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Published ahead of print on January 17, 2008, doi:10.1164/rccm.200709-1387OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 720-729, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200709-1387OC

Original Article

Toll-like Receptor 2 Down-regulation in Established Mouse Allergic Lungs Contributes to Decreased Mycoplasma Clearance

Qun Wu1, Richard J. Martin1, Spencer LaFasto1, Benjamin J. Efaw1, John G. Rino1, Ronald J. Harbeck1 and Hong Wei Chu1

1 Department of Medicine, National Jewish Medical and Research Center, and the University of Colorado Health Sciences Center, Denver, Colorado

Correspondence and requests for reprints should be addressed to Hong Wei Chu, M.D., National Jewish Medical and Research Center, 1400 Jackson Street, Room A639, Denver, CO 80206. E-mail: chuhw{at}njc.org


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Respiratory Mycoplasma pneumoniae (Mp) infection is involved in asthma pathobiology, but whether the established allergic airway inflammation compromises lung innate immunity and subsequently predisposes patients with asthma to Mp infection remains unknown.

Objectives: To test whether the established allergic airway inflammation compromises host innate immunity (e.g., Toll-like receptor 2 [TLR2]) to hinder the elimination of Mp from the lungs.

Methods: We used mouse models of ovalbumin (OVA)-induced allergic airway inflammation with an ensuing Mp infection, and cultures of mouse primary lung dendritic cells (DCs) and bone marrow–derived DCs.

Measurements and Main Results: Lung Mp clearance in allergic mice and TLR2 and IL-6 levels in lung cells, including DCs as well as cultured primary lung DCs and bone marrow–derived DCs, were assessed. The established OVA-induced allergic airway inflammation, or the prominent Th2 cytokines IL-4 and IL-13, inhibited TLR2 expression and IL-6 production in lung cells, including lung DCs, and eventually led to impaired host defense against Mp. Studies in IL-6 knockout mice indicated that IL-6 directly promoted Mp clearance from the lungs. IL-4– and IL-13–induced suppression of TLR2 was mediated by inhibiting nuclear factor-{kappa}B activation through signal transducer and activator of transcription 6 (STAT6) signaling pathway.

Conclusions: The established OVA-induced allergic airway inflammation impairs TLR2 expression and host defense cytokine (e.g., IL-6) production, and subsequently delays lung bacterial clearance. This could offer novel therapeutic strategies to reinstate TLR2 activation by using TLR2 ligands and/or blocking IL-4 and IL-13 to ameliorate persisting respiratory bacterial infections in allergic lungs.

Key Words: asthma • lung • innate immunity • bacterial clearance



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Mycoplasma pneumoniae infection is involved in asthma pathobiology, but whether the established allergic airway inflammation compromises lung innate immunity and subsequently predisposes patients with asthma to M. pneumoniae infection remains unknown.

What This Study Adds to the Field
Established allergic airway inflammation impairs lung, including dendritic cell–dependent, innate host defense, leading to delayed bacterial clearance from the lungs.

 
Mycoplasma pneumoniae (Mp), the major cause of community-acquired pneumonia, has been recognized as a contributing factor in both stable asthma and asthma exacerbations (1, 2). Our previous clinical study has demonstrated that 42% of patients with chronic stable asthma had evidence of airway Mp infection by means of polymerase chain reaction specific for the Mp P1 adhesin gene or the gene encoding the 16S ribosomal ribonucleic acid (1). In addition to our findings, clinical studies from other investigators also suggest a role of Mp in asthma pathobiology. For example, Gil and coworkers isolated Mp in throat swabs of 24.7% of their adult patients with asthma by culture (3). Similarly, Chang and colleagues have found that 36.5% of their pediatric patients with asthma had Mp infection as determined by serology (4). Moreover, treatment with macrolide antibiotics in patients with asthma and Mp infection was shown to significantly improve lung function, and to reduce the severity of respiratory symptoms, further supporting a role of Mp infection in asthma pathogenesis (5–7). However, it remains unclear why asthmatic airways are susceptible to Mp infection.

Previous human studies have suggested that deficient expression of antimicrobial peptides (e.g., LL-37, β-defensins 2 and 3) may account for the increased susceptibility to Staphylococcus aureus infection in patients with atopic dermatitis (8, 9). However, the involved molecular mechanisms remain unclear.

Toll-like receptors (TLRs) are pattern-recognition receptors expressed on many cell types, including dendritic cells (DCs) and airway epithelial cells, to recognize invading microorganisms and to induce innate immune responses and subsequent acquired immunity (10). For example, DCs produce various proinflammatory cytokines (e.g., IL-6 and IL-12) upon TLR ligand stimulation (11–13). IL-6 is a pivotal proinflammatory cytokine to prime host defense against typical bacterial infection, and its production is predominantly TLR2 dependent (14–18). Recently, TLR2 expression was shown to be locally impaired in patients with nasal polyposis (19). Several single nucleotide polymorphisms (SNPs) in TLR2 coding regions have been identified in patients with asthma and were linked to a diminished cell response to TLR2 agonists (20). Interestingly, farmers' children carrying the TLR2 –16934A allele were shown to have three times higher morbidity from asthma than those carrying the TLR2 –16934T allele (21). Our previous work has demonstrated that TLR2 is critical for innate immune responses to Mp in mice: (1) Mp-induced host defense cytokine (e.g., IL-6) production in nonallergic mice is largely dependent on TLR2 and (2) in both nonallergic and allergic settings, TLR2-deficient mice have impaired lung Mp clearance compared with wild-type mice (22, 23). These studies strongly suggest that reduction or lack of TLR2 function in vivo may decrease immune protection from pathogens (e.g., Mp) containing TLR2 ligands, and thus contributes to chronic asthma or disease exacerbations. Nevertheless, the mechanisms underlying a deficient TLR2 function in asthma or other allergic diseases have not been well explored.

