’ of 48% in patients with IBD, witha higher prevalence in patients with CD (53%) versus those with
ulcerative colitis (37%) and an even higher prevalence of chronicsinonasal disease in patients with CD with obstructive bowelcomplications (68% vs 27%), with 23% of these patientsreporting CRS.144 Another study that examined this associationfound a similar prevalence of CRS in patients with IBD as inthe general population, although the proportion of patients withCRS having nasal polyposis was higher among patients withIBD.145 There are no studies linking genetic polymorphisms inNOD1, NOD2, or NOD pathway genes with CRS.
Microbial recognition through bitter taste receptorsLee et al146 recently discovered that one of the bitter taste re-
ceptors, T2R38, is activated by a quorum-sensing moleculefrom Paeruginosa associated with biofilm formation. Bitter tastereceptors are a family of G protein–coupled receptors that signalby inducing a transient intracellular calcium flux and stimulatingciliary beat frequency. Activation of the receptor induces
production of NO and increases ciliary beat frequency in sinusepithelial cells. A common polymorphism (TAS2R38 variant)was discovered that is associated with reduced signaling, NO pro-duction, and ciliary beat frequency and increased growth of Paer-uginosa in air-liquid cultures of human airway epithelial cells.The effect of TAS2R38 on the killing of Paeruginosawas shownto be NO dependent. The TAS2R38 genotype correlated with thepresence of sinonasal gram-negative infection in patients withCRS, suggesting amechanistic link between a deficiency in innatesignaling and increased bacterial infection.
Adaptive immune antimicrobial signaling
mechanisms: IL-17A and IL-22 signaling pathwaysIL-17A and IL-22 are ‘‘signature’’ cytokines of TH17 cells
involved in host defense against extracellular pathogens,including fungi, bacteria, and some parasites.139 IL-22 is consid-ered an ‘‘essential guardian of mucosal immunity against
FIG 2. Salient features of host-microbial interactions involved in triggering innate immune responses in
patients with CRS. TLR signaling pathways induce proinflammatory cytokine and chemokine production.
Bitter taste receptor is activated by a quorum-sensing molecule from P aeruginosa and stimulates produc-
tion of NO, which then stimulates mucocociliary clearance and has direct antimicrobial effects. Depiction of
the intracellular TLR signaling pathways was adapted from the IAVI Report (http://www.iavireport.org/Back-
Issues/Pages/IAVI-Report-9(4)-TollBridgetoImmunity.aspx). Upper right inset (left panel), SEM of bacterial
biofilm showing characteristic glycocalyx and water channels. The photograph was used with permission
from Sanclement et al.29 Right panel, CSLM image (363 magnification) of a patient with CRS stained with
the BacLight LIVE/DEAD kit (Invitrogen, Molecular Probes, Carlsbad, Calif) demonstrating a bacterial biofilm
comprised of many intensely fluorescing live bacteria organized in clusters (large arrow). Small arrowsdesignate the larger live and dead epithelial cells. Used with permission from Psaltis et al.18 The bitter taste
receptor depicted in the small middle insetwas adapted from Fenech C, Patrikainen L, Kerr DS, Grall S, Liu Z,
Laugerette F, et al. Ric-8A, a Ga protein guanine nucleotide exchange factor potentiates taste receptor
extracellular bacteria in the lung and gut.’’147 In intestinal epithe-lial cells IL-22 stimulates the production of a wide variety of anti-bacterial proteins, stimulates mucin 1 production underinflammatory conditions, and enhances epithelial regenerationwith goblet cell restitution.148 IL-22 deficiency in mice is associ-ated with a more severe form of gram-negative enteric infectionwith Citrobacter rodentium, with increased intestinal epithelialdamage, systemic bacterial burden, and mortality. In this modelIL-22 mediates production of Reg family antimicrobial proteins,including RegIIIb and RegIIIc, by colonic epithelial cells. Both Tcells, including CD41 cells and innate lymphoid cells, and den-dritic cells produce IL-22.149-151 A subset of CD41 helper T cellsthat produce IL-22 but not IL-17 has been identified in humansubjects and termed TH22 cells.151
IL-22 also protects the lungs from gram-negative Klebsiellapneumonia infection.152 In conjunction with IL-17A or IL-17F,IL-22 synergistically induces expression of hBD-2 and S100A9and additively enhances the expression of S100A7 andS100A8.153 IL-22–mediated effects on airway epithelial cellsrequire signaling through the IL-22 receptor (IL-22R) and activa-tion of signal transducer and activator of transcription 3.154 Il-23,produced mainly by phagocytes and dendritic cells,155 is a crucialupstream regulator of IL-22 and IL-17 production.152,156
Although IL-22 and IL-17A act synergistically or additively topromote the expression of genes involved in mucosal defense,TH17 and TH22 cells likely serve different roles inmucosal immu-nity and autoimmunity.147,154
IL-17A and IL-22 also have an established role in antifungalimmunity at mucosal surfaces,157 which would suggest thatdefects in the signaling pathways of these cytokines in the sinusmucosa might be associated with difficulty handling fungal infec-tion. However, this has not been studied.
