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Keywords:

  • intermittent allergic rhinitis;
  • Nalp3;
  • NLRs;
  • Nod1;
  • Nod2

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

To cite this article: Bogefors J, Rydberg C, Uddman R, Fransson M, Månsson A, Benson M, Adner M, Cardell LO. Nod1, Nod2 and Nalp3 receptors, new potential targets in treatment of allergic rhinitis? Allergy 2010; 65: 1222–1226.

Abstract

Background:  Recently, a new set of pattern-recognition receptors, the nucleotide-binding oligomerization domain (Nod)-like receptors (NLRs), have emerged. Their activation, either by allergens or microbes, triggers an inflammatory response. The knowledge about NLRs in human airways is limited.

Aim of the study:  To investigate presence of NLRs in the human nose of healthy individuals and patients with intermittent allergic rhinitis outside and during pollen season.

Methods:  The expression of Nod1, Nod2, and Nalp3 in nasal biopsies was determined with real-time RT-PCR and immunohistochemistry. Cultured primary human nasal epithelial cells (HNECs) were analyzed using real-time RT-PCR and flow cytometry to further verify the presence of NLRs in the epithelium.

Results:  Immunohistochemical analysis revealed presence of Nod1, Nod2, and Nalp3 in the nasal epithelium. This was corroborated in cultured HNECs. Patients suffering from symptomatic allergic rhinitis exhibited lower Nod1 and Nalp3 mRNA levels than both controls and patients during pollen season. Nod2 expression was found in all specimens tested, but no differences were seen between the three groups.

Conclusion:  Nod1, Nod2, and Nalp3 receptors were found to be present in the human nose. The expression of Nod1 and Nalp3 were down-regulated during pollen season among patients with allergic rhinitis. This opens up for new insights and novel therapeutic strategies in inflammatory airway disease.

Allergic rhinitis is the most common form of noninfectious rhinitis and its association with an IgE-mediated immune response against allergens is obvious (1). However, allergens are not always the sole trigger of the inflammatory reaction. Several studies suggest a synergistic effect between virus infection and allergen exposure (2). This is to some extent also true for bacterial colonization of the airways (3). Both viruses and bacteria trigger an inflammatory response by activating pattern-recognition receptors (PRRs) in the host (4).

Nucleotide-binding oligomerization domain (Nod)-like receptors (NLRs) are a newly discovered family of PRRs that consists of more than 20 intracellular members (4). They can be divided into several subfamilies, among them the NODs and the NALPs, depending on their N-terminal domains (5). The most well-known members of the NOD subfamily are Nod1 and Nod2, both sensing different subunits of bacterial peptidoglycans. Nalp3 has recently been described to interact with aluminum adjutants (alum), which are currently being used in allergy vaccination (6–8). Nod1, Nod2, and Nalp3 have been found in various epithelial and monocytic cells (9–11). Based on studies of polymorphisms, Nod1 and Nod2 have been implicated in the pathogenesis of inflammatory bowel disease (9). The same type of studies has shown that Nod2 appears to be associated with risk for developing allergic rhinitis, atopic dermatitis, and asthma (10–12). Nalp3 has been genetically linked to several autoinflammatory disorders (10). Despite the genetic association of NLRs with inflammatory airway diseases, not much is known about the presence of NLRs in the human airways. Therefore, the present study is aimed to characterize the expression of Nod1, Nod2, and Nalp3 in the human nose of healthy individuals and patients with allergic rhinitis.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Subjects and study design

The study included 20 nonsmoking patients (nine women) with birch and/or grass pollen-induced intermittent allergic rhinitis with a positive skin prick test (SPT) and a history of seasonal allergic rhinitis for at least 2 years. Ten nonsmoking healthy individuals (four women) were recruited as controls. The controls were all symptom-free with no history of allergic rhinitis and displayed a negative SPT. SPTs were executed with a standard panel of 10 common airborne allergens (ALK, Copenhagen, Denmark).

Nasal biopsies were obtained from patients with allergic rhinitis outside season (November to February) and during the birch or the grass pollen season. The control subjects were sampled outside season. Nasal biopsies were obtained from the inferior turbinate after topical application of local anesthesia. The study was reviewed and approved by the Ethics Committee of the Medical Faculty, and an informed consent was obtained from all participants.

Isolation of primary human nasal epithelial cells (HNECs)

Isolation of primary HNECs was accomplished using nasal brushings as described previously. The cells were cultured in airway epithelial cell growth medium supplemented with 0.4% bovine pituitary extract, 10 ng/ml epidermal growth factor, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 5 μg/ml epinephrine, 6.7 ng/ml triiodothyronine, 10 μg/ml transferrin, 0.1 ng/ml retinonic acid (PromoCell, Heidelberg, Germany), and 100 U/ml penicillin–streptomycin (Gibco, Grand Island, NY, USA). The epithelial cells were maintained in collagen-coated tissue culture flasks at 37°C in a humidified 5% CO2 air atmosphere. Cells from passage 1–3 were used and they all stained positive for epithelial cell adhesion molecule (EpCAM) (> 90%), an adhesion molecule specific for epithelial cells (13).