Prominent T-helper type 2 (Th2) cytokines IL-4 and IL-13 are essential to the development of allergic responses. Mueller and colleagues have shown that IL-4/IL-13 down-regulated TLR4 expression in a human intestinal epithelial cell line, but they did not illustrate any involved molecular mechanism (24). Signal transducer and activator of transcription 6 (STAT6), a member of the STAT family, has been shown to regulate the transcription of several IL-4/IL-13–responsive genes, such as CD23 and E-selectin (25–28). Furthermore, the TLR2 gene promoter contains one STAT consensus sequence and two nuclear factor (NF)-{kappa}B binding sites for binding corresponding transcription factors to regulate TLR2 gene expression (29–31).

In the current study, we hypothesize that the established ovalbumin (OVA)-induced allergic airway inflammation, or the prominent Th2 cytokines IL-4 and IL-13, inhibits TLR2 expression and IL-6 production to impair the host defense against Mp, which involves STAT6 activation and subsequent inhibition of NF-{kappa}B activity. Therefore, we assessed the effects of the established OVA-induced allergic airway inflammation, or IL-4 and IL-13, on TLR2 expression and IL-6 production in lung cells, including lung DCs, and lung Mp clearance in allergic mouse models. In addition, we demonstrated the protective role of IL-6 against Mp using IL-6 knockout (IL-6–/–) and wild-type mice. Furthermore, we verified the negative modulation of IL-4/IL-13 in NF-{kappa}B–mediated TLR2 expression and IL-6 production via STAT6 signaling in bone marrow–derived DCs (BMDCs).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
See the online supplement for further details of methods.

Animals
Female BALB/c mice, STAT6–/– mice on a BALB/c background, C57BL/6 mice, and IL-6–/– mice on a C57BL/6 background (8–12 wk) were obtained from the Jackson Laboratory (Bar Harbor, ME), and covered by a protocol approved by the Institutional Animal Care and Use Committee at National Jewish Medical and Research Center.

Mp Infection in Established Allergic and Nonallergic Mouse Models
BALB/c mice were sensitized twice in a span of 14 days with an intraperitoneal injection of 20 µg OVA (Sigma, St. Louis, MO) in 2.25 mg aluminum hydroxide (Pierce, Rockford, IL). Two weeks after the last sensitization, mice were challenged with 1% aerosolized OVA for 20 minutes once daily for 3 consecutive days. Two days after the last challenge, mice were inoculated intranasally with 50 µl Mp (strain FH, ATCC 15531; American Type Culture Collection, Manassas, VA) at 1 x 107 cfu or 50 µl saline (32), and killed 7 days later to examine lung Mp load and TLR2 expression.

In some allergic mice, a TLR2 ligand (Pam3CSK4, 15 µg/mouse; InvivoGen, San Diego, CA) or phosphate-buffered saline (PBS) was intranasally administrated 2 hours after each OVA challenge and 1 day before Mp. In addition, some allergic mice were intranasally treated 2 hours before each OVA challenge and 1 day before Mp with the following: (1) 100 µg bovine serum albumin (BSA; Sigma), (2) 50 µg anti-mouse IL-4 (11B11; eBioscience, San Diego, CA) and 50 µg anti-mouse IL-13 (R&D Systems, Minneapolis, MN), and (3) 50 µg rat IgG1 (eBioscience) and 50 µg rat IgG2b (R&D Systems). OVA-naive BALB/c mice were treated with 100 µg BSA as the nonallergic controls. One day after the last treatment, mice were inoculated intranasally with Mp at 1 x 107 cfu and killed after 7 days to examine lung Mp load and TLR2 and IL-6 expression.

OVA-naive IL-6–/– and C57BL/6 mice were inoculated intranasally with 50 µl Mp at 1 x 107 cfu and killed after 1, 3, and 7 days to examine lung Mp load.

Bronchoalveolar Lavage and Lung Tissue Processing
The lung was lavaged with 1 ml saline. Cell-free bronchoalveolar lavage (BAL) fluid was stored at –80°C for cytokine analysis. BAL cell cytospins were stained with the Diff-Quick Stain Kit (IMEB, Inc., San Marcos, CA) for cell differential counts. Left lung was homogenized in PBS to perform Mp culture. Right lung lobes were used for RNA extraction or enzymatic digestion to obtain single-cell suspension (33).

Isolation and Culture of Pulmonary CD11c+ DCs
Lung DCs were isolated from single-cell suspensions of OVA-naive BALB/c mice using a CD11c+ (N418) cell isolation kit (Miltenyi Biotec, Auburn, CA). Flow cytometry confirmed the purity of DCs (>=95%). The isolated lung DCs were treated with recombinant mouse IL-4 and IL-13 proteins (R&D Systems), followed by Mp, Pam3CSK4, or PBS treatment as indicated in the legend for Figure 5. Cells and supernatants were harvested for TLR2 flow cytometry and IL-6 ELISA (R&D Systems), respectively.

