Pattern recognition receptors

The innate immune system recognizes specific molecular structures known as pathogen-associated molecular patterns (PAMPs), which are conserved and considered essential for the survival of pathogens. The responses of the innate immune system against these PAMPs signal the adaptive immune system to develop memory and subsequent longer-lasting immune responses.

Toll proteins were originally discovered in the fruit fly Drosophila and led to the identification of a family of transmembrane pattern recognition receptors known as Toll-like receptors (TLRs) in humans. TLRs are transmembrane glycoproteins with an extracellular N -terminal leucine-rich domain and an intracellular C -terminal domain, known as the Toll/interleukin (IL)-1 receptor (TIR) domain because it shows homology with the IL-1 receptor. TLRs are differentially expressed between cells, with some expressed on the cell surface and others expressed intracellularly within endosomes and the endoplasmic reticulum ( Table 1 ). The recognition of various PAMPs by the TLRs induces a cascade of downstream signaling to induce inflammatory cytokine signaling that can be the Th1, Th2, or type I interferon (IFN)-γ cytokine profile. TLR signaling pathways are complex but can be essentially categorized as myeloid differentiation primary response (MyD88)–dependent or TIR-containing adaptor-inducing IFN-γ–dependent pathways. The MyD88-dependent pathway is used by all TLRs except for TLR3.

Research in the area of TLRs and rhinology has shown mRNA expression of TLRs in cultured nasal epithelial cells and sinonasal biopsies from patients with CRS. Immunostaining of sinonasal biopsy specimens has shown that TLR-2 is localized to the stroma deep to the epithelial surface, whereas TLR-4 is predominantly in the submucosal seromucinous glands. Patients with both allergic and nonallergic rhinitis show reduced mRNA expression of TLR-2 in turbinate tissue biopsies compared with controls. TLR-2 and -4 mRNA expression was reduced in patients with nasal polyp with fungal and bacterial colonization, suggesting a possible relationship between fungus and TLR expression in nasal polyps. Stimulation of TLR-2 and -4 increased the in vitro phagocytic ability of neutrophils and eosinophils against Aspergillus conidia , suggesting that manipulation of TLRs may aid in the clearance of fungi.

The genome of DNA viruses contains unmethylated CpG DNA that are DNA motifs recognized by TLR-9. Host CpG DNA, however, is highly methylated and therefore host DNA does not stimulate TLR activation. This mechanism is useful to promote tolerance of normal cells. Use of CpG-oligonucleotides in a nasal mucosal explant model in vitro showed some possibility in reducing the expression of the cytokine IL-5.

Other innate immune pattern recognition receptors have been discovered recently. These include retinoic acid–inducible–like receptors (RLRs) and nucleotide binding and oligomerization domain (NOD)–like receptors (NLRs). Although little is known about the molecules in the field of rhinology, other areas of scientific research have provided some insight into their possible function. RLRs are intracellular receptors important for detecting RNA derived from RNA and DNA viruses. NLRs are another family of intracellular receptors consisting of 23 proteins in humans. These proteins consist of a central NOD and a C -terminal domain with leucine-rich repeats.

In summary many questions remain regarding the role of TLRs, RLRs, and NLRs and their role in CRS. These include defining their expression in different subgroups of CRS, and how they respond to various bacteria, fungi, biofilms, and in association with other innate immune peptides. Research is being conducted in these areas.

Cystic fibrosis and chronic rhinosinusitis

Cystic fibrosis is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane regulator ( CFTR ) gene, with the most common being the Δ F508 mutation. Cystic fibrosis is a recognized genetic predisposition condition associated with CRS because of its mucociliary dysfunction and chronic bacterial or fungal colonization. Nasal polyps are present in approximately 40% of patients with cystic fibrosis, but the polyps exhibit predominantly neutrophilic, rather than eosinophilic, inflammation. Recently, Pinto and colleagues identified eight individuals with CRS within an isolated small group of people who practice a communal lifestyle and share common environmental exposures. Linkage analysis and DNA genome screening were used on these eight individuals, who were also related to each other. A strong linkage signal among these individuals was identified at 7q31.1 to 7q32.1, which is near the CFTR gene.

