Fig. 1 Total cell and eosinophil counts in BALF decreased significantly after Pam3CSK4 treatment
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Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
1.Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
2.Experimental Animal Center, School of Medicine, Hangzhou Teacher’s College, Hangzhou 310018, China
Published: 2008-04 ,
Received: 11 December 2007 ,
Accepted: 04 February 2008
Cite this article
Cheng Zhou, Xiao-dong Kang, Zhi Chen. A synthetic Toll-like receptor 2 ligand decreases allergic immune responses in a mouse rhinitis model sensitized to mite allergen. [J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) 09(4):279-285(2008)
Cheng Zhou, Xiao-dong Kang, Zhi Chen. A synthetic Toll-like receptor 2 ligand decreases allergic immune responses in a mouse rhinitis model sensitized to mite allergen. [J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) 09(4):279-285(2008) DOI: 10.1631/jzus.B0730029.
Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
It has been proposed that activation of Toll-like receptors (TLRs) plays crucial roles in the polarization of adaptive immune responses. A synthetic Toll-like receptor 2 (TLR2) ligand, Pam3CSK4, has been reported to modulate the balance of Th1/Th2 responses. We evaluated the modulation effect of Pam3CSK4 on allergic immune response in a mouse rhinitis model sensitized to house dust mite allergen (HDM). Mice were sensitized and challenged with Dermatophagoides farinae allergen (Der f), and then the allergic mice were treated by Pam3CSK4. Nasal allergic symptoms and eosinophils were scored. Der f-specific cytokine responses were examined in the splenocytes and bronchoalveolar lavage fluid (BALF). Serum level of total IgE was also detected. After establishing a mouse allergic rhinitis model with HDM, we have showed that Pam3CSK4 treatment not only ameliorated the nasal allergic symptoms remarkably but also decreased the eosinophils and total inflammation cells in BALF significantly. Analysis of cytokine profile found that IFN-γ released from either BALF or stimulated splenocytes increased markedly in Pam3CSK4-treated mice, while IL-13 decreased significantly. Moreover, serum level of total IgE was significantly lower in Pam3CSK4-treated mice than in the untreated. Thus, in an allergic rhinitis mouse model developed with HDM, Pam3CSK4 was shown to exhibit an antiallergic effect, indicating its potential application in allergic diseases.
The incidence of allergic diseases increases rapidly during last 20 years, especially in developed countries. It was explained by the hygiene hypothesis, that is, exposure to microbes or their products earlier in life results in decreased atopic sensitization (Liu,
Recently, the Toll-like receptor (TLR) family, a phylogenetically conserved structure in innate immunity, attracted considerable interest from immunologists. The components mainly derived from the microbes, named as pathogen-associated molecular patterns (PAMPs), are recognized by TLRs. At least ten members of TLRs have been identified in human, expressed in both innate and adaptive immune systems. Triggering of TLRs by different microbial components was able to modulate the adaptive immune response in host. Originally it was thought that the signaling via TLRs usually preferred to enhancement of Th1 response (Schnare et al.,
TLR2 recognizes components from a variety of microorganisms including lipoproteins, peptidoglycan and lipoteichoic acid mainly from Gram-negative bacteria. Analysis of TLR2-deficient mice showed that TLR2 is critical to the recognition of peptidoglycan and lipoproteins (Takeda et al.,
Six- to eight-week-old female BALB/c mice were purchased from the Experimental Animal Center of Zhejiang University (Hangzhou, China). They are all kept under pathogen-free condition in the Laboratory of Animal Facility, Medical College of Zhejiang University.
Dermatophagoides farinae (Der f) crude body extract (Medical Acarology Laboratory of Medical School, Fudan University, Shanghai, China) was used as the allergen, which was lyophilized and stored in 4 °C and dissolved in PBS before use. Synthetic bacterial lipopeptide Pam3CSK4 (InvivoGen, San Diego, California, USA) was prepared in sterile PBS.
Mice were divided into three groups: control, allergic rhinitis, and Pam3CSK4 treatment. Each group contains 6 mice. Mice were sensitized via intraperitoneal injection (IP) with 100 µg of Der f and 1 mg of alum in 0.2 ml of PBS. A boosting injection was given on Day 7 with the same reagents. On Days 14~20, mice were challenged with Der f (1 µg/µl, 20 µl per mouse) via intranasal instillation for consecutively 7 d. For the control group, mice were sensitized and challenged with PBS in place of Der f. In the Pam3CSK4 treatment group, the mice were sensitized and challenged in the same way as in the allergic rhinitis group, but Pam3CSK4 (100 µg per mouse) was injected intraperitoneally once 1 d before the final allergen inhalant. Der f-allergic and negative control mice were given PBS via the same route.
Splenocytes were isolated from mice after sacrificed on Day 21. They were cultured in RPMI-1640 medium containing 10% FCS at 2×106 cells/ml with Der f (50 µg/ml) stimulation at 37 °C and 5% CO2. After 72 h incubation, the supernatants of cells were collected and the concentrations of IL-13 and IFN-γ were assayed by commercial ELISA kits according to procedure protocols (R & D Systems, Minneapolis, MN, USA).