Generation and Culture of BMDCs
BMDCs were generated from OVA-naive STAT6–/– and wild-type BALB/c mice by culturing bone marrow cells as previously described, with modifications (34). On Day 8, flow cytometry confirmed that 85% or more of nonadherent cells were CD11c positive. BMDCs were treated similarly to the pulmonary CD11c+ DCs. Cells and supernatants were harvested for TLR2 flow cytometry and IL-6 ELISA, respectively. To measure NF-{kappa}B p65 activity, nuclear proteins were extracted for ELISA-based NF-{kappa}B p65 activation assay (Active Motif, Carlsbad, CA) (22).

Flow Cytometry
Lung single-cell suspensions, or cultured primary lung DCs and BMDCs, were blocked with anti-mouse CD16/CD32 (Fc{gamma}III/II receptor) (2.4G2; BD Biosciences, San Jose, CA); stained with allophycocyanin (APC)-conjugated anti-mouse CD11c (HL3; BD Biosciences), fluorescein isothiocyanate–conjugated anti-mouse I-A/I-E (2G9; BD Biosciences), phycoerythrin (PE)-conjugated anti-mouse TLR2 (6C2; eBioscience), or PE-conjugated rat IgG2b (BD Biosciences); and analyzed on a BD FACSCalibur flow cytometer (BD Biosciences). Details on the gating and analysis strategies are reported in the online supplement. TLR2 levels in mouse lung single-cell suspensions are expressed as geometric mean for the mean fluorescence intensity (MFI) of TLR2 protein and the percentage of DCs (CD11c+I-A/I-E+) expressing TLR2 after subtracting the background of the corresponding isotype control staining. For cultured lung DCs and BMDCs, TLR2 levels on cells with various treatments are expressed as the percentage change of TLR2 MFI over the corresponding PBS controls.

Real-time Reverse Transcriptase–PCR
Real-time reverse transcriptase–PCR was performed as previously described (23). The mouse IL-6 (GenBank accession number BC132458; National Institutes of Health, Bethesda, MD) primers and probe were as follows: forward primer, 5'-ACACAT GTTCTCTGGGAAATCGT-3'; reverse primer, 5'-AAGTGCATCATCGTTGTTCATACA-3'; probe, 5'-TGAGAAAAGAGTTGTGCAATGGCAATTCTG-3'.

Statistical Analysis
Normally distributed data are presented as means ± SD and compared using the Student t test between the two groups. Nonparametric (nonnormally distributed) data are expressed as medians with interquartile (25–75%) ranges and compared using the Wilcoxon rank sum test between the two groups. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Established OVA-induced Allergic Airway Inflammation Impairs Lung Mp Clearance and TLR2 Expression
In our preliminary time course (Days 3, 7, 28, and 56 post–Mp infection) studies in OVA-induced mouse allergic airway inflammation models, we found that the impact of allergic inflammation on lung Mp clearance was the most significant on Day 7 postinfection. Thus, we chose the 7-day post-Mp model to study the mechanisms responsible for impaired Mp clearance associated with the established allergic airway inflammation. As shown in Figure 1A, on Day 7 post–Mp infection (Day 9 after the last OVA challenge) allergic mice had significantly higher levels of lung tissue Mp burden (sixfold) than nonallergic mice.


Figure 1
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  		Figure 1. Increased lung tissue Mycoplasma pneumoniae (Mp) load and reduced Toll-like receptor 2 (TLR2) expression in allergic mice. Mp or saline intranasal inoculation in nonallergic and ovalbumin (OVA)-allergic mouse models on Day 7 post-Mp or saline (n = 7–8/group) was performed as described in METHODS. (A) Lung tissue Mp load. (B) Left: TLR2 expression (the mean fluorescence intensity [MFI]) on whole (mixed cell types) lung cells; right: representative histograms of flow cytometric analysis of TLR2 protein expression on whole lung cells from indicated groups (blue = isotype control; red = TLR2 antibody). (C) Left: the percentages of CD11c+ dendritic cells (DCs) expressing TLR2 in whole lung cells; right: representative scatter graphs of flow cytometric analysis of TLR2 expression on gated CD11c+I-A/I-E+ DCs from indicated groups. The boxes in A, and in the left panels of B and C show interquartile range (25–75%); the thick horizontal line indicates the median; the vertical axis extends from the minimum to the maximum value.

 
Because our previous study in nonallergic mice suggests a pivotal role of TLR2 signaling in Mp clearance (22), we examined if the established allergic airway inflammation modulated TLR2 expression on whole lung cells and lung DCs. As shown in Figure 1B, in nonallergic mice, Mp infection significantly up-regulated TLR2 expression on whole lung cells compared with saline controls. In contrast, in allergic mice, Mp infection failed to increase whole lung cell TLR2 protein expression. In the presence of Mp, TLR2 protein levels on whole lung cells of allergic mice were about threefold lower than those in nonallergic mice. Furthermore, similar to what was seen on whole lung cells, in nonallergic mice Mp infection also significantly increased lung DCs expressing TLR2 compared with the saline controls (2.3-fold; Figure 1C). However, unlike nonallergic mice, allergic mice did not have an increase in TLR2-positive lung DCs after Mp infection. Instead, after the infection, allergic mice manifested significantly less TLR2-positive lung DCs than nonallergic mice (2.1-fold; Figure 1C).