Previous studies have shown that carriers with a cystic fibrosis mutation have a higher prevalence of CRS compared with the general population. Similarly, pediatric patients with CRS who did not meet the criteria for a diagnosis of cystic fibrosis were screened for and found to have a higher prevalence of cystic fibrosis variant mutations than would be expected in a general population. Other innate immune defects have been identified in cystic fibrosis. Epithelial cells use CFTR to internalize Pseudomonas aeruginosa and thereby remove it from the airway surface. Experts believe that in patients with cystic fibrosis, epithelial cells are unable to remove P aeruginosa because of mutations in the CFTR gene. These patients also show reduced activity of antimicrobial peptides in lung surface fluid, which is believed to be caused partly by the high salt conditions in cystic fibrosis. Further studies are required to investigate the possibility that carriers of variant mutations in the CFTR gene who do not have clinical cystic fibrosis are predisposed to the development of recalcitrant CRS.

Cystic fibrosis and chronic rhinosinusitis

Cystic fibrosis is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane regulator ( CFTR ) gene, with the most common being the Δ F508 mutation. Cystic fibrosis is a recognized genetic predisposition condition associated with CRS because of its mucociliary dysfunction and chronic bacterial or fungal colonization. Nasal polyps are present in approximately 40% of patients with cystic fibrosis, but the polyps exhibit predominantly neutrophilic, rather than eosinophilic, inflammation. Recently, Pinto and colleagues identified eight individuals with CRS within an isolated small group of people who practice a communal lifestyle and share common environmental exposures. Linkage analysis and DNA genome screening were used on these eight individuals, who were also related to each other. A strong linkage signal among these individuals was identified at 7q31.1 to 7q32.1, which is near the CFTR gene.

Previous studies have shown that carriers with a cystic fibrosis mutation have a higher prevalence of CRS compared with the general population. Similarly, pediatric patients with CRS who did not meet the criteria for a diagnosis of cystic fibrosis were screened for and found to have a higher prevalence of cystic fibrosis variant mutations than would be expected in a general population. Other innate immune defects have been identified in cystic fibrosis. Epithelial cells use CFTR to internalize Pseudomonas aeruginosa and thereby remove it from the airway surface. Experts believe that in patients with cystic fibrosis, epithelial cells are unable to remove P aeruginosa because of mutations in the CFTR gene. These patients also show reduced activity of antimicrobial peptides in lung surface fluid, which is believed to be caused partly by the high salt conditions in cystic fibrosis. Further studies are required to investigate the possibility that carriers of variant mutations in the CFTR gene who do not have clinical cystic fibrosis are predisposed to the development of recalcitrant CRS.

Mucociliary function and innate immunity

The mucociliary clearing action of epithelium in the sinonasal tract is a key innate immune function and the primary defense mechanism against inhaled particles. This function occurs through the production of mucus, which traps inhaled particles, followed by the active process of ciliary beating, transporting the trapped mucus and particles toward the pharynx where they are swallowed. The sinonasal mucosa is lined by ciliated respiratory epithelium with goblet cells that produce mucus.

Messerklinger is credited with showing the distinct pattern of flow of mucociliary clearance and mucus in each sinus. The sinuses are dependent on healthy mucociliary clearing to transport mucus, bacteria, fungus, and other material from the sinus. Stripping of the mucosa has been shown to result in abnormal mucosal regeneration, thus removing this essential innate immune function. This finding has been the basis for the mucosa preservation approach of endoscopic sinus surgery. CRS is associated with decreased mucociliary clearing, with demonstration of reduced saccharin transit time. Impaired mucociliary clearing leads to stasis of the mucus and secretions, theoretically leading to ongoing colonization and potentially inflammation. However, more than 3 months after endoscopic sinus surgery, the mucociliary clearing showed significant improvement with regeneration of cilia, suggesting that impaired mucociliary clearing was secondary to the chronic inflammation rather than was the primary defect.

The anatomy, physiology, and biochemistry of mucociliary clearing have been reviewed previously. Briefly, mucociliary clearing is dependent on the properties of mucus and coordinated beating action of the cilia, and uncertainty still exists as to whether a decrease in mucociliary clearing in patients with CRS is caused by the mucus, ciliary dysfunction, or both. Cilia beat spontaneously at a basal ciliary beat frequency (CBF), a function that is dependent on intracellular ATP and increases in response to various stimuli. Ciliary beating increases in response to fever, stress, exercise, adrenergic, cholinergic, and purinergic stimuli.

A study by Chen and colleagues showed a significant difference in basal and stimulated CBF of sinonasal mucosal explants between patients who had CRSwNP and controls. In another study, the same investigators compared controls and patients with CRSsNP using cholinergic and adrenergic stimulation to determine if the stimulating effect seen with ATP was unique to purinergic stimuli. The most significant effect with impairment of stimulated CBF was seen in the CRSsNP group. Tissue dissociation was performed to isolate the epithelial cells and maintained ex vivo in submersion. Daily observation of basal and stimulated CBF found that 72% of these cultures resumed ciliary action within 36 hours, suggesting that the blunting of ciliary effects was caused by chronic inflammation and that epithelial cells isolated from this chronic inflammatory environment have a chance of regaining normal ciliary function.

