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Toll-Like Receptor 4 or 2 Agonists Decrease Allergic Inflammation

Respiratory and Critical Care Division and Laboratory of Immunogenetics and Transplantation, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Patricia W. Finn, Respiratory and Critical Care Division, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. E-mail: pwfinn{at}rics.bwh.harvard.edu

	Abstract

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Toll-like receptors (TLRs) recognize highly conserved microbial molecular patterns, such as found in endotoxin. This study tested whether TLR4 and TLR2 stimulation in vivo would modulate subsequent adaptive (allergic) immune responses. We analyzed the effects of pulmonary administration of a TLR4 agonist, lipid A (LpA), and two TLR2 agonists, peptidoglycan (Ppg) and PamCys, in a murine model of allergic inflammation. The TLR agonists were administered during allergen sensitization or challenge. Both TLR agonists decreased the allergen-induced pulmonary recruitment of eosinophils when administered at sensitization or challenge. When given before sensitization, the TLR4 and TLR2 agonists decreased additional allergen-induced parameters of inflammation (pulmonary eosinophilia, bronchoalveolar lavage IL-13, total serum IgE, and airway hyperresponsiveness). Interestingly, TLR4 and TLR2 agonists decreased the number of CD4⁺ cells in the lung. Also, at the site of local allergen stimulation, the draining thoracic lymph nodes, allergen-induced lymphocyte proliferation, and IL-13 secretion were decreased by administration of LpA and Ppg. These data provide a distinct example of the modulation of adaptive (allergic) responses by non–antigen-dependent stimuli. Our findings also demonstrate that both TLR4 and TLR2 agonists decrease allergic responses, supporting the concept that exposure to bacterial components under defined conditions may protect against allergic disease.

Key Words: innate • immunity • asthma • Toll-like receptor 4 • Toll-like receptor 2 • allergy

	Introduction

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Mammalian Toll-like receptors (TLRs) recognize microbial products and initiate innate immune responses (1–3). Recent interest has focused on the concept that stimulation of innate immunity through the TLRs can modulate subsequent adaptive immune responses. The example most frequently described is that endotoxin ligation of TLR4 increases the expression of CD80 and CD86 costimulatory molecules, a pathway critical for maximal antigen-dependent T cell activation (1, 4, 5). At least 11 TLRs have been identified (6), and in terms of pulmonary responses, TLR4 and TLR2 signals are recent topics of interest. Both TLR4 and TLR2 are abundant in the lung, and both have ligands associated with recurrent pulmonary exposure, e.g., endotoxin lipopolysaccharide (LPS) for TLR4, and gram-positive bacteria for TLR2, respectively.

Epidemiologic studies have detected an association between higher exposure to endotoxin during the first year of life and a lower prevalence of asthma in childhood (7). Others have shown that TLR4 polymorphisms can modify endotoxin response in asthma (8). Previous studies in murine models indicate that TLR4 activation in the context of allergen exposure can promote decreases in allergic responses, although increased responses have also been observed, depending on the model, dose, timing, and murine strain (9–13). The role of TLR2 in allergic pulmonary inflammation in vivo is less well defined. Children of farmers found to have a decreased risk of developing allergies have increased levels of TLR2 mRNA (14). Also, genetic variations in TLR2 have been found to be major determinants of the susceptibility to asthma and allergies in children of farmers (15). The effects of TLR4 and TLR2 agonists in allergic inflammation under the same conditions have not been well characterized previously.

In this study, we tested whether TLR4 and TLR2 stimulation in vivo would modulate subsequent allergic responses. Using a murine model of allergic inflammation, we examined the allergic effects that might be influenced by these two different bacterial components and their relationship to allergic inflammation.

	MATERIALS AND METHODS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Mice
Six- to 8-wk-old BALB/c and C57BL/6J male mice were purchased from Jackson Laboratory (Bar Harbor, ME). The mice were maintained according to the guidelines of the Committee on Animals of the Harvard Medical School and the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources National Research Council.

Ovalbumin Sensitization and Challenge
Mice were sensitized and challenged with the allergen ovalbumin (OVA) as previously described (16–18). Briefly, OVA mice were sensitized via intraperitoneal injection with 10 µg of chicken OVA (Sigma, St. Louis, MO), and 1 mg of Al(OH)₂ (alum; Sigma) in 0.2 ml of phosphate-buffered saline (PBS; Sigma), followed by a boosting injection on Day 7 with the identical reagents. PBS mice received 1 mg of alum in 0.2 ml of PBS without OVA. On Days 14–20, mice received aerosolized challenges with 6% OVA or PBS, respectively, for 20 min/d via an ultrasonic nebulizer (Model 5000; DeVilbiss, Somerset, PA). All groups were killed at Day 21 and analyzed for the allergic parameters described below.

