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* Department of Clinical Chemistry and Molecular Diagnostics, Germany;
Institute of Immunology, Hospital of the Philipps-University, Marburg, Germany; and
Institute of Immunology, College of Veterinary Medicine, University of Leipzig, Leipzig, Germany
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The inverse relationship between microbial load in childhood and later development of allergic diseases has led to the hygiene hypothesis (4). According to this hypothesis, frequent exposure to microbial products results in a predominant TH1 phenotype, whereas lack of such interactions could promote TH2-driven allergic diseases. In this regard, it has been shown that TH1 cells are able to directly or indirectly counteract TH2 immunity. One mode of action is via IFN-
(5).
Therefore, creating a milieu which prevents or suppresses the development of TH2 cells could be an approach toward modulating the allergic immune response. The development of T effector responses is under close control of cells of the innate immune system. A critical role for the Th cell development is described for dendritic cells (DCs).4 On the one hand, production of IL-4 and IL-10 by DCs biases Th cell development to TH2 cells. On the other hand, IL-12-producing DCs act as strong TH1 inducers (6). A molecular basis for DC activation has been provided with the identification of the TLR which recognize various conserved microbial structures termed pathogen-associated molecular patterns. Various bacterial TLR ligands such as unmethylated CpG motif-containing DNA and LPS have been shown to stimulate production of IL-12 in host cells and consequently down-regulate TH2 responses in animal models of allergic airway inflammation (7, 8).
A critical question still left unanswered is the capacity of viral TLR ligands to interfere with allergic sensitization or already established allergic airway inflammation. dsRNAs or ssRNAs are produced as intermediates during viral replication and are recognized by TLR-3 and TLR-7, respectively (9, 10). Because epidemiological studies reported an inverse relation between systemic infections with hepatitis A virus, measles virus, or others (11, 12, 13, 14), it was the aim of this study to evaluate the effect of systemic TLR-3 or TLR-7 ligand application on allergic sensitization and on allergic airway inflammation in a mouse model of experimental asthma.
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Materials and Methods |
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Pathogen-free 6- to 8-wk-old female BALB/c mice were obtained from Harlan Winkelman. IL-12p35–/– mice were generated as described and backcrossed into BALB/c background (15, 16). TLR-3/-7 double-deficient mice were generated by mating TLR-3 with TLR-7-deficient BALB/c mice (17, 18). All animals were kept under standard housing conditions. All animal studies were performed under the approval of the local authorities.
Mice were sensitized to OVA (OVA grade VI; Sigma-Aldrich) by three i.p. injections of 10 µg of OVA adsorbed to 150 µg of aluminum hydroxide (inject alum; Pierce) on days 1, 15, and 22 followed by four challenges with 1% (w/v) OVA aerosol on days 26–29 (protocol 1) as previously described (19). The endotoxin concentration of the used OVA solutions was under detection level as assessed by Limulus lysate assay (Pyrochrome). To investigate the effect of TLR-3 and TLR-7 activation during sensitization polycytidylic-polyinosinic acid (p(I:C); 200 µg of p(I:C) dissolved in 200 µl of PBS) or R-848 (50 µg of R-848 dissolved in 100 µl of PBS) were delivered by i.p. or s.c. injections, respectively, given at least 24 h before each of the OVA i.p. injections. A second protocol was used to assess the effect of TLR activation on already established experimental asthma in mice (protocol 2). BALB/c mice were sensitized to OVA as described above and allergic airway inflammation was induced by four OVA aerosol challenges on days 26–29. OVA aerosol rechallenges were performed on days 47 and 48. p(I:C) or R-848 were administered by i.v. or s.c. injections on days 41, 43, 45, 47, and 48, respectively. To investigate the role of IL-12 in mediating the effects of either R-848 or p(I:C) protocol 2 was performed using IL-12p35–/– mice. Additionally, IL-10-blocking Abs (anti-IL-10 Ab; MAB417; R&D Systems) were applied into IL-12p35–/– mice by two i.p. injections (125 µg of Ab dissolved in 100 µl of sterile PBS) on days 39 and 44. A rat IgG1 isotype (125 µg) Ab (MAB005; R&D Systems) was used as control. All analyses were performed 24 h after the last OVA aerosol challenge.
