TLR2 Agonist Ameliorates Established Allergic Airway Inflammation by Promoting Th1 Response and Not via Regulatory T Cells1
http://www.100md.com
免疫学杂志 2005年第12期
Abstract
TLRs are primary sensors of both innate and adaptive immune systems, where they play a pivotal role in the response directed against structurally conserved components of pathogens. Synthetic bacterial lipopeptide Pam3CSK4 is a TLR2 agonist capable of modulating Th1 and Th2 responses. This study examines the therapeutic effect of Pam3CSK4 in established airway inflammation in a murine model of asthma. In mice previously sensitized and challenged with OVA, Pam3CSK4 given i.p. markedly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid. Pam3CSK4 therapy was associated with a reduction in OVA-induced IL-4 and IL-5 secretion from thoracic lymph node culture, airways inflammation, bronchial hyperresponsiveness, and serum levels of IgE. Pam3CSK4 therapy was also associated with an increase in OVA-induced IFN-, IL-12, and IL-10 production. However, the anti-inflammatory effect of Pam3CSK4 was independent of IL-10 or TGF-, but was critically dependent on IL-12, the production of which by dendritic cells was enhanced by Pam3CSK4 in vitro. Our results provide direct evidence that Pam3CSK4 could represent a novel therapeutic agent in allergic airways disease.
Introduction
Asthma is a chronic inflammatory condition of the airways characterized by airways hyperresponsiveness (AHR),3 inflammatory infiltrates in the bronchial wall containing eosinophils, and elevated serum IgE levels. Th2 lymphocytes are thought to play a key role in the initiation and perpetuation of this airway inflammation (1, 2, 3). The prevalence of asthma and allergies has increased dramatically over the last 20 years in developed countries, which cannot be explained by changes in genetic predisposition. Environmental factors, especially in industrialized countries, are now thought to be responsible for this rapid increase in asthma (4, 5).
Respiratory infections have been linked to asthma in both a preventative and an exacerbating role (4, 5). Hence, investigating the system that initially recognizes the respiratory tract pathogens seems imperative to understanding this inflammatory disease. TLRs are pattern recognition receptors that act as primary sensors of microbial products. There are now 10 TLRs identified in humans, found in both the innate and the adaptive immune system, where they play a pivotal role in the response directed against distinct, structurally conserved components of pathogens (6, 7). Administration of low dose intranasal LPS in a murine model of asthma resulted in an increase in the Th2-mediated allergic phenotype, which is dependent on TLR4 (8). Immunomodulatory oligodeoxynucleotides and LPS can reduce allergic inflammation in experimental models of asthma via the activation of TLR9 and TLR4, respectively (8, 9, 10). To date, TLR9 is the only TLR that has been shown to reverse established airway inflammation in both acute and chronic murine models of asthma (10, 11).
Recently, it has been demonstrated that TLR2 polymorphism is a major determinant of the susceptibility to asthma and allergies in children of farmers in Germany (12). TLR2 recognizes microbial components by forming a heterodimer with TLR1 or TLR6 (13), and previous studies have examined the effect of the TLR2 agonists synthetic bacterial lipopeptide (Pam3CSK4) and peptidoglycan (PGN) in murine models of asthma. The results were contradictory. When administered during the sensitization period, early TLR2 agonist treatment was reported to worsen Th2-mediated asthma (14, 15). In contrast, TLR2 activation immediately before the intranasal challenge was found to reduce allergic airways inflammation (16). It is important to note, however, that although PGN was thought to activate the TLR2/6 heterodimer, recent work suggests that lipoprotein and lipoteichoic acid contamination of PGN may be responsible for its TLR2 activation (17). All studies to date have examined TLR2 activation before the airway challenge in these models (14, 15, 16), and none has examined the therapeutic potential of TLR2 agonists in established inflammation.
We have investigated the therapeutic effect of the synthetic TLR2 agonist, Pam3CSK4, on established airways inflammation. We report in this study that Pam3CSK4 profoundly attenuated an established OVA-specific asthma in mice. This therapeutic effect was critically dependent on IL-12, but not IL-10 or TGF-, and was accompanied by an enhanced specific IFN--producing Th1 response. Our data therefore provide direct evidence that Pam3CSK4 or other TLR2 agonists could have potential as a new treatment of clinical asthma.