Future work investigating immunomodulatory agents that can stimulate CBF or improve ciliogenesis (eg, topical retinoic acid ) will improve understanding and treatment of CRS.

Cigarette smoking, innate immunity, and chronic rhinosinusitis

Smoking is a well-recognized adverse factor in CRS associated with worse outcomes after endoscopic sinus surgery. A retrospective chart review and complex multiple linear logistic statistical analysis found that patients who ever smoked had a 43-fold risk for developing nasal polyps. Smokers and nonsmokers undergoing septoplasty surgery were recently compared. The group that did not smoke had significantly shorter mucociliary clearing times. Cigarette smoke is known to cause airway epithelial hyperplasia, submucosal gland hypertrophy, goblet cell hyperplasia, epithelial permeability, and squamous cell metaplasia. The effects of cigarette smoke may be caused by mainstream, side stream, or exhaled smoke; gaseous, particulate, or aqueous phase components; or individual chemical compounds. The chemical compounds in cigarette smoke include nicotine, tar, ammonia, carbon monoxide, formaldehyde, acrolein, acetone, benzopyrenes, hydroxyquinone, nitrogen oxides, and cadmium. Cigarette smoke extract at high concentrations causes cytotoxicity of primary human nasal epithelial cells. Cigarette smoke condensates (particle phase) and extracts (volatile phase) significantly impaired ciliogenesis in an in vitro mouse nasal epithelium culture model. These findings may explain the poorer outcomes seen in patients who continue to smoke after surgery.

The effects of cigarette smoke on the innate immune system are not as well studied, but several recent papers suggest that cigarette smoke suppresses the innate immune function of sinonasal epithelial cells. Cantin and colleagues showed that cigarette smoke exposure using gaseous and aqueous extracts can suppress CFTR mRNA and protein expression in airway cell lines in vitro. Therefore, they surmised that cigarette smoke might induce a form of CFTR suppression. CFTR is important for internalization of P aeruginosa , and cigarette smoke may affect CFTR function in patients who do not have cystic fibrosis, therefore impairing the innate immune system.

TLR-4 mRNA expression was found to be down-regulated in nasal brushings of smokers and in a dose-dependent fashion in vitro in an airway epithelial cell line. The effects of acrolein, an aldehyde compound in cigarette smoke, on primary culture of human sinonasal epithelial cells were recently studied. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) showed that IL-8 and human ß-defensin 2 mRNA and protein expression decreased in a dose-dependent manner because of acrolein. Unfortunately, this study did not include a normal control group for comparison.

Smoking has a substantial effect on reducing surfactant protein (SP)-D levels in bronchoalveolar fluid (BALF), with former smokers having reduced levels compared with those who never smoked, suggesting that cigarette smoke may have a long-lasting effect on SP-D even after they have stopped smoking. Further research is required to determine if a similar reduction in SP levels are seen in sinonasal mucosa of smokers.

In summary, these studies indicate that various components of cigarette smoke can affect the innate immune response in many ways. Most studies on cigarette smoke and innate immunity study the effects from an acute exposure rather than chronic exposure standpoint. Studies on innate immunity in CRS usually do not stratify patients who smoke as a separate subgroup for study. However, the effects of cigarette smoking on innate immunity seem significant, and therefore a need exists to determine what these are.

Antimicrobial peptides, biofilms, and chronic rhinosinusitis

Numerous antimicrobial peptides are expressed in the sinonasal and lower airway epithelium. These include lactoferrrin, lysozyme, cathelicidins, defensins, SP-A and -D, acid mammalian chitinase, collectins, serum amyloid A, and secretory leukocyte proteinase inhibitor. Lysozyme, lactoferrin, and secretory leukocyte proteinase inhibitor are the most abundant antimicrobial peptides in nasal secretions. The antimicrobial peptides are secreted in response to microbes, thus inhibiting or directly lysing the microorganism through endogenous antibiotics. These peptides also have other immune functions and can attract and activate effector cells of the innate and adaptive immune system. For example, the human cathelicidin LL-37 is chemotactic for neutrophils, monocytes, and T cells. Conversely, these peptides can also suppress or modulate the adaptive immune system. SP-D has been shown to inhibit lymphocyte proliferation, cause a shift from a Th1 to Th2 cytokine response, and regulate dendritic cell responses.