Preparation and cell culture of splenic mononuclear cells (MNCs)
MNCs were isolated from spleens of OVA-sensitized mice. A total of 2 x 106 cells/well were incubated with only OVA (50 µg/ml) or together with R-848 (1 µg/ml) or p(I:C) (100 µg/ml) for 72 h as described previously (20). Cell-culture supernatants were collected for cytokine detection.
Assessment of leukocyte distribution in bronchoalveolar lavage (BAL) and peritoneal lavage (PL) fluids
BAL was performed as described previously (19). For PL collection, 12 h after i.p. injection of p(I:C) or R-848, animals were sacrificed and 5 ml of ice-cold PBS was injected in the abdominal cave. After 2 min, PL fluids were collected. Total number of leukocytes was determined by using a Casy TT cell counter (Schaerfe System). BAL cells were differentially stained with Diff-Quick (Dade Diagnostics). Cell-free lavage fluids were stored at –20°C for further cytokine analysis.
Lung histology
Lungs were fixed and periodic-acid Schiff (PAS) was stained for light microscopy as described previously (19). Goblet cell hyperplasia was assessed in PAS-stained lung sections and expressed as cells per 100-µm basement membrane according to Foster et al. (21).
Assessment of airway responsiveness to metacholine (MCh)
The airway responsiveness to MCh (acetyl-
-methylcholine chloride; Sigma-Aldrich) was measured on the basis of the alteration of the midexpiratory airflow in response to increasing doses of inhaled MCh as described previously (22). Midexpiratory airflow (EF50) was measured using headout body plethysmography. Briefly, the system consists of a glass-made headout body plethysmograph which is attached to an aerosol exposure chamber (Forschungsstaetten; Medical School Hannover, Hannover, Germany). The mouse was positioned in the headout body plethysmograph while the head of the animal protruded through a neck collar (9-mm ID, dental latex dam; Roeko) into the aerosol exposure chamber, which was ventilated by continuous airflow of 200 ml/min.
For airflow measurement, a calibrated pneumotachograph (PTM 378/1.2; Hugo Sachs Elektronic) and a differential pressure transducer (8T-2; Gaeltec) coupled to an amplifier (HSE-IA; Hugo Sachs Elektronic) were attached to the top port of each plethysmograph. For each animal, the amplified analog signal from the pressure transducer was digitized via an A/D converter (DT301 PCI; Data Translation; Marlboro) at a sampling rate of 2000/sec. Notocord hem 3.5 was used for data calculation.
Mice were exposed to MCh aerosols with gradient concentrations (12.5, 25, 50, 75, 100, 125 mg/ml) while EF50 was assessed continuously. The MCh concentration that caused a 50% reduction in baseline EF50 (MCH50) is used as a parameter for airway constriction.
Measurements of cytokine levels in BAL and PL fluids
In cell-free lavage fluids, IL-5 was measured by ELISA (BD Biosciences) as described previously (22). IL-4, IL-2, IFN-, IL-10, and IL-12p70 were measured using cytometric bead arrays (CBA; BD Biosciences) according to manufacturer’s protocol.
Measurements of OVA-specific Ig titers in serum samples
OVA-specific IgE, IgG1, and IgG2a concentrations were determined by ELISA technique as described previously (23).
Statistics
Results are presented as mean values ± SEM unless stated otherwise. ANOVA test was performed to determine the level of significance between the different animal groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BALB/c mice were sensitized to and aerosol-challenged with OVA as described in Materials and Methods. This protocol resulted in high levels of OVA-specific IgE and IgG1 Abs and slightly enhanced production of OVA-specific IgG2a paralleled by goblet cell hyperplasia and allergic airway inflammation as characterized by large numbers of eosinophils and lymphocytes in BAL fluids. p(I:C) application during sensitization suppressed the production of OVA-specific Abs in each of the Ig isotypes analyzed. Titers of OVA-specific IgE and IgG1 Abs were also decreased after R-848 application but, in contrast to p(I:C), IgG2a levels were increased as compared with the OVA group (Table I).