TLR4 or 2 Agonist Administration
A TLR4 agonist, lipid A (LpA) (Salmonella minnesota; Sigma); a TLR2 agonist, peptidoglycan (Ppg) (Staphylococcus aureus; Sigma); or a TLR2 agonist, PamCys (synthetic lipopeptide Pam3Cys-Ser-Lys4; EMC microcollections, Tübingen, Germany) was administered intratracheally to OVA- or PBS-sensitized and -challenged mice at the time points shown in RESULTS. The doses of TLR agonists used were as follows: LpA (7.5, 25, 75 µg), Ppg (7.5, 75, 150, 225 µg), and PamCys (7.5 µg).

Specificity of Reagents
For proliferation assays, splenocytes from TLR4-deficient mice (C.C3-Tlr4^Lps-d) from Jackson Laboratory and TLR2 knockout (KO) mice (kind gift from Dr. Douglas Golenbock, University of Massachusetts Medical Center, Worcester, MA) were analyzed. The positive control, the mitogen concanavalin A (Con A, 10 µg/ml), induced a positive proliferative response (stimulation index [SI]: stimulated cpm/unstimulated cpm) > 3 in wild-type, TLR4-deficient, and TLR2 KO mice. In contrast, LpA did not induce positive responses in TLR4-deficient mice, and Ppg did not induce positive responses in TLR2 KO mice (SI < 3 at doses of 1, 10, or 100 µg/ml, respectively; data not shown). In addition, as OVA contamination can potentially modify allergic responses (19), analysis of OVA by the limulus assay revealed low concentrations (< 0.01 EU/ml = 0.002ng/ml) that did not significantly change lymphocyte proliferation of unprimed splenocytes (data not shown).

Bronchoalveolar Lavage Analysis
Each mouse underwent bronchoalveolar lavage (BAL), as previously described (17, 18). BAL cells were pelleted and the supernatant was stored at –80°C. Cells were resuspended in RPMI (5 x 10⁵ cells/ml). Slides for differential cell counts were prepared with Cytospin (Shandon, Pittsburgh, PA) and fixed and stained with Diff-Quik (Dade Behring, Newark, DE). For each sample, an investigator blinded to the treatment groups performed two counts of 100 cells.

Cytokines
BAL interleukin (IL)-13 and interferon (IFN)- were measured by ELISA according to manufacturer's specifications (R&D Systems, Minneapolis, MN). Briefly, samples of BAL fluid were aliquoted in duplicate into 96-well plates (50 ml/well) pre-coated with antibody to specific cytokines and assayed according to the manufacturer's instructions. Optical density was measured at 450 nm. Cytokine concentrations were determined by comparison with known standards.

Serum IgE
Blood was centrifuged at 13,000 rpm for 10 min to recover serum. Total serum IgE levels were determined by ELISA as previously described (17). Total serum IgE concentrations were calculated by using a standard curve generated with commercial IgE standard (BD PharMingen, San Diego, CA).

Determination of Airway Hyperresponsiveness
Airway hyperresponsiveness (AHR) was assessed using whole body plethysmography 4 h after the final aerosol challenge. Mice were placed in individual chambers and increasing doses of methacholine were nebulized into the chambers via an inlet at concentrations of 20, 40, and 80 mg/ml for 2 min similar as previously described (16). Readings were averaged over 5 min after the nebulization. The whole body plethysmography system measures changes in box pressure during expiration and inspiration, peak expiratory and peak inspiratory pressures (PEP and PIP, respectively), inspiratory time (Ti), expiratory time (Te), and relaxation time (Tr = time of the pressure decay to 36% of total box pressure during expiration), and generates a value called enhanced Pause (Penh = PEP/PIP x [(Te-Tr)/Tr]) directly correlating with airway resistance (20). Penh analysis was performed only in BALB/c mice. The BALB/c strain exhibit significant correlation between Penh and classical measures of lung resistance (24), as we have previously used (17, 18, 21–23).