Materials and Methods
BMDCs were generated as described previously (20, 21). The cells were cultured at 2 x 105 cell/ml with 0.3 μM OVA323–339 peptide and Pam3CSK4 (10 or 100 μg/ml) for 12 h. Supernatants were collected and stored at –70°C for cytokine analysis. The residual cells were then washed and cultured with 1 x 106 CD4+ T cells, 0.3 μM OVA323–339 peptide, and Pam3CSK4 (10 or 100 μg/ml) for an additional 12 h. T cells were purified from the lymph nodes of OVA TCR transgenic (DO11.10) by negative selection using magnetic beads (MACS; Miltenyi Biotec) (22). Supernatants were collected and stored at –70°C for cytokine analysis.
ELISA
Murine IL-4, IL-5, IL-12, IFN- (BD Biosciences), and IL-10 (BioSource International) were assayed using paired Abs. The lower limit of detection for IL-4, IL-5, IL-10, and IL-12 was 10 pg/ml, and that for IFN- was 40 pg/ml. Total serum IgE was measured using an OptEIA ELISA kit (BD Biosciences); the lower limit of detection was 6 ng/ml.
Statistical analysis
ANOVA was used to compare differences between multiple groups. Statistical significance between individual groups was then examined by Student’s t test. A value of p < 0.05 was considered significant, and p < 0.01 was deemed highly significant.
Results
Pam3CSK4 reverses established OVA-induced airways inflammation
To investigate the therapeutic potential of Pam3CSK4 in reversing inflammation in allergic asthma, we used an OVA-induced murine asthma model. BALB/c mice were primed and boosted with OVA as described in Materials and Methods. All mice were challenged i.n. on 3 consecutive days beginning on day 25. Pam3CSK4 (100 μg/mouse) was administered i.p only once on day 25, 26, or 27. Pam3CSK4 was given 2 h after challenge (Fig. 1A). Thus, mice treated with Pam3CSK4 on day 27 alone would have been challenged on the previous 3 days with OVA. Untreated OVA-sensitized and -challenged mice produced the expected airway hypersensitivity. Pam3CSK4 treatment showed a profound reduction in the BAL total cell count, eosinophilia, and Penh value (Fig. 1B). Histological analysis demonstrated a reduction in the inflammatory infiltrates in the peribronchial and perivascular areas of the murine lungs treated with Pam3CSK4 (Fig. 1C). This decrease in airway hypersensitivity was observed on all days that Pam3CSK4 was administered. Even mice that had received all three i.n. OVA challenges showed a profound decrease in airways inflammation when Pam3CSK4 was administered 2 h after the last i.n. allergen dose (see day 27 data). This beneficial effect of Pam3CSK4 was sustained for at least 5 days after treatment (data not shown). These results demonstrate that Pam3CSK4 could be a novel therapeutic agent for established asthma. The dose of Pam3CSK4 used was determined by in vivo titration. Although 25 μg of Pam3CSK4 was found to significantly reduce airway hypersensitivity, 100 μg consistently produced highly significant attenuation of AHR (data not shown). Hence, 100 μg was selected for all subsequent experiments.
Immunological analysis of the effect of Pam3CSK4
To investigate the immunological mechanism involved in Pam3CSK4 treatment, lymphoid cells were harvested from the Pam3CSK4-treated and untreated control mice and cultured with OVA in vitro. Pam3CSK4 did not affect T cell proliferation against OVA in vitro (data not shown). However, lymphoid cells from Pam3CSK4-treated, OVA-sensitized/challenged mice produced significantly more IFN- and IL-10 compared with those from untreated, OVA-sensitized/challenged mice (Fig. 2A). In contrast, Pam3CSK4 treatment led to a marked reduction in IL-4 and IL-5 synthesis by lymphoid cells from OVA-sensitized/challenged mice. Serum obtained from mice after Pam3CSK4 treatment showed a marked reduction in the concentration of total IgE Ab. OVA-specific IgE also demonstrated a decrease after Pam3CSK4 therapy (data not shown). The level of serum IL-12 was greatly increased (up to 15 ng/ml) as early as 2 h after Pam3CSK4 injection (Fig. 2B). Thus, there appears to be a skewing of the Th2 to Th1 response immediately after Pam3CSK4 treatment.