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The next experiments addressed the question whether these marked effects would also be observed in a model of experimental asthma with an already established pulmonary pathology. After the first set of allergen challenges, a secondary inflammatory response was induced by repeated OVA aerosol rechallenges. One day before and at the day of the secondary challenges, p(I:C) or R-848 were administered. The secondary response was characterized by high numbers of eosinophils and lymphocytes in BAL fluids and in bronchial tissues. This was accompanied by the development of AHR and increased mucus production as indicated by goblet cell hyperplasia of the airway epithelium (Fig. 2). In contrast, the inflammatory response was markedly reduced in p(I:C)- or R-848-treated animals as shown by lower numbers of eosinophils and lymphocytes in BAL fluids and lung tissues (Fig. 2, A, D, and E). Strikingly, goblet cell hyperplasia was completely absent in these mice (Fig. 2F). The reduction in eosinophils was paralleled by markedly suppressed levels of IL-5 in BAL fluids (Fig. 2B). Concentrations of IL-10 in the BAL fluid were not significantly changed after TLR ligand application (Fig. 2C). IL-4, IL-12, IL-13, IFN-, and eotaxin levels in the BAL fluid were below the detection limits. Assessment of lung function revealed nearly normalized airway responsiveness to MCh as compared with mice of the OVA group (Fig. 2G). These findings were associated with a suppression of the TH2-related production of OVA-specific IgE and IgG1. Similar to the effect on prevention of experimental asthma application of R-848 augmented TH1-related OVA-specific IgG2a Ab levels. This effect was not observed following p(I:C) application (Table II).
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The i.p. injection of either p(I:C) or R-848 resulted in enhanced production of IL-12p70 and even higher levels of IFN- in PL fluids of wild-type (wt) BALB/c mice (Table III). p(I:C) had a significantly stronger effect on IL-10 production than R-848. The induction of these cytokines appeared to be specific for TLR-3 or TLR-7 activation, respectively, because neither administration of p(I:C) nor R-848 stimulated production of IL-10, IL-12, or IFN- in PL fluids of TLR-3/-7 double-deficient animals (Table III). The results from these experiments suggest that R-848 and p(I:C) might affect T cell responses through different pathways. To further delineate these phenotypes, we analyzed the cytokine production of MNC after in vitro OVA restimulation. Briefly, MNC cultures from OVA-sensitized BALB/c mice were stimulated with OVA alone or in combination with R-848 or p(I:C). Seventy-two hours later, levels of IFN-, IL-2, IL-4, IL-5, IL-10, and IL-12p70 were analyzed in cell-culture supernatants. Dose-response experiments revealed concentrations of 1 µg/ml R-848 and 100 µg/ml p(I:C) to be the most effective (data not shown). Stimulation of splenic MNCs with OVA alone resulted, as expected, in high levels of IL-2, IL-10, IL-4, and IL-5 in parallel to relatively low levels of IFN- and IL-12p70. In the presence of R-848 alone, the levels of IL-10, IL-12, and IFN- increased, whereas p(I:C) alone had no effect on any cytokine production. Coapplication of OVA and R-848 or p(I:C) inhibited the production of IL-2, IL-4, and IL-5, but continued to trigger IL-10. Furthermore, concentrations of IL-12p70 and IFN- were significantly increased after coapplication of OVA and R-848. Thus, R-848 suppressed TH2 cell responses and induced a TH1-like cytokine response. p(I:C) showed similar results on the suppression of TH2 cytokines, but TH1 cytokines were not stimulated (Fig. 3).
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It has been demonstrated that some TLR ligands trigger the production of IL-12 in APCs, thereby mediating the suppression of TH2 responses. To test the contribution of IL-12 to the immunomodulatory effects of R-848 and p(I:C) on established experimental asthma, IL-12p35–/– mice were studied. IL-12p35–/– mice sensitized to OVA developed the typical asthma pathology with allergic airway inflammation as indicated by a marked influx of eosinophils and lymphocytes into the airways together with pronounced inflammation of the airway walls dominantly consisting of eosinophils. In contrast to wt animals, eosinophils and lymphocytes appeared in lower numbers in BAL fluids. Nevertheless, OVA-sensitized and -challenged IL-12p35–/– mice demonstrated development of goblet cell hyperplasia and they were more sensitive to MCh than nonsensitized IL-12p35–/– animals, reflecting development of AHR.