Immunohistochemistry

The morphological localization of Nod1, Nod2, and Nalp3 was determined according to standardized protocols. Nasal biopsies from healthy controls were fixed in 4% formaldehyde, embedded in paraffin and sliced into 3-μm sections. The slides were incubated over night at 4°C with primary Abs against Nod1 (pAb), Nod2 (2D9), and Nalp3 (nalpy3-b) from Abcam (Cambridge, UK), diluted 1 : 10, 1 : 25, and 1 : 50, respectively. Subsequently, the sections were incubated with HRP-labeled polymer and 3,3′-diaminobenzidine (DAB) for Ab detection. As negative controls, N-series Universal Negative Control Reagents or Ab diluent (DakoCytomation, Copenhagen, Denmark) were utilized.

Real-time RT-PCR

RNA was extracted from homogenized nasal biopsies. cDNA synthesis was carried out using the Omniscript Reverse Transcriptase kit (Qiagen, Hilden, Germany) and Oligo(dT) primer (DNA-Technology, Århus, Denmark) in a Mastercycler personal PCR machine (Eppendorf AG, Hamburg, Germany) at 37°C for 1 h. Quantitative real-time PCR assays were performed using the Smartcycler II detection system (Cepheid, Sunnyvale, CA, USA) and 20 ng of cDNA was applied for each PCR reaction. The following intron-overspanning primers for detection of Nod1, Nalp3, and β-actin were used: Nod1 fwd 5′-gtggacaacttgctgaagaatgac-3′ and rev 5′-ctgtaccaggtccagaattttgc-3′; Nalp3 fwd 5′-gcgatcaacaggagagaccttta-3′ and rev 5′-gctgtcttcctggcatatcaca-3′; β-actin fwd 5′-gccaaccgcgagaagatg-3′ and rev 5′-acggccagaggcgtacag-3′. The reaction was executed in 95°C for 15 min, followed by 46 cycles at 94°C for 30 s and 55°C for 60 s (initially 66°C, followed by a 2°C decrease in the first six cycles). For detection of Nod2 and β-actin, probes were purchased from Applied Biosystems (Foster City, CA, USA) and used according to the manufacture’s instruction. The thermal cycler was followed as initial set up for 10 min at 95°C, 45 cycles of denaturation at 95°C for 15 s each and thereafter annealing/extension at 60°C for 1 min. The amplification plots of Nod1, Nod2, and Nalp3 can be seen in Fig. 1. The relative amount of mRNA for the NLRs was determined by subtracting the threshold cycle (CT) values of the genes with the CT value for the housekeeping gene β-actin (ΔCT) and expressed in relation to 100 000 mRNA molecules of β-actin (100 000 × 2−ΔCT).

image

Figure 1.  Nasal biopsies were analyzed for mRNA expression with primers, Nod1 and Nalp3, and probes, Nod2, using real-time PCR. The amplification plots of (A) Nod1, (B) Nod2, and (C) Nalp3 are shown.

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FACS analysis

HNECs were analyzed for expression of NLR proteins on a Coulter Epics XL flow cytometer (Beckman Coulter, Marseille, France) and gated based on forward and side scatter properties. Data were analyzed with the Expo32 ADC analysis software (Beckman Coulter). To detect the intracellular NLRs, the IntraPrep™ Permeabilization Reagent kit was used according to instructions of the manufacturer (Beckman Coulter). Unlabeled Nod2 and Nalp3 mAbs were detected using the Alexa Fluor 488 mouse IgG1 labeling kit (Molecular Probes, Eugene, OR, USA). The rabbit anti-human Nod1 pAb was identified using a secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG pAb. Cells were incubated with Abs for 20 min in RT, thereafter washed and resuspended in phosphate buffered saline (PBS).

Statistical analysis

All data are expressed as mean ± SD, and n equals the number of subjects. For normally distributed data statistical analysis was performed using one-way anova with Tukey’s multiple comparison post-test, whereas nonparametric Kruskal-Wallis test with Dunn’s multiple comparison post-test was used for data that were not normally distributed. A P-value of < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nasal biopsies exhibited strong epithelial immunostaining for Nod1 and Nod2, and a somewhat weaker staining for Nalp3 (Fig. 2). Along with this, HNECs showed mRNA expression of Nod1 and Nod2, whereas levels of Nalp3 were considerably lower (Fig. 3A). Flow cytometry analysis revealed a clear presence of Nod1 proteins and a lower expression of Nod2 and Nalp3 (Fig. 3B).

image

Figure 2.  Nasal biopsy sections from healthy controls were stained with Abs against (A) Nod1, (B) Nod2, and (C) Nalp3. (D) As negative control N-series Universal Negative Control Reagent was used. For localization, sections were stained with 3,3′-diaminobenzidine (DAB) (darker staining) and analyzed by microscopy (magnification 100×).