Pam3CSK4 enhances IL-12 and IFN- synthesis in vitro
To investigate the cell source and mechanism by which Pam3CSK4 induced IL-12 production and Th1 cell development, we cultured BMDCs with graded doses of Pam3CSK4 in vitro. In response to Pam3CSK4, BMDCs produced significant levels of IL-12 (Fig. 3A). IL-10, IFN-, IL-4, and IL-5 were not detected in any appreciable amount (data not shown). The cells were then washed and cultured with highly purified CD4+ T cells from OVA-TCR transgenic mice (DO10.11) in the presence of OVA peptide. CD4+ T cells cultured in the presence of BMDCs and Pam3CSK4 produced significant IFN-, but little or no detectable IL-4 (Fig. 3B). These results demonstrate that Pam3CSK4 is capable of inducing DCs to produce IL-12, which, in turn, enhanced the development of Th1 cells and the production of IFN-.
The therapeutic effect of Pam3CSK4 is IL-12 dependent
We then investigated the role of IL-12 in the therapeutic effect of Pam3CSK4. Mice were sensitized and challenged with OVA as described above and injected i.p. with anti-mouse IL-12 or control rabbit IgG 2 h before i.n. challenge on day 27. Pam3CSK4 was administered 2 h later. Fig. 4 shows that the therapeutic effect of Pam3CSK4 was completely abrogated by anti-IL-12 Ab. This was clearly demonstrated by the reversal of eosinophilia and IgE, IFN-, IL-4, and IL-5 synthesis (Fig. 4A). Mice injected with anti-IL-12 Ab showed lung inflammatory cellular infiltration indistinguishable from that of mice sensitized, but untreated with Pam3CSK4 (Fig. 4B). To confirm the role of IL-12 in this model, we sensitized and challenged IL-12 KO mice with OVA and treated them with Pam3CSK4 as described above. Although wild-type control mice showed the expected beneficial effect of Pam3CSK4 treatment, IL-12 KO mice did not (Fig. 5). IL-12 KO mice exhibited the same degree of eosinophilia and IL-4 synthesis (Fig. 5A) and lung cellular infiltration (Fig. 5B) as the untreated IL-12 KO or wild-type mice. These results therefore clearly demonstrate that the therapeutic effect of Pam3CSK4 in asthma is critically dependent on the preferential induction of IL-12.
The therapeutic effect of Pam3CSK4 is IL-10 and TGF- independent
The enhancing effect of Pam3CSK4 on IL-10 production in vivo and in vitro suggests a potential role of regulatory T cells in the beneficial outcome of Pam3CSK4 treatment. IL-10 and TGF- have been implicated as the effectors of several subclasses of regulatory T cells (23, 24, 25, 26). To investigate this possibility, mice were sensitized and challenged with OVA in the standard protocol. The animals were injected i.p with anti-mouse IL-10R Ab, anti-TGF- Ab, or control normal IgG 2 h before i.n. OVA challenge on day 27. Pam3CSK4 therapy (day 27) was administered 2 h after the last i.n. challenge. These Abs and the amounts administered are routinely found to be effective in abrogating CD4+CD25+ regulatory T cells in our own laboratory (26). Neither anti-IL-10R Ab nor anti-TGF- Ab had any effect on BAL cell counts, eosinophilia, OVA-induced cell cytokine production by lymphoid cells, or inflammatory cell infiltrate in the lungs (Fig. 6). These results indicate that the therapeutic effect of Pam3CSK4 in airway hypersensitivity observed is unlikely to be mediated by regulatory T cells.
Discussion
Our study demonstrates an impressive therapeutic role of Pam3CSK4 in a murine asthma model. The beneficial effect was immediate and sustained. The effect of Pam3CSK4 appears to be based at least in part on its ability to induce IL-12 synthesis by DCs. The heightened levels of IL-12, in turn, enhance a specific Th1 response and decrease Th2 activity. The therapeutic effect is independent of IL-10 and TGF- and, by extension, is unlikely to involve regulatory T cells.