Flow Cytometric Analysis
Flow cytometry was performed on whole-lung homogenates. Briefly, after homogenates were digested with 5 mg of DNase and 10 mg of collagenase in 10 ml of RPMI-media, cells were washed twice with PBS with 5% fetal calf serum (wash buffer). Samples were counted, and placed to obtain 1 x 10⁶ cells per well times the number of wells in each experiment. Each cell sample was suspended in 1 ml of wash buffer and incubated for 30 min at 4°C with saturating concentrations of CD4⁺ (L3T4 cat 553729, rat IgG2b k; FITC BD Biosciences, San Jose, CA) antibody. Samples were washed twice with wash buffer, fixed by resuspension in 4% paraformaldehyde, and stored in the dark at 4°C. Samples were analyzed on a Coulter Cytomics FC 500 Series Flow Cytometry Systems (Beckman Coulter, Fullerton, CA), and fluorescence was detected by using FL1 (FITC). Lymphocytes were gated according to size in a forward and side scatter plot. Listmode data were analyzed with Beckman Coulter Software. The CD4⁺ cells were analyzed, and proportions were calculated based on the lymphocyte population.

Thoracic Lymphocyte Proliferation
The draining thoracic lymph nodes (TLN) were harvested from experimental groups as previously described (21) and were restimulated in vitro with OVA (0.5 mg/ml) or Con A (10 µg/ml), and incubated for 48 h. Con A induced a positive proliferative response in OVA and PBS mice ([H³]thymidine-measured radiation cpm of 407,673.2 and 199,657.2, respectively) (data not shown). Supernatants were harvested and analyzed for IL-13 secretion (R&D Systems).

Statistical Analysis
Statistical analysis of normally distributed data was performed by t test, and nonparametric data were analyzed by a Mann-Whitney test (Sigma Stat 3.0 software). Data are reported as means ± SEM. Statistical significance was defined by P < 0.05.

	RESULTS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Analysis of Allergen-Induced Eosinophilia after Pulmonary Administration of a TLR4 or TLR2 Agonist
The influence of pulmonary administration of a TLR4 agonist, LpA, the bioactive component of LPS, was examined in a murine model of allergic inflammation. We measured allergen-induced BAL eosinophilia in BALB/c mice. Consistent with allergic inflammation, OVA-sensitized and -challenged mice (OVA mice) had a significant increase in BAL eosinophilia as compared with PBS mice (P < 0.05, Table 1). Analysis of total cell numbers showed a similar trend (data not shown).

OVA mice treated with a TLR2 agonist, peptidoglycan (Ppg), either 1 d before (Ppg –1) or 1 d after (Ppg +1) sensitization manifested a significant decrease in the percentage of BAL eosinophils as compared with mice given OVA alone (P < 0.05, Table 1). OVA mice treated with Ppg 1 d after the first aerosolized challenge (Ppg +15) also had a significant decrease in the percentage of BAL eosinophils as compared with mice given OVA alone (P < 0.05, Table 1). Taken together, these data indicate that allergen-induced pulmonary eosinophilia is suppressed by the administration of either LpA or Ppg at sensitization or challenge.

TLR4 or TLR2 Agonists Decrease Lymphocyte Activation In Vivo
We previously defined a critical role for lymphocyte activation in an OVA model of allergic inflammation (21–23). In this study, we examined the effects of TLR agonists on lymphocyte activation. We performed flow cytometric analysis of lung cells from OVA mice treated with either LpA or Ppg. As expected (21), CD4⁺ cells were increased in the OVA mice as compared with PBS mice (Figure 1). Administration of either a TLR4 or TLR2 agonist to OVA mice decreased the number of CD4⁺ cells. To examine the functional effects of TLR4 or TLR2 agonists on lymphocyte activation, we analyzed allergen-specific OVA lymphocyte proliferation and cytokine secretion. We previously showed that allergen-specific lymphocyte proliferation and secretion of Th2 cytokines (e.g., IL-4) are increased in draining thoracic lymph nodes (TLN) from OVA mice as compared with PBS mice (22). Here we show that OVA-specific TLN proliferation was decreased in OVA mice that had received either a TLR4 or a TLR2 agonist in vivo (Figures 2A and 2B). In addition, allergen-induced IL-13 secretion was also decreased in these groups (Figures 2C and 2D).

View larger version (46K): [in this window] [in a new window]   Figure 1. TLR4 and TLR2 agonists decrease CD4+ cells. Cell homogenates from whole lung were prepared from OVA or PBS allergen-sensitized and -challenged BALB/c mice, which received either lipid A (LpA) or peptidoglycan (Ppg) 1 d before sensitization. Lymphocyte subsets were analyzed by flow cytometry for the expression of CD4+ cells based on side scatter. A minimum of 10,000 events were acquired in the CD4+ gate. CD4+ = % CD4+ cells.