SP-A and -D are members of the collectin family of proteins involved in innate immunity. Deficiency of SP-A and -D in patients with cystic fibrosis is believed to result in increased frequency of bacterial colonization, infections, and inflammation. In the study by Postle and colleagues, the levels of SP-D protein in BALF were significantly decreased in patients with cystic fibrosis (median, 0.1 μg/mL) compared with the acute infection (12.17 μg/mL) and control (641 μg/mL) groups.

SP-A and -D mRNA and protein have been identified in the sinonasal mucosa of healthy controls and patients with CRS. Woodworth and colleagues showed that SP-A and -D mRNA levels were up-regulated in the CRS and cystic fibrosis groups. Ooi and colleagues showed that SP-D protein levels were undetectable using ELISA or immunohistochemistry in patients with allergic fungal rhinosinusitis (AFRS) and were decreased in those with CRS relative to the normal controls.

SP-A, -D, and tumor necrosis factor α protein levels were investigated in sinus mucosal biopsies from cystic fibrosis, AFRS, and controls using semiquantitative Western blot analysis. The cystic fibrosis group had reduced SP-A, -D, and tumor necrosis factor α protein levels compared with controls but higher levels than the AFRS group.

BALF collected from patients with chronic obstructive pulmonary disease (COPD) and healthy control subjects was subjected to multivariate linear regression analysis. This study showed reduced SP-D levels in the group with COPD but higher levels in those using inhaled corticosteroids. The investigators evaluated the effects of dexamethasone on type II alveolar epithelial cells from rat lungs and found that SP-D mRNA and protein, but not SP-A, increased after 4 days of culture with dexamethasone (10 ng/mL). Experts speculate that corticosteroids in CRS may boost SP-D levels and be a potent anti-inflammatory treatment.

In summary, several studies have shown reduced levels of secreted SP-A and -D in patients with cystic fibrosis. However, no studies have investigated secreted SP levels in CRS. One study showed reduced secreted SP-D levels in CRS in response to fungal extract challenge compared with normal controls, but that was a mucosal explant in vitro model. Further research is required to investigate the levels of SPs produced and secreted in patients with CRS compared with normal controls, and to study the effects of corticosteroids on SP-A and -D levels in larger studies with sinus biopsies and human cell cultures.

Biofilms are organized communities of microbial cells encased in a self-produced exopolysaccharide matrix and irreversibly attached to an inert or living surface. Most bacteria exist in this biofilm form, with only 1% residing in the free-floating or planktonic form, which is the form that clinicians commonly culture. Through intercellular signaling called quorum sensing , planktonic bacteria are believed to undergo genotypic and phenotypic alterations, converting them into the more robust and resistant biofilm form. This form has a much slower growth and metabolic rate, and in its dormant state can evade the host’s immune system.

Biofilms have been implicated as a potential cause of CRS in a subset of patients, with these structures identified in sinus biopsies taken from patients undergoing sinus surgery. The study by Psaltis and colleagues also found that patients with biofilms seemed to have more clinical and endoscopic evidence of ongoing postoperative inflammation, and speculated that these structures may explain the recalcitrant nature of CRS. This study also showed that the presence of fungus was associated with an adverse outcome after endoscopic sinus surgery. Using the modified Calgary Biofilm Detection Assay, Prince and colleagues showed that 28.6% (45/157 samples) of bacteria cultured in sinuses from bacterial swabs of patients with CRS undergoing endoscopic sinus surgery can form biofilms in vitro. The most common organism cultured was Staphylococcus aureus , followed by polymicrobial infections and P aeruginosa . More recently, pan-fungal and specific bacterial fluorescent in situ hybridization probes showed the presence of fungus within bacterial biofilms of patients with AFRS, CRS and eosinophilic mucin rhinosinsitis.

Topical treatments are an evolving area of interest in the medical management of refractory CRS that has failed to respond to standard treatments. Their use partly evolved from experience with topical tobramycin in treating chronic Pseudomonas infections in patients with cystic fibrosis, in whom a much higher dose of antibiotic can be delivered locally to achieve a higher minimal inhibitory concentration without causing adverse effects usually associated with systemic administration.

Research on topical treatments has focused mainly on biofilms and fungus. For example, regular treatment with topical mupirocin for 7 days has been shown to have the most significant effect on inhibiting biofilm formation in a sheep model of Staphylococcal biofilm sinusitis.