To date, no definite defect in IL-17A or IL-22 signaling hasbeen identified in patients with CRS. Using a genome-wideassociation study, Endam et al158 identified a polymorphism inIL-22R in association with refractory CRS, but no functionalstudies were performed to explore this relationship. ReducedIL-22R1 expression in sinus mucosal epithelial cells was foundin patients with recalcitrant NPs, and a relationship was found be-tween reduced expression of IL-22R1 and a higher rate of recur-rence of NPs after sinus surgery.159
Role of bacteria or fungi in maladaptive TH2
responses in patients with CRSIt has long been recognized that CRS is a disease in which the
local tissue inflammatory response might be strongly biasedtoward TH2 inflammation despite a lack of systemic evidence forallergic disease. This is particularly true in patients withCRSwNP160 but also true, to a lesser degree, in patients withCRSsNP.161-164 There is evidence that links colonizing microor-ganisms to this maladaptive TH2 ‘‘local allergic’’ response in pa-tients with CRSwNP (schematized in Fig 3 [F3-4/C]). This has been shownin cultured airway epithelial cells and dispersed T lymphocytesfrom NPs.
Fungi are commonly detected in the attached mucus of sinustissues in patients with CRS47,165 and can induce eosinophil acti-vation and degranulation.166 Certain fungi, particularly Alterna-ria and Candida species, were shown to induce production ofIL-5 and IL-13, as well as IFN-g, in peripheral blood lymphocytesfrom patients with CRS. It was hypothesized that this maladaptiveresponse would account for an eosinophilic mixed TH1/TH2mucosal immune response.165 Fungal allergens also elicit modestproduction of IL-5 and IL-13 from dispersed NP T lympho-cytes.167 However, a recent study did not demonstrate consistentIL-5 production in response to Alternaria species in patients withCRS from Utah.168 A small randomized placebo-controlled trialof intranasal amphotericin B reported improvement in radio-graphic mucosal thickening, nasal endoscopy, and eosinophilicinflammation.169 However, 2 other randomized placebo-controlled trials with intranasal antifungal treatment did notdemonstrate a significant clinical benefit.170,171 In addition, anti-fungal treatment did not alter the cytokine or chemokine produc-tion profiles of nasal inflammatory cells.172
Mucosal colonization with S aureus has been found in 64% ofpatients with CRSwNP compared with roughly 30% of healthysubjects or patients with CRSsNP.173 In a study of 13 patientswith massive polyposis, 55% of patients were found to haveenterotoxin-producing S aureus in the nasal mucus adjacent topolyps.174 It was further shown that T lymphocytes isolatedfrom the polyps showed a skewing of Vb use with enrichmentfor Vb known to respond to staphylococcal superantigens.174
This finding was later confirmed by another group.175,176 IgE an-tibodies directed against staphylococcal superantigens were
FIG 3. Normal host response to microbial infection (left panel) versus maladaptive TH2 response
(right panel). The maladaptive TH2 response has been best demonstrated in patients with NPs and pertains
found in NP homogenates in 27.8% of patients with NPs and53.8% of patients with NPs with coexisting asthma.173 Finally,staphylococcal enterotoxin B (SEB) was found to induce robustproduction of IL-5 and IL-13 in dispersed NP T lymphocytes.167
These studies suggest that colonizing S aureus might be a majordriver of the local TH2 inflammatory response in patients withCRSwNP.
Staphylococcal superantigens, such as SEB, have been shownto impair oral tolerance and promote allergy in a murine model offood allergy.177 SEB has also been shown to augment allergicinflammation in a murine model of eosinophilic CRS, leadingto formation of nasal polyposis.55
acteristic of patients with CRSwNP, investigations were under-taken to ascertain whether TH2-type inflammation modulatedinnate immune function. Ramanathan et al130 first demonstratedthat cultured primary nasal epithelial cells (PNECs) from HCsand patients with CRSwNP express surface TLR9 and respondto CpG (a TLR9 agonist) by increasing production of hBD-2and IL-8. They found that TLR9 expression on PNECs from pa-tients with CRSwNP was reduced by 50% compared to controlPNECs. Culturing control PNECs in the presence of the TH1 cyto-kine IFN-g increased TLR9 expression by 49%, whereasculturing in the presence of the TH2 cytokines IL-4 or IL-13decreased TLR9 expression by 46.6%. Because this group previ-ously reported reduced epithelial expression of IL-22R1 in pa-tients with recalcitrant NPs, it would be interesting to knowwhether TH2 cytokines would induce this effect.