View larger version (21K): [in this window] [in a new window]   Figure 2. TLR4 and TLR2 agonists decrease allergen-induced lymphoproliferation and cytokine secretion. BALB/c mice were sensitized and challenged with OVA and received either LpA or Ppg 1 d before sensitization (OVA + LpA, OVA + Ppg, respectively). The draining thoracic lymph nodes from these groups were harvested and restimulated in vitro in the presence or absence of OVA (0.5 mg/ml; A and B). Concavalin A (Con A) was used as positive control and the values were as follows: for A, OVA 407,673.2 cpm and OVA + LpA 438,090.9 cpm; for B, OVA 272,376.8 cpm and OVA + Ppg 334,047.2 cpm. Proliferation was determined by [3H]thymidine incorporation. IL-13 secretion in the cell supernatant was measured by ELISA (C and D).

View larger version (28K): [in this window] [in a new window]   Figure 3. Pulmonary administration of a TLR4 agonist decreases allergic immune responses. Mice were sensitized and challenged with OVA as described in MATERIALS AND METHODS. LpA was administered intratracheally 1 d before allergen sensitization at doses of 7.5 µg and 25 µg. Cell counts were determined by differential staining of cells isolated from BAL fluid. Total serum IgE and BAL cytokines (R&D Systems) were measured by ELISA. (A) BAL eosinophilia; (B) total serum IgE; (C) BAL IL-13; (D) BAL IFN-; (E) a measurement of airway resistance by Penh. These data are representative of two experiments. Data are shown as geometric mean ± SEM (n = 3–6 per group). PBS versus OVA (P < 0.05), *OVA versus OVA + LpA (P < 0.05).

A TLR2 Agonist Decreases Allergen-Induced Pulmonary Eosinophils, Total Serum IgE, BAL IL-13, and AHR
Analysis of a TLR2 agonist was pursued in the same allergic model with Ppg administration before allergen sensitization. Local pulmonary administration of Ppg to OVA-treated mice significantly suppressed allergen-induced pulmonary eosinophilia, total serum IgE, BAL IL-13, and AHR (Figures 4A–4C, 4E). BAL IFN- was at the level of detection and was modestly increased in OVA mice that had received Ppg (Figure 4D). To test the ability of Ppg to maximally decrease allergic responses, we used higher doses of Ppg (150, 225 µg) in these studies than those used for analysis of eosinophilia (Table 1).

View larger version (25K): [in this window] [in a new window]   Figure 4. Pulmonary administration of a TLR2 agonist decreases allergic immune responses. Mice were sensitized and challenged with OVA as described in MATERIALS AND METHODS. Ppg was administered intratracheally 1 d before allergen sensitization at doses of 150 µg and 225 µg. Cell counts were determined by differential staining of cells isolated from BAL fluid. Total serum IgE and BAL cytokines (R&D Systems) were measured by ELISA. (A) BAL eosinophilia; (B) total serum IgE; (C) BAL IL-13; (D) BAL IFN-; (E) a measurement of airway resistance by Penh. These data are representative of two experiments. Data are shown as geometric mean ± SEM (n = 3–6 per group). PBS versus OVA (P < 0.05), *OVA versus OVA + LpA (P < 0.05).

View larger version (12K): [in this window] [in a new window]   Figure 5. Two different TLR2 agonists decrease allergen-induced BAL eosinophilia and IL-13 response. Mice (C57BL/6) were sensitized and challenged with OVA as described in MATERIALS AND METHODS. Peptidoglycan (Ppg, 7.5 µg) or PamCys (7.5 µg) were administered intratracheally 1 d before allergen sensitization. (A) Cell counts were determined by differential staining of cells isolated from BAL fluid. (B) IL-13 was measured by ELISA (R&D Systems). Data are shown as geometric mean ± SEM (n = 3–6 per group). PBS versus OVA (P < 0.05), *OVA versus OVA + Ppg/PamCys (P < 0.05).