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image

Figure 3.  Primary human nasal epithelial cells (HNECs) were analyzed for expression of Nod1, Nod2, and Nalp3. (A) Expression of NLR mRNA using real-time RT-PCR analysis. The values are expressed in relation to the housekeeping gene β-actin as 100 000 × 2−ΔCT and presented as mean ± SD (n = 3). (B) Intracellular staining with Abs against NLRs (open histogram), appropriate isotype controls (light shaded histogram) or secondary Abs (dark shaded histogram). Data show one representative out of three independent experiments.

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We proceeded to investigate if the NLR expression was affected by pollen season by comparing data obtained from nasal biopsies from three groups: healthy controls and patients with allergic rhinitis during and outside season. Real-time RT-PCR demonstrated that patients during season exhibited a significantly lower mRNA level of Nod1 than controls and patients outside season (Fig. 4A). For Nod2, no differences in expression levels were found between the three groups (Fig. 4B). Lastly, the expression of Nalp3 was significantly lower in the allergic group both outside and during season than among the healthy individuals (Fig. 4C).

image

Figure 4.  Biopsies from healthy controls (n = 10) and patients with intermittent allergic rhinitis outside (n = 10) and during pollen season (n = 10) were analyzed for mRNA expression of (A) Nod1, (B) Nod2, and (C) Nalp3 using real-time RT-PCR. Data are expressed in relation to the housekeeping gene β-actin as 100 000 × 2−ΔCT and presented as mean ± SD, where the horizontal bar represents the mean, (*P < 0.05, **P < 0.01, ***P < 0.001).

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study demonstrates the presence of Nod1, Nod2, and Nalp3 in the human nasal epithelium. A marked down-regulation of Nod1 and Nalp3 expression is seen during pollen season among patients with a history of allergic rhinitis, suggesting the involvement of these receptors in the pathophysiology of inflammatory airway disease.

The nasal epithelial cells are uniquely positioned at the interface between ‘inside versus out,’ making them prime candidates for orchestrating immune responses by using tightly regulated and specifically localized set of pattern recognition receptors (PPRs). So far, Toll-like receptors (TLRs) are the best characterized PRRs. The present demonstration of Nod1, Nod2, and Nalp3 broadens the possibilities for synergistic effect between microbes and allergen. Whereas the TLRs represent the archetype of the transmembrane PRR with an extracellular ligand binding domain, the NLR family seems to play a pivotal role for the recognition of intracellular pathogens.

In previous studies, we have demonstrated how nasal application of TLR ligands causes nasal symptoms per se and reinforces inflammatory airway symptoms induced by allergen (14–16). Our general impression is that the nasal expression of TLRs increases during symptomatic allergic rhinitis. However, in analogy with the presently described down-regulation of Nod1 and Nalp3, we have found TLR3 to be down-regulated in eosinophils from bone marrow and peripheral blood during symptomatic allergic rhinitis. The reason for the TLR3 down-regulation is probably complex involving systemically activated feedback loops, whereas we believe that the Nod1 and Nalp3 down-regulation might be more directly related to the inflammatory disease. This idea is based on the strong association between polygenic inflammatory diseases and polymorphisms in the NLR genes (9–12). This vast spectrum of genetic variants in the NLR genes is most likely the result from a long and continuous adaptation to microbes in our milieu. By the same token, these variants may predispose to chronic diseases like asthma and allergic rhinitis under altered environmental conditions. Thus, the genetics and functional genomics of NLRs demonstrate that the host recognition of pathogen-associated molecules is context-dependent (17). Differences in cellular interpretation can either lead to a ‘protective’ inflammation or to induction of a chronic debilitating inflammation. The down-regulation of Nod1 and Nalp3 presently seen during the pollen season might prevent inflammatory incitements, thereby facilitating the resolution of the allergen-induced response. Against this background it is important to acknowledge that although the majority of patients with allergic rhinitis have controlled symptoms during treatment, many patients are still insufficiently controlled. Novel treatment strategies are therefore needed (18). The discovery of NLRs in the human nose points toward novel possible treatment targets.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The study was financially supported by the Swedish Medical Research Council, the Swedish Heart-Lung Foundation, the Swedish Association for Allergology. The authors thank Ann Reutherborg and Ingegerd Larsson for skilful technical assistance during the course of this study as well as Anna Karin Bastos and Josefine P Riikonen for logistic support.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References