TLR2 Ligand Administration before the Establishment of Allergic Airway Inflammation Promotes Mp Clearance from Allergic Lungs
The observation that reduced TLR2 expression was coupled with impaired Mp clearance in allergic mice led us to hypothesize that the established allergic airway inflammation may dampen Mp clearance through down-regulating TLR2 function. To test this hypothesis, we inoculated a TLR2 agonist (Pam3CSK4) into mice before the establishment of allergic airway inflammation to reinstate TLR2 function. As shown in Figure 2A, Pam3CSK4 markedly decreased lung tissue Mp burden, which was significantly lower than that in Mp-infected allergic mice without Pam3CSK4 treatment. As expected, in allergic mice with an ensuing Mp infection, Pam3CSK4 notably up-regulated TLR2 protein expression on whole lung cells (Figure 2B) as well as lung DCs (Figure 2C) compared with PBS pretreated controls. In addition, Pam3CSK4 significantly inhibited BAL eosinophils compared with PBS pretreatment in allergic mice with an ensuing Mp infection (5 ± 4 x 104 vs. 14 ± 7 x 104/ml, P = 0.03).


Figure 2
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  		Figure 2. Toll-like receptor 2 (TLR2) ligand stimulation before the establishment of allergic airway inflammation promotes Mp clearance in allergic lungs. Ovalbumin (OVA)-allergic mouse models with or without Pam3CSK4 pretreatment on Day 7 post-Mp (n = 7–8/group) were performed as described in METHODS. (A) Lung tissue Mycoplasma pneumoniae (Mp) load; (B) TLR2 expression (the mean fluorescence intensity [MFI]) on whole lung cells; and (C) the percentages of CD11c+ lung dendritic cells (DCs) expressing TLR2. The boxes show interquartile range (25–75%); the thick horizontal lines indicate the median; the vertical axes extend from the minimum to the maximum value. PBS = phosphate-buffered saline.

 
Blocking IL-4 and IL-13 in Allergic Lungs before Mp Infection Restores TLR2 Expression and Mp Clearance
To examine whether IL-4 and IL-13 contribute to down-regulated TLR2 expression in allergic lungs, we blocked IL-4 and IL-13 function by using neutralizing antibodies in allergic lungs with an ensuing Mp infection. As shown in Figures 3A and 3B, with Mp infection, TLR2 protein expression on whole lung cells and lung DCs in allergic mice was significantly lower than that in nonallergic mice. However, as compared with rat IgG (isotype control), administration of anti–IL-4 and anti–IL-13 antibodies before the establishment of allergic airway inflammation significantly increased whole lung cell and lung DC TLR2 protein expression levels similar to those of Mp-infected nonallergic mice. Moreover, lung tissue Mp levels were significantly decreased in allergic mice pretreated with anti–IL-4 and anti–IL-13 antibodies compared with corresponding controls (5 [2–6] x 102 vs. 29 [12–60] x 102 cfu/left lung, P = 0.01). In addition, blocking IL-4 and IL-13 in allergic mice before an ensuing Mp infection reduced BAL eosinophils as compared with rat IgG control (4 ± 2 x 104 vs. 15 ± 7 x 104/ml, P = 0.02).


Figure 3
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  		Figure 3. Blocking IL-4 and IL-13 in allergic lungs before Mycoplasma pneumoniae (Mp) infection restores Toll-like receptor 2 (TLR2) expression. Ovalbumin (OVA)-allergic mouse models with or without pretreatment of IL-4 and IL-13 blocking antibodies before Mp infection on Day 7 post-Mp (n = 4–7/group) were performed as described in METHODS. (A) TLR2 expression (the mean fluorescence intensity [MFI]) on whole lung cells and (B) the percentages of CD11c+ lung dendritic cells (DCs) expressing TLR2. The boxes show interquartile range (25–75%); the thick horizontal lines indicate the median; the vertical axes extend from the minimum to the maximum value. BSA = bovine serum albumin.

 
IL-6 Is Critical to Lung Mp Clearance, and Blocking IL-4 and IL-13 in Allergic Lungs before Mp Infection Increases Lung IL-6 Expression
IL-6 is a critical cytokine in immune responses to typical bacterial infections (14–18). However, the role of IL-6 in atypical bacterial infection and clearance, such as Mp, has not been examined. Our current study demonstrated that, in OVA-naive wild-type (IL-6+/+) mice, IL-6 protein in BAL fluid was significantly induced upon Mp infection on Day 1 as compared with that in saline control animals (206 ± 90 pg/ml vs. undetectable, P < 0.05), but disappeared on Days 3 and 7 postinfection. However, after Mp infection, the impaired bacterial clearance in IL-6–/– mice compared with wild-type mice not only occurred on Day 1 (41 [39–54] x 104 vs. 26 [21–41] x 104 cfu/left lung, P = 0.04) but also persisted for 3 days (13 [9–15] x 104 vs. 0.8 [0.03–6] x 104 cfu/left lung, P = 0.02), and in particular for 7 days (Figure 4A).