Immediately after anesthesia, BALF was obtained as described in the following. Briefly, the right lung was lavaged in situ using two aliquots of 0.8 ml of PBS. The lavage of each mouse was kept on ice, then the BALF was centrifuged at 1500 r/min for 5 min, and the supernatant was aliquoted and stored at −70 °C for the detections of IFN-γ and IL-13. The BALF cell pellet was then resuspended in 1 ml of PBS and counted using a hemocytometer. The slides were made using a Cytospin (Shandon, UK) and were stained with Giemsa. Total cells and eosinophils in BALF were counted.
The levels of total IgE in mouse sera were detected by ELISA kit (BD Bioscience, San Jose, California, USA).
The numbers of sneezing and nose rubbing motions during 30 min after the final allergen challenge were recorded in each experimental group.
The severity of allergic rhinitis was evaluated by two nasal symptoms, sneezing and nose rubbing. In our pilot experiments, the allergic symptoms exacerbated gradually since Day 14 after allergen challenge, and reached the peak during Days 20~30. Therefore, we administered Pam3CSK4 intraperitoneally once 1 d before final allergen challenge. As shown in Table
Numbers of sneezing | Numbers of nose rubbing | |
---|---|---|
Der f-allergic rhinitis group |
30.0±5.7 |
19.8±4.2 |
Pam3CSK4 treatment group |
16.3±4.1 |
12.2±2.8 |
PBS group | 4.6±1.6 | 2.3±1.2 |
P<0.001, as compared with PBS group
P<0.01, as compared with Der f-allegic group
Every group contained 6 mice
Since the inflammatory cells, especially eosinophils, play a crucial role in allergic diseases, immediately after anesthesia, the BALF of each mouse was obtained. The BALF total cells and eosinophils were counted. The results show that either the number of total cells or eosinophils was significantly higher in the Der f-allergic rhinitis mice than in PBS mice (Fig.
Fig. 1 Total cell and eosinophil counts in BALF decreased significantly after Pam3CSK4 treatment
Pam3CSK4 was given to allergic mice once 1 d before the last intranasal challenge. **P<0.001, as compared with PBS mice; ∆P<0.01, as compared with untreated Der f-allergic mice
The role of TLR2 signaling in allergic inflammation is a current debating topic. How TLR2 stimulation modulates the balance of Th1/Th2 immune responses is not well defined, maybe varying with the allergic model, strain, dose and timing. To characterize the polarization of the immune response after Pam3CSK4 treatment, IFN-γ and IL-13 were measured as indications of Th1 and Th2 responses, respectively. In BALF, IFN-γ could not be detected in both Der f-allergic rhinitis and PBS mice, whereas detected in 5 out of 6 mice of Pam3CSK4 treatment group (Fig.
Fig. 2 Pam3CSK4 enhanced IFN-γ and inhibited IL-13 productions. (a) IFN-γ in BALF; (b) IL-13 in BALF; (c) IFN-γ released from splenocytes stimulated with Der f; (d) IL-13 released from splenocytes stimulated with Der f
**P<0.001 and *P<0.01, as compared with untreated Der f-allergic rhinitis mice. ND: Not detected
IgE is the central antibody in the induction of allergy and a consequence of an extreme Th2 immune response. In order to know whether Th1 response induced by Pam3CSK4 could down-regulate the subsequent IgE synthesis, we detected the total IgE in mouse sera. The results show that serum total IgE levels were elevated in both the Der f-allergic group and the Pam3CSK4 treatment group as compared with the control group (P<0.001, Fig.
Fig. 3 The total serum IgE decreased remarkably after Pam3CSK4 treatment
**P<0.001, as compared with PBS mice; ∆P<0.05, as compared with untreated Der f-allergic rhinitis mice
The central goal in immunotherapy for allergic diseases is to develop more effective, safer and long-lasting therapeutical strategy. To reach this goal, scientists focus their interest on modulation of the skewed Th2 response toward a more balance Th1/Th2 response, since allergic diseases are strongly associated with Th2 immune response (Zhang et al.,
Although ovalbumin is the most commonly used allergen to develop animal models of allergy, we chose HDM to develop our animal model. We believe that the establishment of allergic animal model sensitized to HDM offers more clinical utility in studying the aetiology and treatment of inhalation allergies (Sarpong et al.,
The findings in our study strongly suggest that allergic rhinitis sensitized to HDM is inhibited by Pam3CSK4, since the allergic symptoms, airway eosinophils, serum total IgE and IL-13 reduced significantly in Pam3CSK4 treatment group compared with the allergic rhinitis group, while IFN-γ increased. Previous studies have evaluated the effect of TLR2 signaling on allergic diseases, but the results are conflicting. In our model, triggering of TLR2 with Pam3CSK4 showed a beneficial antiallergic effect on allergic rhinitis, which is consistent with several previous studies (Akdis et al.,
It was proposed that the increase of allergic diseases in the industrialized world has often been related to a decline in infections during childhood, since a reduction in the overall microbial burden and low stimulation of TLRs will result in weak Th1 imprinting and unrestrained Th2 responses that presumably allow an increase in allergy (Liu,
In summary, based on a mouse allergic rhinitis model with the house dust mite allergen that developed in the current studies, we have clearly showed that the stimulation of TLR2 with Pam3CSK4 was able to decrease not only allergic symptoms but also the Th2 response in allergic mice, suggesting its therapeutic effect on allergic diseases. Further studies need to be done to evaluate the persistence of its antiallergic effect, potential side effects, and effects related with various timing and route of administration in animal models.
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