Is there an inappropriate or heightened response to
colonizing ‘‘commensal’’ organisms in patients
with CRS?Epithelial surfaces have adapted specialized mechanisms for
coping with potential intruders. These not only protect the hostfrom infection but also provide beneficial effects in terms ofdigestion (in the gut) and immune maturation (gut and respiratorytract).Mechanisms have also evolved to copewith ‘‘commensals’’in the gut and presumably also in the oral mucosa and nasalepithelium, where bacterial colonization is normal. It has beensuggested that CRS might have a proinflammatory response tocommon colonizing organisms, such as coagulase-negativeStaphylococcus species.178 Could it be that the ‘‘proinflamma-tory’’ state of CRS relates to a lack of tolerance to seeminglyinnocuous ‘‘commensal’’ organisms? We do not know, but thisrepresents an exciting area for future investigation.
SUMMARY/CONCLUSIONSThere is limited information indicating that patients with CRS
have an increased incidence of rhinovirus infection and somesuggestion that they might have an exaggerated response to viralinfection, but there is no evidence for persistent viral infection inthe majority of cases. It would be instructive to map the timecourse and innate immune response to experimental rhinovirusinfection and ascertain whether patients with CRS have increasedsusceptibility, increased viral burden, or an inappropriate innateimmune response.
A role for bacterial infection or colonization in CRS patho-genesis is well established. Bacteria are cultured and bacterialbiofilm is present in the majority of patients undergoing surgicalintervention. Diseased sinus tissue also has an increased bacterialburden, most convincingly demonstrated for S aureus, and bacte-rial biofilmwith other organisms, including Paeruginosa, is oftenpresent. The majority of patients with CRS also have fungal hy-phae detectable in mucus extracted from diseased sinuses, andfungal biofilm is detectable in some cases, usually in associationwith bacterial biofilm. The presence of bacterial biofilm, particu-larly polymicrobial biofilm or biofilm containing S aureus, hasprognostic value and is associated with more severe sinus diseasepreoperatively and worse symptom and nasal endoscopy scoresafter surgery.
Mucociliary clearance is essential for normal sinus function.Active disease is associated with a reduction in mucociliaryclearance but normalization of mucociliary clearance afterclearance of infection and restoration of normal sinus. There isno evidence for a primary defect in mucociliary clearance toaccount for CRS, except in the distinct clinical syndrome of PCD.
A defect in local host innate immunity has long been suggestedas a pathogenic mechanism of CRS to account for CRS,especially because systemic immune function is normal in thevast majority of cases. Decreased levels of the antimicrobialproteins, most notably lactoferrin, have been found most often insinus secretions, whereas levels of other antimicrobial proteinsand peptides have been reported to be normal.
Multiple innate pattern recognition receptor pathways, notablyTLRs and intracellular NOD receptors and bitter taste receptors,are critical in host/microbial interactions in the sinuses. Noprimary defects have been found in these pathways to account forrefractory CRS, although a 50% reduction in the expression ofTLR9 has been found in patients with refractory NPs.
A downregulation of epithelial innate immunity by maladap-tive TH2 tissue inflammation has been demonstrated in patientswith recalcitrant CRSwNP. In vitro studies have shown thatIL-4 and IL-13 decrease TLR9 expression on cultured epithelialcells by nearly 50% and also reduce expression of hBD-2 andSP-A. Colonizing fungi might play a role in maladaptive TH2 re-sponses and thereby promote tissue eosinophilia. Colonizationwith enterotoxin-producing S aureus is a driver of the local TH2response in patients with CRSwNP because it induces a localexpansion of T lymphocytes and production of enterotoxin-specific IgE antibodies.
To date, an effective means of restoring host-microbialbalance and mitigating disease in patients with CRS remainselusive. There are few studies on the effectiveness of medicaltreatment alone at eradicating bacterial infection and bacterialbiofilm in patients with CRS. Clinical trials of antifungal rinseshave been generally unsuccessful. Surgical removal of diseasedsinus tissue with restoration of sinus ventilation and use ofculture-directed antibiotics remain the best approaches totreatment. There is hope that further elucidation of the geneticunderpinnings of CRS and the host-microbial interactionspresent will provide greater insight into disease pathogenesisand more effective treatment.