Figure 4
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  		Figure 4. IL-6 is essential to lung Mycoplasma pneumoniae (Mp) clearance. (A) Lung tissue Mp load on Day 7 from Mp infection models in IL-6–/– and wild-type (IL-6+/+) mice (n = 5/group) as described in METHODS (B) lung tissue IL-6 mRNA relative levels on Day 7 post-Mp were measured by using real-time reverse transcriptase–polymerase chain reaction in ovalbumin (OVA)-allergic mouse models with or without pretreatment of IL-4 and IL-13 blocking antibodies before Mp infection (n = 4–7/group) as described in METHODS The boxes show interquartile range (25–75%); the thick horizontal lines indicate the median; the vertical axes extend from the minimum to the maximum value. BSA = bovine serum albumin.

 
Upon Mp infection, lung tissue IL-6 mRNA levels in allergic mice were significantly lower than those in nonallergic mice (Figure 4B). Administration of anti–IL-4 and anti–IL-13 antibodies before the establishment of allergic airway inflammation markedly increased lung tissue IL-6 mRNA levels as compared with rat IgG control. As expected, IL-6 protein was undetectable in any allergic or nonallergic mice after 7 days of infection.

IL-4 and IL-13 Decrease Mp-induced TLR2 Expression and IL-6 Production in Primary Lung DCs
To determine if IL-4 and IL-13 directly inhibit TLR2 expression and IL-6 production, primary lung DCs from OVA-naive BALB/c mice were isolated and treated in vitro with IL-4 and IL-13 or BSA (nonspecific protein control) before Mp, Pam3CSK4, or PBS controls. As shown in Figure 5A, IL-4/IL-13 alone significantly reduced TLR2 protein expression in lung DCs compared with BSA controls. Similar to in vivo data, Mp or Pam3CSK4 alone markedly up-regulated TLR2 protein expression compared with PBS controls. However, IL-4/IL-13 treatment before Mp or Pam3CSK4 completely blocked the up-regulation of TLR2, and instead resulted in a decrease of TLR2 expression as compared with BSA controls.


Figure 5
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  		Figure 5. IL-4 and IL-13 decrease Mycoplasma pneumoniae (Mp)–induced Toll-like receptor 2 (TLR2) expression and IL-6 production in primary lung dendritic cells (DCs). Isolated primary lung DCs were resuspended in RPMI 1640 medium with 10% fetal bovine serum (FBS), seeded at 2 x 105 cells/well into a 96-well culture plate, treated with IL-4/IL-13 (10 ng/ml each) or BSA (20 ng/ml) 2 hours before Mp infection (1 cfu/cell), Pam3CSK4 (10 ng/ml) or phosphate-buffered saline (PBS), and further incubated for 24 hours. Cells were examined for TLR2 expression (the mean fluorescence intensity [MFI]) by flow cytometry (A), and culture supernatants were collected for IL-6 ELISA (B). Data are from three independent experiments and expressed as means ± SD. ND = non-detectable.

 
As shown in Figure 5B, IL-6 protein was undetectable in cell supernatants treated with PBS or IL-4/IL-13 alone. In contrast, Mp or Pam3CSK4 alone induced robust IL-6 production. In line with TLR2 protein expression, a significant reduction of IL-6 protein was also found in DCs treated with IL-4/IL-13, followed by Mp or Pam3CSK4. In addition, similar to the in vivo study, Pam3CSK4 pretreatment before IL-4/IL-13 significantly reinstated TLR2 protein expression and IL-6 production upon Mp administration compared with PBS pretreated controls (data not shown).

IL-4 and IL-13 Activate STAT6 to Inhibit Mp-induced TLR2 Expression and IL-6 Production Mediated by NF-{kappa}B
In our preliminary experiments, IL-4/IL-13 stimulation in wild-type BMDCs was shown to induce phosphorylation (activation) of transcription factor STAT6 as determined by immunoblotting (data not shown). In the current study, BMDCs from OVA-naive STAT6–/– and wild-type (STAT6+/+) mice were used to investigate the molecular mechanisms by which IL-4/IL-13 down-regulated TLR2 expression and IL-6 production. Here, we demonstrated that the in vitro response patterns of TLR2 expression and IL-6 production in BMDCs were essentially the same as those in primary lung DCs after Mp or Pam3CSK4 in the absence or presence of IL-4/IL-13.

As shown in Figure 6A, as compared with corresponding controls, IL-4/IL-13 alone significantly reduced, whereas Mp or PamCSK4 alone markedly increased, TLR2 protein expression on wild-type BMDCs. IL-4/IL-13 pretreatment before Mp or Pam3CSK4 significantly reduced the induction of TLR2 protein expression on wild-type BMDCs. However, the inhibitory effect of IL-4/IL-13 on baseline, and Mp- or Pam3CSK4-induced TLR2 expression disappeared in STAT6–/– BMDCs.