R-848 and p(I:C) application had differential effects on the phenotype of experimental asthma in IL-12p35–/– animals that were OVA sensitized and challenged following protocol 2 of Materials and Methods. Both TLR ligands markedly prevented the strong increase in OVA-specific IgE Abs, but had no effect on OVA-specific IgG1 Ab titers (Table IV). This was accompanied by a strong increase in OVA-specific IgG2a Abs following R-848 administration but not in the p(I:C)-treated group. In terms of airway inflammation, most of the inflammation related parameters such as tissue inflammation (as revealed by histology), presence of goblet cells, numbers of BAL lymphocytes, BAL IL-5 levels, and airway hyperreactivity were still observed in R-848- or p(I:C)-treated IL-12p35–/– mice. An exception was a marked reduction of the influx of eosinophils into the airway lumen which was still observed in TLR-3 or TLR-7 ligand-treated IL-12p35–/– mice. In both groups, the amount of BAL lymphocytes revealed a slight reduction (Fig. 4). Thus, the suppressing effects of R-848 and p(I:C) on mucosal inflammation, AHR, and IL-5 production seem to be mediated by IL-12, whereas the influx of eosinophils into the airways occurred in an IL-12-independent fashion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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(25, 26, 27). In the present study, we demonstrated that 1) systemic application of TLR-3 as well as of TLR-7 ligands during the sensitization phase almost completely protected BALB/c mice from allergic sensitization and, subsequently, from the development of experimental asthma. 2) Administration of p(I:C) as well as of R-848 following elicitation of a primary inflammatory response markedly prevented the development of a secondary response, which 3) depend on the production of IL-12 and IL-10.
In response to both p(I:C) and R-848, we observed the production of IL-10 which has potent anti-inflammatory effects as also described for TLR-2 activation by Candida albicans (28). Furthermore, increased concentrations of IL-12 and IFN- were also detected in peritoneal lavage fluids after p(I:C) or R-848 administration. We found a similar cytokine profile when MNCs from OVA-sensitized animals were in vitro restimulated with the allergen in presence of p(I:C) or R-848. IL-12 is an important inducer of TH1 responses and is predominantly produced by DCs, monocytes, and macrophages (29, 30, 31). DCs stimulated with various TLR ligands such as LPS or CpG readily produce IL-12 (32). We observed IL-12 production by peritoneal macrophages in response to i.p. injection of p(I:C) or R-848 suggesting that certain effects of TLR-ligands on the secondary immune response to inhaled allergen are at least partly mediated by IL-12. This conclusion is supported by experiments with mice genetically deficient for the IL-12p35 subunit and, therefore, being unable to produce bioactive IL-12p70. In these animals, p(I:C) as well as R-848 application were ineffective in preventing/suppressing allergic airway inflammation and goblet cell hyperplasia. IL-12 has been shown to drive the development of naive CD4-positive T cells toward a TH1 phenotype and it promotes the production of IFN- by CD4-positive T cells (33). Therefore, induction of a TH1 response that counterbalances the TH2-type allergic immune response could be responsible for the observed effects of R-848 and p(I:C).
p(I:C) and R-848 had different effects on OVA-specific Ab production. Both, p(I:C) as well as R-848 markedly reduced the production of OVA-specific IgE and IgG1. But in contrast to p(I:C), administration of R-848 did not result in diminished, but did result in enhanced production of OVA-specific IgG2a indicating the induction of an OVA-specific TH1 response. It has been shown that IL-12 increases serum IgG2a levels and suppresses IgG1 and IgE response and IL-4 switches the Ab production toward IgG1 and IgE in mice and IgE in humans (34, 35). An allergen-specific TH1 response could have been induced IL-12 following R-848 application and may counteract the allergen-specific TH2 response via induction of IFN- production (36). The different effects of p(I:C) and R-848 on the production of OVA-specific Abs as well as on cytokine production in cell culture may be based on the differential expression of TLR-3 and TLR-7 on T cells and APCs (37, 38). Because murine T cells express TLR-7 but not TLR-3, R-848 could have direct effects on T cells, while p(I:C) has not. These could include a direct shift from IL-4-producing TH2 cells into IFN--producing T cells as it has been reported for R-848 and human T cells in vitro (39).