In a human study it has been shown that IL-12 administration could reduce blood and sputum eosinophilia, but did not significantly change AHR (27). It is known that both IFN- and IL-12 can reverse Th2-mediated AHR and inflammation in murine models of allergic airways inflammation (28). However, it has also been reported that high levels of IFN- and IL-12 could promote established Th2-induced eosinophilic inflammation, and that a strong Th1 response (independent of Th2 involvement) could induce AHR (29, 30, 31). This is perhaps not surprising, because at high concentrations, IFN- is a well-recognized proinflammatory cytokine in its own right. In as much as Th2 cytokines are a dominant detrimental feature in airways diseases, the key therapeutic approach is perhaps to achieve a balance between Th2 and Th1 cytokines, where there is a decrease in Th2 cytokines without a large increase in the Th1 response. The role of TLR2 signaling in the induction of Th1 and Th2 cell development remains controversial. TLR2 activation has been reported to lead to the initiation of both Th1 and Th2 differentiation (15, 32, 33). It is likely that whether TLR signaling activates the Th1 or the Th2 pathway depends on the timing (relative to specific antigenic stimulation), dose, and nature of TLR2 agonists and the genetic background of the responding hosts. In our model, although there is a mild increase in Th1 cytokines, the levels do not appear to be high enough to exacerbate AHR or increase the Th1-dominant IgG2a Ab subtype (data not shown). Recent reports investigating the effect of TLR2 agonists in the sensitization phase and the effect of a TLR2/4 agonist in the challenge phase of murine models of asthma have also shown that TLR activation could lead to a beneficial decrease in the Th2 phenotype, with a mild nondetrimental increase in the Th1 response (34, 35).
The effect of Pam3CSK4 was rapid. AHR attenuation was evident 24 h after i.p. administration. This was accompanied by a rapid reduction in the serum IgE level, which was also reported by other investigators (11). The mechanism of this rapid IgE reduction is unclear. However, it should be noted that the half-life of rodent serum IgE can be as short as 12 h (36, 37). The i.p. route used in our study was designed to influence the systemic immunological response and to avoid a potential local proinflammatory effect of Pam3CSK4 on other cell types, including macrophages, neutrophils, mast cells, and NK cells. Indeed, we have found that i.n. inoculation of Pam3CSK4 in our model consistently exacerbated AHR (data not shown). Experiments are currently being designed to address the mechanism of the disease-exacerbating effect of i.n. Pam3CSK4.
The enhancing effect of Pam3CSK4 on IL-10 production raised the possibility that regulatory T cells may play a role in the beneficial outcome of Pam3CSK4 treatment. Many investigators have observed that the function of regulatory T cells (CD4+CD25+, Tr1, and Th3) is dependent on IL-10 and TGF- (23, 24, 25, 26). It is also known that regulatory T cells express TLRs, and their suppressive function can be enhanced by TLR activation (38, 39). Using blocking Abs, we demonstrate in this study that the anti-inflammatory effect of Pam3CSK4 is independent of IL-10 or TGF-, and hence, regulatory T cells are unlikely to play an important role in the therapeutic effect of Pam3CSK4.
The beneficial effect of Pam3CSK4 and its mechanism of action appear to be akin to those of the TLR9 agonist, CpG oligonucleotides. TLR9 activation also led to an increase in IL-12 production by DCs and an enhancement of IFN- synthesis by T cells, resulting in decreased Th2 cell differentiation and attenuated eosinophilic airways inflammation (11). However, it has been reported that CpG could enhance the risk of aggravation of autoimmune disorder in immunocompetent hosts (40). Thus, TLR2 agonists, such as synthetic Pam3CSK4, may represent additional and alternative potential reagents for controlling allergic diseases.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Medical Research Council, the Wellcome Trust, the National Asthma Campaign, the European Commission, and the Scottish Chief Scientist’s Office.
2 Address correspondence and reprint requests to Dr. Foo Y. Liew or Dr. Damo Xu, Division of Immunology, Infection, and Inflammation, University of Glasgow, Western Infirmary, Glasgow, Scotland, United Kingdom G11 6NT. E-mail address: f.y.liew{at}clinmed.gla.ac.uk or d.xu{at}clinmed.gla.ac.uk
3 Abbreviations used in this paper: PGN, peptidoglycan; AHR, airways hyperresponsiveness; BAL, bronchoalveolar lavage; BMDC, bone marrow-derived DC; DC, dendritic cell; i.n., intranasally; KO, knockout; Penh, enhanced pause.
Received for publication February 14, 2005. Accepted for publication April 12, 2005.