Figure 6
Figure 6
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  		Figure 6. IL-4 and IL-13 dampen Toll-like receptor 2 (TLR2) expression and IL-6 production by inhibition of nuclear factor (NF)-{kappa}B activation through STAT6 signaling. Bone marrow–derived dendritic cells (BMDCs) were generated from STAT6–/– and wild-type (STAT6+/+) mice, resuspended in RPMI 1640 medium with 10% fetal bovine serum (FBS), seeded at 106 cells/well into 24-well culture plates, treated with IL-4/IL-13 (10 ng/ml each) or bovine serum albumin (BSA) (20 ng/ml) 2 hours before Mycoplasma pneumoniae (Mp) infection (1 cfu/cell), Pam3CSK4 (10 ng/ml), or phosphate-buffered saline (PBS), and further incubated for 24 hours. Cells were used to determine TLR2 expression (the mean fluorescence intensity [MFI]) by flow cytometry (A), and culture supernatants were harvested for IL-6 ELISA (B). For NF-{kappa}B p65 assay, BMDCs were seeded at 4 x 106 cells/well into 6-well culture plates, treated with IL-4/IL-13 (10 ng/ml each) or BSA (20 ng/ml) 2 hours before Mp infection (1 cfu/cell), Pam3CSK4 (10 ng/ml), or PBS, and then further incubated for 4 hours. Nuclear proteins were extracted for NF-{kappa}B p65 assay (C). Data are from three independent experiments and expressed as means ± SD.

 
Likewise, as shown in Figure 6B, IL-6 protein levels were undetectable in STAT6–/– or wild-type BMDCs treated with PBS or IL-4/IL-13 alone. Mp or PamCSK4 itself significantly increased IL-6 production in both STAT6–/– and wild-type BMDCs. Moreover, in wild-type BMDCs, IL-4/IL-13 pretreatment significantly reduced IL-6 induction after Mp or PamCSK4 compared with BSA controls. However, in STAT6–/– BMDCs, IL-4/IL-13 completely lost their inhibitory effects on Mp- or Pam3CSK4-induced IL-6 production.

To further decipher the molecular mechanisms involved in IL-4/IL-13–mediated inhibition of TLR2 function, we examined NF-{kappa}B activation upon stimulation with Mp or Pam3CSK4 in the absence or presence of IL-4/IL-13 since previous studies suggest a crucial role of NF-{kappa}B activation in TLR2 expression (29–31). We found that Mp infection in wild-type BMDCs significantly induced NF-{kappa}B p65 levels in nuclear proteins compared with PBS controls. Moreover, compared with DMSO (dimethyl sulfoxide) pretreatment, a 2-hour pretreatment with CAPE (an NF-{kappa}B activation inhibitor dissolved in DMSO, 10 µM) before Mp markedly reduced Mp-induced TLR2 expression and IL-6 production by 55 and 97%, respectively (data not shown). As shown in Figure 6C, in wild-type BMDCs IL-4/IL-13 itself only resulted in a minor decrease of nuclear NF-{kappa}B p65 levels as compared with BSA controls. Furthermore, IL-4/IL-13 pretreatment significantly reduced Mp- or Pam3CSK4-induced NF-{kappa}B activation. In contrast, IL-4/IL-13 did not exhibit any inhibition of Mp- or Pam3CSK4-induced NF-{kappa}B activation in STAT6–/– BMDCs.


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Interactions of the innate and adaptive immune systems in allergic diseases, including asthma, are critical in reshaping the disease process. In allergy and infection research, previous work has centered on the impact of respiratory infections on asthma exacerbations or allergic responses. In contrast, limited information is available with regard to the regulatory role of the established allergic airway inflammation in host innate immunity to respiratory infections. Therefore, our study has addressed a unique question to fill a major gap in the current research topics regarding asthma and respiratory infections. That is, does the established allergic airway inflammation compromise host innate immunity to hinder the elimination of an infectious organism?

Our current study has revealed that the established OVA-induced allergic airway inflammation impairs TLR2 expression and host defense cytokine (e.g., IL-6) production, and subsequently delays lung Mp clearance. First, we have confirmed previous reports that the host defense against typical bacteria (e.g., S. aureus and Pseudomonas aeruginosa) can be inhibited by allergic inflammation in humans and in mice (8, 9, 35). Second, we have verified our previous findings that allergic airway inflammation delays Mp clearance from lungs in both C57BL/6 and BALB/c mice (23, 32). However, unlike previous studies, we have provided the in-depth molecular mechanisms (i.e., TLR2 expression and IL-6 production) to explain why allergic airways are susceptible to Mp infection, which will likely lead to novel therapeutic approaches aimed at improving the impaired bacterial clearance in allergic lungs. Specifically, we have demonstrated that pretreating allergic mice with a TLR2 agonist (Pam3CSK4) or with IL-4– and IL-13–neutralizing antibodies before Mp infection normalized lung Mp clearance via restoring TLR2 expression and subsequent IL-6 production.

One of our novel findings is related to the role of TLR2 and IL-6 in Mp clearance in the established allergic airway inflammation. We, for the first time, found that IL-6 is crucial to atypical bacterial clearance in that IL-6–/– mice had an increased lung Mp burden compared with wild-type mice. Further, Mp-induced IL-6 production is largely dependent on TLR2, as our previous study has demonstrated that a TLR2 blocking antibody significantly decreased Mp-induced IL-6 protein levels in BAL fluid of OVA-naive BALB/c mice (22). Moreover, in our preliminary experiments, TLR2 ligands (e.g., Pam3CSK4 and Mp-derived lipoproteins) did not induce IL-6 production in TLR2–/– BMDCs (at 24 and 48 h post-treatment). The fact that down-regulated TLR2 expression, less IL-6 production and impaired Mp clearance in allergic mice can be restored by anti–IL-4 and anti–IL-13 antibodies further supports the role of the TLR2/IL-6 pathway in host defense against Mp infection in allergic lungs. Although IL-6 protein levels were undetectable on Day 7 postinfection in allergic mice, the cytokine profile in DCs after in vitro infection seems to reflect the pattern of early in vivo cytokine induction in response to Mp infection. In this study, Mp- or Pam3CSK4-induced TLR2 expression and IL-6 production were significantly diminished by IL-4/IL-13 preincubation in primary lung DCs and BMDCs. Collectively, insufficient IL-6 production resulting from down-regulated TLR2 expression in allergic mice may be one of the pivotal mechanisms responsible for impaired Mp clearance.