After p(I:C) as well as R-848 application, airway eosinophilia persisted in IL-12p35–/– mice and the production of IL-10 was enhanced. Therefore, we hypothesized that IL-10 participates in the development of the beneficial effects in addition to IL-12. Indeed, neutralizing anti-IL-10 Abs abolished the inhibition of airway eosinophilia in IL12p35–/– mice. IL-10 is a potent anti-inflammatory cytokine that inhibits the production of several inflammatory proteins. IL-10 inhibits survival of eosinophils and the activation of TH2 cells (40). Furthermore, IL-10 suppresses innate immune responses by dampening responses to many microbial stimuli including those recognized by certain TLRs (41). In addition, IL-10 is also a potent inhibitor of adaptive T cell-mediated immune responses (42). The protective as well as suppressive effects of TLR-3 or TLR-7 ligand application on allergic TH2-type immune responses may be based on the anti-inflammatory effects of IL-10.
Both the anti-inflammatory effects of IL-10 and an Ag-specific TH1 response may have inhibiting effects on the allergen-specific TH2 response. Thus, by reducing IL-4 and IL-5 production in T cells, differentiation of allergen-specific TH2 cells, influx of eosinophils into the airways, as well as production of allergen-specific IgE and IgG1 could be suppressed.
In accordance with Moisan et al. (43) and Quarcoo et al. (44), we also observed protective effects of TLR-7 activation against the development of experimental asthma. In extension of these observations, we demonstrated that activation of TLR-7 further has the potential to significantly improve already established experimental asthma and that IL-12 as well as IL-10 are required for the mediation of these effects. Recently published data point toward a novel pathway which could explain the production of high levels of IL-12 and IL-10 (and IFN-) in the absence of measurable TH1 activities and any TH1-related lung pathology. These effects have been related to Th type 1-like regulatory cells developing in the presence of IL-10 and IL-12. Because these TH1-type regulatory cells are able to suppress allergic airway inflammation as well as AHR in a mouse model of allergic asthma by producing IL-10 as well as IFN- (45), we cannot exclude a regulatory role of them in our model.
In conclusion, systemic application of TLR-3 as well as of TLR-7 ligands revealed effects that not only protected from the development of experimental asthma but also reduced asthma pathology when allergic airway inflammation has already been established. Even though TLR-3 or TLR-7 ligands have different effects on the production of Ag-specific Abs, both require IL-12 to drive their effects on experimental asthma in mice.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by the Deutsche Forschungsgemeinschaft (Transregio 22) and the P.E. Kemkes Foundation (16/04). 
2 S.S. and M.W. contributed equally to this work and appear in alphabetical order.
3 Address correspondence and reprint requests to Dr. Michael Wegmann, Biomedizinisches Forschungszentrum, Abteilung für Klinische Chemie und Molekulare Diagnostik, Hans-Meerwein-Strasse, 35033 Marburg, Germany. E-mail address: wegmann{at}med.uni-marburg.de
4 Abbreviations used in this paper: DC, dendritic cell; p(I:C), polycytidylic-polyinosinic acid; MNC, mononuclear cell; BAL, bronchoalveolar lavage; PL, peritoneal lavage; MCh, metacholine; PAS, periodic-acid Schiff; CBA, cytometric bead array; AHR, airway hyperresponsiveness; wt, wild type.
Received for publication April 18, 2006. Accepted for publication March 29, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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. J. Immunol. 166: 207-217. 
/ signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection. Proc. Natl. Acad. Sci. USA 100: 10872-10877. by TCR-V-expressing T-cell subsets in allergen-sensitized mice. Am. J. Respir. Cell Mol. Biol. 14: 36-43. [Abstract]
B by Toll-like receptor 3. Nature 413: 732-738. [Medline] TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN- production is IFN--dependent. Int. Immunol. 5: 1119-1128. , and IFN- by mouse dendritic cell subsets. J. Immunol. 166: 5448-5455. represses IL-4 expression via IRF-1 and IRF-2. Immunity 17: 703-712. [Medline]-producing cells. J. Allergy Clin. Immunol. 111: 380-388. [Medline]This article has been cited by other articles:
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