References
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Eisenbarth, S. C., D. A. Piggott, J. W. Huleatt, I. Visintin, C. A. Herrick, K. Bottomly. 2002. Lipopolysaccharide-enhanced, Toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J. Exp. Med. 196: 1645-1651.(Manish Patel, Damo Xu2, P)
TLRs are primary sensors of both innate and adaptive immune systems, where they play a pivotal role in the response directed against structurally conserved components of pathogens. Synthetic bacterial lipopeptide Pam3CSK4 is a TLR2 agonist capable of modulating Th1 and Th2 responses. This study examines the therapeutic effect of Pam3CSK4 in established airway inflammation in a murine model of asthma. In mice previously sensitized and challenged with OVA, Pam3CSK4 given i.p. markedly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid. Pam3CSK4 therapy was associated with a reduction in OVA-induced IL-4 and IL-5 secretion from thoracic lymph node culture, airways inflammation, bronchial hyperresponsiveness, and serum levels of IgE. Pam3CSK4 therapy was also associated with an increase in OVA-induced IFN-, IL-12, and IL-10 production. However, the anti-inflammatory effect of Pam3CSK4 was independent of IL-10 or TGF-, but was critically dependent on IL-12, the production of which by dendritic cells was enhanced by Pam3CSK4 in vitro. Our results provide direct evidence that Pam3CSK4 could represent a novel therapeutic agent in allergic airways disease.
Introduction
Asthma is a chronic inflammatory condition of the airways characterized by airways hyperresponsiveness (AHR),3 inflammatory infiltrates in the bronchial wall containing eosinophils, and elevated serum IgE levels. Th2 lymphocytes are thought to play a key role in the initiation and perpetuation of this airway inflammation (1, 2, 3). The prevalence of asthma and allergies has increased dramatically over the last 20 years in developed countries, which cannot be explained by changes in genetic predisposition. Environmental factors, especially in industrialized countries, are now thought to be responsible for this rapid increase in asthma (4, 5).
Respiratory infections have been linked to asthma in both a preventative and an exacerbating role (4, 5). Hence, investigating the system that initially recognizes the respiratory tract pathogens seems imperative to understanding this inflammatory disease. TLRs are pattern recognition receptors that act as primary sensors of microbial products. There are now 10 TLRs identified in humans, found in both the innate and the adaptive immune system, where they play a pivotal role in the response directed against distinct, structurally conserved components of pathogens (6, 7). Administration of low dose intranasal LPS in a murine model of asthma resulted in an increase in the Th2-mediated allergic phenotype, which is dependent on TLR4 (8). Immunomodulatory oligodeoxynucleotides and LPS can reduce allergic inflammation in experimental models of asthma via the activation of TLR9 and TLR4, respectively (8, 9, 10). To date, TLR9 is the only TLR that has been shown to reverse established airway inflammation in both acute and chronic murine models of asthma (10, 11).
Recently, it has been demonstrated that TLR2 polymorphism is a major determinant of the susceptibility to asthma and allergies in children of farmers in Germany (12). TLR2 recognizes microbial components by forming a heterodimer with TLR1 or TLR6 (13), and previous studies have examined the effect of the TLR2 agonists synthetic bacterial lipopeptide (Pam3CSK4) and peptidoglycan (PGN) in murine models of asthma. The results were contradictory. When administered during the sensitization period, early TLR2 agonist treatment was reported to worsen Th2-mediated asthma (14, 15). In contrast, TLR2 activation immediately before the intranasal challenge was found to reduce allergic airways inflammation (16). It is important to note, however, that although PGN was thought to activate the TLR2/6 heterodimer, recent work suggests that lipoprotein and lipoteichoic acid contamination of PGN may be responsible for its TLR2 activation (17). All studies to date have examined TLR2 activation before the airway challenge in these models (14, 15, 16), and none has examined the therapeutic potential of TLR2 agonists in established inflammation.
We have investigated the therapeutic effect of the synthetic TLR2 agonist, Pam3CSK4, on established airways inflammation. We report in this study that Pam3CSK4 profoundly attenuated an established OVA-specific asthma in mice. This therapeutic effect was critically dependent on IL-12, but not IL-10 or TGF-, and was accompanied by an enhanced specific IFN--producing Th1 response. Our data therefore provide direct evidence that Pam3CSK4 or other TLR2 agonists could have potential as a new treatment of clinical asthma.