PATIENTS WITH CRSOne study using sensitive molecular techniques (PCR) found
no evidence for Mycoplasma pneumoniae, Chlamydia pneumo-niae, or Legionella pneumophila in mucosal samples from 11 pa-tients with CRS.E1Q11 Recent studies used pyrosequencing topotentially overcome issues of detection of nonculturable bacteriain mucosal samples from patients with CRS; did not detectMyco-plasma, Ureaplasma, and Chlamydia species; and found Legion-ella, Mycobacterium, and Nocardia species in only rarecases.E2,E3
Tichenor et alE4 identified 8 patients with CRS with refractorysymptoms who had positive cultures for atypical mycobacteriaover a 10-year period. Mycobacterium avium molecular finger-prints clonally related to the patients’ isolates were also identifiedin the plumbing of 3 of 8 households sampled, suggesting that atleast in these 3 cases, indoor plumbing and sinus irrigations mighthave been responsible for the infection.
Solyar et alE5 reported on a series of 37 patients with refractoryCRS who had a positive mycobacterial culture over a 7-yearperiod. The most frequent isolates were Mycobacterium absces-sus (57.1%), Mycobacterium avium-intracellulare complex(14.3%), and Mycobacterium chelonae (14.3%). It was reportedthat 5.3% of outpatient-acquired and 2.6% of inpatient-acquiredcultures were positive for atypical mycobacteria. Some evidenceof improvement in CRS was noted after treatment with antimyco-bacterial antibiotics. These studies suggest that atypical myco-bacterial infection is rare in patients with CRS but should besought in patients with refractory CRS.
TECHNIQUES FOR BACTERIAL BIOFILM
DETECTION IN PATIENTS WITH CRSSee Table E2 for a summary of studies of bacterial or fungal
biofilm in patients with CRS.SEM and TEM have the advantage of high magnification and
are the only techniques that provide ultrastructural confirmationof biofilm presence. Technical artifacts can arise because ofsample dehydration and surface distortion. Disadvantages ofSEM include small sample size, potentially introducing samplingartifacts. CSLM has the advantage that specimens can be imagedwithout fixation or dehydration, and specific bacteria or fungi canbe stained with fluorescent markers. The FISH assay uses eitheruniversal bacterial probes, such as EUB338,E6 or species-specificprimers based on unique sequences in the 16S ribosomal RNAgene. The LIVE/DEADBacLight Bacterial Viability Kit (Invitro-gen, Carlsbad, Calif) uses 2 nucleic acid stains: green-fluorescentSYTO 9 stain, which labels both live and dead bacteria, and red-fluorescent propidium iodide, which penetrates only bacteria orcells with damaged membranes. CSLM is used to confirm thepresence of biofilm on the mucosal surface. No bacteria-specificprobes are used.
A direct comparison of techniques in a sheep model ofrhinosinusitis concluded that CSLM is more objective fordocumenting biofilm presence than SEM or TEM because ofthe inherent flaws, sampling error, and subjectivity involved inSEM and TEM.E7 Another study determined that the BacLightmethod was more specific than CSLM/FISH for biofilm detectionin sinus tissues.E8,E9 Hochstim et alE6 reported that bacterial bio-film can be detected by using routine hematoxylin-eosin staining,with good agreement with FISH/CSLM as the control standard.
However, this technique is suspect because of its reliance on iden-tification of small basophilic bacterial clusters on the epithelialsurface. A concern is whether any of the techniques other thanSEM or TEM are sufficiently rigorous to define biofilm becausethey do not provide ultrastructural confirmation of the presenceof biofilm.
BIOFILM-FORMING CAPACITY IN VITROThe Calgary Biofilm Assay is an in vitro test used to assess the
ability of a bacterial isolate to form biofilm. The bacteria arecultured on a 96-well plate with round pegs positioned over theplate. A semiquantitative analysis using uptake of crystal violetin the biofilm has been developed.E10 The LIVE/DEAD BacLightBacterial Viability Kit (see above) can also be used to assess bio-film formation in vitro on 98-well chamber slides.E11 Princeet alE12 used the Calgary Biofilm Detection Assay to examinein 159 patients with CRS with mucopurulence and found that28.6% of patients had biofilm-forming capacity. Polymicrobialcultures, P aeruginosa, and/or S aureus comprised 71% of sam-ples. Postsurgical cases had a higher prevalence of biofilm-forming capacity than surgery-naive patients (30.7% vs 15%).
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E1. Lee RE, Kaza S, Plano GV, Casiano RR. The role of atypical bacteria in chronic
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Molecular characterization of the polymicrobial flora in chronic rhinosinusitis.
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