	DISCUSSION

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

Our findings indicate that a TLR4 agonist, LpA, given during allergic sensitization decreases allergen-induced pulmonary eosinophilia, BAL IL-13, total serum IgE, and AHR. LpA also decreased allergen-induced pulmonary eosinophilia, BAL IL-13, and total serum IgE in an additional strain (C57BL/6, not shown). In addition, later administration of LpA during allergen challenge decreased allergen-induced pulmonary recruitment of eosinophils. TLR4 stimulation has previously been shown, using different protocols, to decrease parameters of allergic inflammation (9–11, 25, 26), whereas others have shown that TLR4 stimulation can increase AHR and promote airway allergic responses, depending on timing, strain, and use of adjuvant (27–29). Here we show that following the same protocol, a TLR4 agonist (and TLR2 agonists) can decrease allergic responses at both sensitization and challenge, at multiple doses, and in two mice strains in the context of a maximal allergic response induced by allergen and adjuvant.

Interestingly, LPS (TLR4 agonist) administered concomitantly with allergen, thus used as an adjuvant, can differentially induce a Th1 or Th2 immune response depending on the dose (10). TLR4 signals, e.g., LPS, are presumed to induce Th1 primarily (e.g., IFN-) and proinflammatory cytokines (e.g., TNF-, IL-6) in the absence of allergen (30, 31). TLR4 agonists could potentially decrease Th2 allergic responses by induction of Th1 responses (9). Other TLR agonists, such as immunostimulatory DNA sequences (CpG DNA motifs; TLR9 agonist), strongly induce a Th1 (IFN-) immune response (e.g., 750 pg/ml) in supernatants from splenocytes (32, 33). In this study, we found that LpA induces the production of IFN- in the BAL of OVA mice at modest levels (e.g., 4.3 pg/ml). We cannot exclude the possibility that these modest levels of IFN- still affect local immune responses in the lung or that these levels are sufficient to skew the immune system toward a Th1 immune response. Also, these data suggest that mechanisms other than predominance of the Th1 subset may be involved in TLR4-mediated suppression of allergic responses. The recent identification of TLR4 on the surface of CD4⁺ cells suggests another pathway by which TLR4 signals may modulate lymphocyte-dependent responses (34, 35). Our data show that administration of LpA decreases the population of CD4⁺ cells in the lung. After LpA administration, the concomitant decrease of allergen-induced lymphocyte proliferation indicates a functional effect of TLR4 agonists on lymphocyte responses. Interestingly, allergen-induced production of IL-13 in the thoracic lymph nodes was also decreased, supporting the concept that TLR4 agonists can modulate antigen-dependent immune responses. Whether TLR4-mediated suppression of allergic responses occurs directly by binding to TLR4 receptors on T cells or indirectly by inducing other mediators is under investigation.

TLR2 interactions in allergic inflammation are a current topic of debate. Our data indicate a suppressive effect of TLR2 agonists in allergic inflammation. A previous study, using a synthetic lipopeptide (TLR2 agonist), also showed a decrease in allergen-induced immune responses (36). Our study supports and complements the concept that TLR2 agonists decrease allergen-induced pulmonary eosinophilia, Th2 cytokine production, and the systemic induction of serum IgE. In contrast, recent reports show that TLR2 agonists used as adjuvants of allergen sensitization can aggravate Th2 responses (37, 38). Our model with OVA allergen and the adjuvant alum indicated that TLR2 signals suppress allergic responses in the context of a maximal allergic response. We showed that pulmonary exposure to Ppg can decrease allergic parameters of inflammation at both allergen sensitization and challenge. To characterize the effects of TLR2 agonists in allergic inflammation, we used another TLR2 agonist (PamCys) and also showed a decrease in allergic inflammation. We also found that the administration of either Ppg or PamCys decreased allergic inflammation in two different strains. Together, these data indicate that a TLR2-mediated decrease in allergic inflammation is neither agonist- nor strain-dependent. Future investigations of the specific lymphocyte populations that may be targets for the effects of TLR4 and 2 agonists will be crucial in the understanding of the specific conditions that can be used to manipulate these pathways to decrease allergic responses.

	Acknowledgments

 
The authors thank Drs. Leon Ting, Yolonda Colson, Rebecca Baron, and Sepideh Amirifeli for their review and thoughtful critique of this manuscript; John Daley and Suzane B. Lazo-Kallanian for their technical support in flow cytometry; and Nancy Voynow and Amy S. Nunn for editorial assistance.

	Footnotes

 
This work was supported by NIH grants HL 56723 and HL 67684 (P.W.F.).

^* These two authors contributed equally to the work presented in this article.

Received in original form December 4, 2003

Received in final form December 1, 2004

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