Materials and Methods
BMDCs were generated as described previously (20, 21). The cells were cultured at 2 x 105 cell/ml with 0.3 μM OVA323–339 peptide and Pam3CSK4 (10 or 100 μg/ml) for 12 h. Supernatants were collected and stored at –70°C for cytokine analysis. The residual cells were then washed and cultured with 1 x 106 CD4+ T cells, 0.3 μM OVA323–339 peptide, and Pam3CSK4 (10 or 100 μg/ml) for an additional 12 h. T cells were purified from the lymph nodes of OVA TCR transgenic (DO11.10) by negative selection using magnetic beads (MACS; Miltenyi Biotec) (22). Supernatants were collected and stored at –70°C for cytokine analysis.
ELISA
Murine IL-4, IL-5, IL-12, IFN- (BD Biosciences), and IL-10 (BioSource International) were assayed using paired Abs. The lower limit of detection for IL-4, IL-5, IL-10, and IL-12 was 10 pg/ml, and that for IFN- was 40 pg/ml. Total serum IgE was measured using an OptEIA ELISA kit (BD Biosciences); the lower limit of detection was 6 ng/ml.
Statistical analysis
ANOVA was used to compare differences between multiple groups. Statistical significance between individual groups was then examined by Student’s t test. A value of p < 0.05 was considered significant, and p < 0.01 was deemed highly significant.
Results
Pam3CSK4 reverses established OVA-induced airways inflammation
To investigate the therapeutic potential of Pam3CSK4 in reversing inflammation in allergic asthma, we used an OVA-induced murine asthma model. BALB/c mice were primed and boosted with OVA as described in Materials and Methods. All mice were challenged i.n. on 3 consecutive days beginning on day 25. Pam3CSK4 (100 μg/mouse) was administered i.p only once on day 25, 26, or 27. Pam3CSK4 was given 2 h after challenge (Fig. 1A). Thus, mice treated with Pam3CSK4 on day 27 alone would have been challenged on the previous 3 days with OVA. Untreated OVA-sensitized and -challenged mice produced the expected airway hypersensitivity. Pam3CSK4 treatment showed a profound reduction in the BAL total cell count, eosinophilia, and Penh value (Fig. 1B). Histological analysis demonstrated a reduction in the inflammatory infiltrates in the peribronchial and perivascular areas of the murine lungs treated with Pam3CSK4 (Fig. 1C). This decrease in airway hypersensitivity was observed on all days that Pam3CSK4 was administered. Even mice that had received all three i.n. OVA challenges showed a profound decrease in airways inflammation when Pam3CSK4 was administered 2 h after the last i.n. allergen dose (see day 27 data). This beneficial effect of Pam3CSK4 was sustained for at least 5 days after treatment (data not shown). These results demonstrate that Pam3CSK4 could be a novel therapeutic agent for established asthma. The dose of Pam3CSK4 used was determined by in vivo titration. Although 25 μg of Pam3CSK4 was found to significantly reduce airway hypersensitivity, 100 μg consistently produced highly significant attenuation of AHR (data not shown). Hence, 100 μg was selected for all subsequent experiments.
Immunological analysis of the effect of Pam3CSK4
To investigate the immunological mechanism involved in Pam3CSK4 treatment, lymphoid cells were harvested from the Pam3CSK4-treated and untreated control mice and cultured with OVA in vitro. Pam3CSK4 did not affect T cell proliferation against OVA in vitro (data not shown). However, lymphoid cells from Pam3CSK4-treated, OVA-sensitized/challenged mice produced significantly more IFN- and IL-10 compared with those from untreated, OVA-sensitized/challenged mice (Fig. 2A). In contrast, Pam3CSK4 treatment led to a marked reduction in IL-4 and IL-5 synthesis by lymphoid cells from OVA-sensitized/challenged mice. Serum obtained from mice after Pam3CSK4 treatment showed a marked reduction in the concentration of total IgE Ab. OVA-specific IgE also demonstrated a decrease after Pam3CSK4 therapy (data not shown). The level of serum IL-12 was greatly increased (up to 15 ng/ml) as early as 2 h after Pam3CSK4 injection (Fig. 2B). Thus, there appears to be a skewing of the Th2 to Th1 response immediately after Pam3CSK4 treatment.