NF-{kappa}B activation regulates many aspects of inflammatory response, including TLR expression and cytokine production (e.g., IL-6, tumor necrosis factor [TNF]-{alpha}). Upon a variety of stimuli, NF-{kappa}B proteins are released from associated IkappaB proteins, and translocate into the nucleus where they bind to {kappa}B consensus sites to up-regulate the transcription of various target genes (36). Transcription factor STAT6 is activated in response to IL-4 and IL-13 through tyrosine phosphorylation, followed by translocation to the nucleus, and binding to specific DNA sequence motifs to trigger the transcription of several IL-4/IL-13–responsive genes such as CD23 and E-selectin (25–28). It has been reported that one STAT consensus sequence and two NF-{kappa}B binding sites are closely positioned in the 5' regulatory region of mouse TLR2 gene promoter, enabling STAT (e.g., STAT6) and NF-{kappa}B to interact directly with each other. The deletion analysis of each NF-{kappa}B site clearly showed that NF-{kappa}B enhances TLR2 promoter activity and subsequently induces TLR2 gene expression in mouse macrophages in response to Mycobacterium avium or cytokines (e.g., IL-15) (29–31). In line with these findings, An and colleagues have shown that inhibition of NF-{kappa}B activation suppressed the up-regulated mRNA expression of TLR2, TLR4, and TLR9 in cultured immature BMDCs in response to corresponding TLR ligands (37). Taken together, TLR2 ligand stimulation activates NF-{kappa}B, which reciprocally induces TLR2 expression.

The molecular mechanisms by which IL-4 and IL-13 suppress gene expression remain poorly understood. A previous study suggests that IL-4 suppresses TNF-{alpha}–induced E-selectin protein via STAT6 signaling pathway in human umbilical vein endothelial cells (28). Briefly, NF-{kappa}B and STAT6 are activated with TNF-{alpha} and IL-4 treatments through distinct signal transduction pathways, leading to the concurrent nuclear translocation of both NF-{kappa}B and STAT6. Furthermore, IL-4–activated STAT6 was shown to compete with NF-{kappa}B for binding to a dual NF-{kappa}B enhancer element on the E-selectin promoter region, thereby acting as a transcriptional repressor of NF-{kappa}B–induced E-selectin expression. In our current study, Mp infection in wild-type BMDCs activated NF-{kappa}B p65, which preceded TLR2 up-regulation and IL-6 production. In addition, IL-4/IL-13 pretreatment in wild-type BMDCs significantly reduced Mp- or Pam3CSK4-induced NF-{kappa}B activation with decreased TLR2 expression and IL-6 production. However, the inhibitory effects of IL-4 and IL-13 on NF-{kappa}B–mediated TLR2 expression and IL-6 production disappeared in STAT6–/– BMDCs, suggesting that IL-4 and IL-13 activate STAT6 to down-regulate Mp-induced TLR2 expression. Our findings are supported by a previous study in that STAT6 was shown to be required for IL-4 to inhibit TLR4 promoter activity in the U-937 monocytic cell line (38). Thus, our findings suggest that NF-{kappa}B is activated after Mp infection. In return, activated NF-{kappa}B could bind to the TLR2 gene promoter for maximal induction of TLR2 expression in response to Mp infection. In contrast, IL-4/IL-13–induced STAT6 activation may antagonize the effects of NF-{kappa}B activation on TLR2 gene expression in the established OVA-induced allergic airway inflammation with an ensuing Mp infection. We propose that, upon IL-4 and IL-13 stimulation, activated STAT6 might compete with NF-{kappa}B for binding to TLR2 gene promoter, thus suppressing TLR2 transcriptional activity. Future experiments are warranted to confirm if IL-4/IL-13–induced STAT6 activation indeed competes with NF-{kappa}B for site occupancy to suppress TLR2 expression and IL-6 production in DCs by performing chromatin immunoprecipitation and transient transfection of TLR2 gene promoter with or without site-directed mutagenesis. Last, we had an interesting observation that STAT6–/– BMDCs showed lower levels of nuclear NF-{kappa}B p65 in response to Mp or Pam3CSK4 compared with wild-type BMDCs. Although the involved mechanisms for STAT6 to regulate immune responses in an infectious setting need to be better defined, we found that STAT6–/– BMDCs had less TLR2 protein expression than wild-type BMDCs.