One area of interest is the possibility of using endogenous antimicrobial peptides as topical treatments for CRS. The cathelicidin LL-37 has antibacterial and antifungal properties and is being explored as a novel approach to treating infections and inflammation. Synthetic LL-37 has recently been shown in vitro to have antibiofilm properties when cultured with P aeruginosa at concentrations below what is required to kill the bacteria. P aeruginosa cultured with 0.5 μg/mL LL-37, which is far below the concentration required to kill or inhibit bacterial growth (1/128 minimal inhibitory concentration), led to a 40% decrease in biofilm mass. An increasing dose-dependent effect was seen, with a maximum of 80% inhibition at 16 μg/mL LL-37 (1/4 minimal inhibitory concentration).

LL-37 was also shown to affect established P aeruginosa biofilms, with loss of the characteristic mature untreated biofilm architecture when 4 μg/mL LL-37 was added and incubated for 2 days after mature biofilm formation was observed. Confocal scanning laser microscopy was used to identify biofilms with the BacLight kit (Invitrogen, Carlsbad, CA, USA) to identify live and dead bacteria, and crystal violet staining and absorbance were used to assess growth of biofilm. LL-37 was further shown to affect biofilm formation through decreasing the attachment of bacterial cells, stimulating twitch motility, and down-regulating the genes responsible for the quorum-sensing system of biofilms (Las and Rhi).

Chennupati and colleagues showed that synthetic LL-37 used in an animal model of biofilm Pseudomonas sinusitis successfully eradicated the biofilm and decreased bacterial counts. However, this occurred at the higher concentration of 2.5 mg/mL, whereas at the lowest concentration of 0.1 mg/mL, evidence of rods was still present on scanning electron microscopy, suggesting biofilm formation, although the highest concentration of LL-37 (2.5 mg/mL) caused increasing inflammation and ciliotoxicity. LL-37 can cause apoptosis in vitro of human airway epithelial cells.

Overhage and colleagues observed significant inhibition of biofilm growth with much lower concentrations of LL-37 compared with the study by Chennupati and colleagues (4 μg/mL vs 2.5 mg/mL, respectively). Different biofilm detection methods may explain the findings, because Overhage and colleagues used confocal scanning laser microscopy, which is reported to be more accurate in detecting biofilms than scanning electron microscopy. Alternatively differences in LL-37 peptide synthesis may explain the differences in concentration findings. Mucus may not be easily distinguished from the biofilm exopolysaccharide matrix, and the rods seen may represent architectural distortion or artifact when using scanning electron microscopy.

Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are major components of gram-negative and -positive bacteria, respectively. LPS and LTA increase epithelial cell thickness and squamous metaplasia, impairing mucociliary clearance. A novel LL-37–derived antimicrobial peptide P60.4-Ac inhibited the ability of LPS or LTA to cause these epithelial changes in an in vitro air–liquid interface culture of primary cells from the sinus mucosa.

In summary, recent in vitro results support LL-37 as a potential therapeutic option against biofilms, but evidence in an animal model of biofilm sinusitis indicates that loss of cilia at high concentration would limit its use as topical therapy.

Lactoferrin is another innate immune molecule known to inhibit P aeruginosa biofilm formation at concentrations again below those that kill or inhibit bacterial growth. Lactoferrin chelates iron, thus stimulating twitching and causing the bacteria to migrate across the surface instead of coalescing to form biofilms. Lactoferrin also has direct bactericidal and fungicidal activity independent of its iron-binding capacity. The mRNA and protein expression of lactoferrin are significantly reduced in patients with CRS and eosinophilic mucus CRS compared with normal controls, suggesting a defect in innate immunity perhaps predisposing to CRS development.

Psaltis and colleagues analyzed patients with CRS for the presence of biofilms and found that patients who tested positive for the presence of biofilm had a significantly reduced lactoferrin expression compared with those who tested negative. The association between lactoferrin down-regulation and presence of biofilms is intriguing and requires further exploration.

Lysozyme is found to be expressed strongly in neutrophils, serous cells of the submucosal glands, and goblet cells. It is an enzyme directed against the peptidoglycan component of the bacterial cell wall, causing cell lysis. It also has anti-fungal properties and acts synergistically with antimycotic agents. More recently, lysozyme was shown to inhibit biofilm formation against Candida on denture acrylic surfaces. Lysozyme precursor C was down-regulated in patients with CRS compared with normal controls using proteomic techniques to analyze aspirated nasal mucus.

Currently, a paucity of research exists on lysozyme in rhinology, despite its being one of the most abundant antimicrobials in nasal fluid and its recently shown antifungal and antibiofilm properties. In summary, evidence is emerging on the use of these peptides for treating CRS, especially in preventing biofilm formation. The antimicrobial properties of naturally occurring compounds, such as honey, may be caused by antimicrobial peptides. Further studies are required to explore the role of these antimicrobial peptides and whether they can be used, either topically or systemically, as disease-modifying therapy in CRS.