Pam3CSK4 enhances IL-12 and IFN- synthesis in vitro
To investigate the cell source and mechanism by which Pam3CSK4 induced IL-12 production and Th1 cell development, we cultured BMDCs with graded doses of Pam3CSK4 in vitro. In response to Pam3CSK4, BMDCs produced significant levels of IL-12 (Fig. 3A). IL-10, IFN-, IL-4, and IL-5 were not detected in any appreciable amount (data not shown). The cells were then washed and cultured with highly purified CD4+ T cells from OVA-TCR transgenic mice (DO10.11) in the presence of OVA peptide. CD4+ T cells cultured in the presence of BMDCs and Pam3CSK4 produced significant IFN-, but little or no detectable IL-4 (Fig. 3B). These results demonstrate that Pam3CSK4 is capable of inducing DCs to produce IL-12, which, in turn, enhanced the development of Th1 cells and the production of IFN-.
The therapeutic effect of Pam3CSK4 is IL-12 dependent
We then investigated the role of IL-12 in the therapeutic effect of Pam3CSK4. Mice were sensitized and challenged with OVA as described above and injected i.p. with anti-mouse IL-12 or control rabbit IgG 2 h before i.n. challenge on day 27. Pam3CSK4 was administered 2 h later. Fig. 4 shows that the therapeutic effect of Pam3CSK4 was completely abrogated by anti-IL-12 Ab. This was clearly demonstrated by the reversal of eosinophilia and IgE, IFN-, IL-4, and IL-5 synthesis (Fig. 4A). Mice injected with anti-IL-12 Ab showed lung inflammatory cellular infiltration indistinguishable from that of mice sensitized, but untreated with Pam3CSK4 (Fig. 4B). To confirm the role of IL-12 in this model, we sensitized and challenged IL-12 KO mice with OVA and treated them with Pam3CSK4 as described above. Although wild-type control mice showed the expected beneficial effect of Pam3CSK4 treatment, IL-12 KO mice did not (Fig. 5). IL-12 KO mice exhibited the same degree of eosinophilia and IL-4 synthesis (Fig. 5A) and lung cellular infiltration (Fig. 5B) as the untreated IL-12 KO or wild-type mice. These results therefore clearly demonstrate that the therapeutic effect of Pam3CSK4 in asthma is critically dependent on the preferential induction of IL-12.
The therapeutic effect of Pam3CSK4 is IL-10 and TGF- independent
The enhancing effect of Pam3CSK4 on IL-10 production in vivo and in vitro suggests a potential role of regulatory T cells in the beneficial outcome of Pam3CSK4 treatment. IL-10 and TGF- have been implicated as the effectors of several subclasses of regulatory T cells (23, 24, 25, 26). To investigate this possibility, mice were sensitized and challenged with OVA in the standard protocol. The animals were injected i.p with anti-mouse IL-10R Ab, anti-TGF- Ab, or control normal IgG 2 h before i.n. OVA challenge on day 27. Pam3CSK4 therapy (day 27) was administered 2 h after the last i.n. challenge. These Abs and the amounts administered are routinely found to be effective in abrogating CD4+CD25+ regulatory T cells in our own laboratory (26). Neither anti-IL-10R Ab nor anti-TGF- Ab had any effect on BAL cell counts, eosinophilia, OVA-induced cell cytokine production by lymphoid cells, or inflammatory cell infiltrate in the lungs (Fig. 6). These results indicate that the therapeutic effect of Pam3CSK4 in airway hypersensitivity observed is unlikely to be mediated by regulatory T cells.
Discussion
Our study demonstrates an impressive therapeutic role of Pam3CSK4 in a murine asthma model. The beneficial effect was immediate and sustained. The effect of Pam3CSK4 appears to be based at least in part on its ability to induce IL-12 synthesis by DCs. The heightened levels of IL-12, in turn, enhance a specific Th1 response and decrease Th2 activity. The therapeutic effect is independent of IL-10 and TGF- and, by extension, is unlikely to involve regulatory T cells.
In a human study it has been shown that IL-12 administration could reduce blood and sputum eosinophilia, but did not significantly change AHR (27). It is known that both IFN- and IL-12 can reverse Th2-mediated AHR and inflammation in murine models of allergic airways inflammation (28). However, it has also been reported that high levels of IFN- and IL-12 could promote established Th2-induced eosinophilic inflammation, and that a strong Th1 response (independent of Th2 involvement) could induce AHR (29, 30, 31). This is perhaps not surprising, because at high concentrations, IFN- is a well-recognized proinflammatory cytokine in its own right. In as much as Th2 cytokines are a dominant detrimental feature in airways diseases, the key therapeutic approach is perhaps to achieve a balance between Th2 and Th1 cytokines, where there is a decrease in Th2 cytokines without a large increase in the Th1 response. The role of TLR2 signaling in the induction of Th1 and Th2 cell development remains controversial. TLR2 activation has been reported to lead to the initiation of both Th1 and Th2 differentiation (15, 32, 33). It is likely that whether TLR signaling activates the Th1 or the Th2 pathway depends on the timing (relative to specific antigenic stimulation), dose, and nature of TLR2 agonists and the genetic background of the responding hosts. In our model, although there is a mild increase in Th1 cytokines, the levels do not appear to be high enough to exacerbate AHR or increase the Th1-dominant IgG2a Ab subtype (data not shown). Recent reports investigating the effect of TLR2 agonists in the sensitization phase and the effect of a TLR2/4 agonist in the challenge phase of murine models of asthma have also shown that TLR activation could lead to a beneficial decrease in the Th2 phenotype, with a mild nondetrimental increase in the Th1 response (34, 35).
The effect of Pam3CSK4 was rapid. AHR attenuation was evident 24 h after i.p. administration. This was accompanied by a rapid reduction in the serum IgE level, which was also reported by other investigators (11). The mechanism of this rapid IgE reduction is unclear. However, it should be noted that the half-life of rodent serum IgE can be as short as 12 h (36, 37). The i.p. route used in our study was designed to influence the systemic immunological response and to avoid a potential local proinflammatory effect of Pam3CSK4 on other cell types, including macrophages, neutrophils, mast cells, and NK cells. Indeed, we have found that i.n. inoculation of Pam3CSK4 in our model consistently exacerbated AHR (data not shown). Experiments are currently being designed to address the mechanism of the disease-exacerbating effect of i.n. Pam3CSK4.
The enhancing effect of Pam3CSK4 on IL-10 production raised the possibility that regulatory T cells may play a role in the beneficial outcome of Pam3CSK4 treatment. Many investigators have observed that the function of regulatory T cells (CD4+CD25+, Tr1, and Th3) is dependent on IL-10 and TGF- (23, 24, 25, 26). It is also known that regulatory T cells express TLRs, and their suppressive function can be enhanced by TLR activation (38, 39). Using blocking Abs, we demonstrate in this study that the anti-inflammatory effect of Pam3CSK4 is independent of IL-10 or TGF-, and hence, regulatory T cells are unlikely to play an important role in the therapeutic effect of Pam3CSK4.
The beneficial effect of Pam3CSK4 and its mechanism of action appear to be akin to those of the TLR9 agonist, CpG oligonucleotides. TLR9 activation also led to an increase in IL-12 production by DCs and an enhancement of IFN- synthesis by T cells, resulting in decreased Th2 cell differentiation and attenuated eosinophilic airways inflammation (11). However, it has been reported that CpG could enhance the risk of aggravation of autoimmune disorder in immunocompetent hosts (40). Thus, TLR2 agonists, such as synthetic Pam3CSK4, may represent additional and alternative potential reagents for controlling allergic diseases.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Medical Research Council, the Wellcome Trust, the National Asthma Campaign, the European Commission, and the Scottish Chief Scientist’s Office.
2 Address correspondence and reprint requests to Dr. Foo Y. Liew or Dr. Damo Xu, Division of Immunology, Infection, and Inflammation, University of Glasgow, Western Infirmary, Glasgow, Scotland, United Kingdom G11 6NT. E-mail address: f.y.liew{at}clinmed.gla.ac.uk or d.xu{at}clinmed.gla.ac.uk
3 Abbreviations used in this paper: PGN, peptidoglycan; AHR, airways hyperresponsiveness; BAL, bronchoalveolar lavage; BMDC, bone marrow-derived DC; DC, dendritic cell; i.n., intranasally; KO, knockout; Penh, enhanced pause.
Received for publication February 14, 2005. Accepted for publication April 12, 2005.
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