We realize that ligation of the IL-4/IL-13 receptor complex also results in activation of several other signaling pathways such as mitogen-activated protein kinases (MAPK), including p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK), in bronchial smooth muscle and epithelial cells (39–43). However, in our preliminary experiments, we found that the ERK signaling pathway is unlikely to be involved in IL-4/IL-13–induced down-regulation of TLR2 expression because pretreatment with an ERK inhibitor (PD98059) failed to reverse the inhibitory effects of IL-4/IL-13 on TLR2 protein expression and IL-6 production in wild-type BMDCs. Other MAPK family members will be investigated in our future experiments.

Although we focused on DCs to understand the mechanisms behind impaired TLR2 expression and bacterial clearance, we realize that other types of lung cells, such as airway epithelial cells, may also serve as critical components of airway mucosal defense mechanisms as they are constitutively equipped with various TLRs and provide an active defense mechanism against invading microbes (44). In our unpublished results, we performed air–liquid interface cultures of mouse primary tracheal epithelial cells from wild-type BALB/c mice to examine the effects of IL-13 on Mp clearance and TLR2 mRNA expression. Similar to what we found in allergic mice, IL-13 pretreatment resulted in a significant increase of Mp levels in apical supernatants from epithelial cells on Day 7 post–Mp infection compared with the PBS pretreated cells (17.4 x 104 vs. 4.5 x 104 cfu/ml, P = 0.049). Furthermore, Mp-induced TLR2 mRNA expression in tracheal epithelial cells was markedly inhibited by up to 89% by IL-13 pretreatment compared with the control (no IL-13). These results suggest that an allergic setting (e.g., IL-13) also down-regulates TLR2 expression in airway epithelial cells, which may consequently dampen Mp clearance in allergic lungs. Taken together, we are able to conclude that reduced lung TLR2 expression in innate cells (i.e., lung DCs, airway epithelial cells) under established allergic airway inflammation or the Th2 prominent cytokines IL-4 and IL-13 may increase airway microbial burden in asthmatic hosts. Thus, maintaining sufficient TLR2 levels in patients with asthma has therapeutic implications in eliminating bacterial infections containing TLR2 ligands (e.g., Mp).

In addition to TLR2, other TLRs or innate mechanisms (e.g., antimicrobial substances) may also contribute to host defense against Mp infection. For example, because the Mp genome has unmethylated CpG motifs (45), it is likely that TLR9 plays a role in Mp-induced proinflammatory cytokine production since Mp-induced inflammatory responses were not completely abolished in TLR2-deficient mice (22). In addition, our recent report has identified that an allergic setting markedly reduces the expression of short palate, lung, and nasal epithelium clone 1 (SPLUNC1; a novel host defense protein derived from large airway epithelial cells), which may in part contribute to the persistent Mp infection in mouse allergic airways (32).

Our present study has focused on the inhibitory role of Th2 cytokines (IL-4 and IL-13) in lung mycoplasma clearance in the established OVA-allergic mice. Because an imbalance in Th1 and Th2 cytokines (e.g., reduced ratio of IFN-{gamma}/IL-4) plays a role in asthma pathogenesis (46, 47), Th1 cytokines may affect lung mycoplasma clearance. Indeed, T-bet (a member of the T-box family of transcription factors) and IFN-{gamma}, two critical components of the Th1 pathway, have been shown to promote mouse lung clearance of Mycoplasma pulmonis (48, 49). Interestingly, in contrary to T-bet and IFN-{gamma}, IL-12 (a Th1-driving cytokine) may prevent Mp clearance from the mouse lung (50, 51). However, the impact of the Th1 pathway on TLR (e.g., TLR2) expression in mice has not been well investigated. Future studies to define the relative importance of Th1 and Th2 cytokines in regulating TLR2 expression and production of host defense mediators in the context of mycoplasma infection are warranted. In addition, we realize that the effects of Th2 cytokines on host defense mechanisms can vary depending on the species of the invading pathogens. For example, Th2 cytokine IL-4 seems to have a minimal role in modulating lung inflammation and bacterial clearance in nonallergic mice infected with M. pulmonis, a rodent, not a human, pathogen (49, 52).

In summary, we have found that the established OVA-induced allergic airway inflammation, or the prominent Th2 cytokines IL-4 and IL-13, reduces Mp-induced TLR2 expression and subsequent production of IL-6, and eventually impairs Mp clearance from the allergic lungs. This cascade was partly mediated by inhibition of NF-{kappa}B activation through STAT6 signaling pathway (Figure 7). This is the first study indicating that attenuation of TLR2-mediated innate immune responses by Th2 cytokines renders the allergic lungs more susceptible to respiratory bacterial (i.e., Mp) infections. Our findings could offer a great potential for developing novel therapeutic strategies (e.g., reinstating TLR2 function using TLR2 ligands and/or blocking IL-4 and IL-13) to ameliorate persisting respiratory bacterial (i.e., Mp) infections in allergic lungs.


Figure 7
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  		Figure 7. Proposed mechanisms involved in impaired Toll-like receptor 2 (TLR2) expression and decreased Mycoplasma pneumoniae (Mp) clearance in the established allergic airways. Sufficient TLR2 expression (i.e., in dendritic cells and epithelial cells) is critical to elicit lung defense including production of proinflammatory cytokines (i.e., IL-6) to maintain normal bacterial clearance against Mp infection (illustrated by red arrow). However, the established allergic airway inflammation, or the prominent Th2 cytokines IL-4 and IL-13, may inhibit nuclear factor (NF)-{kappa}B activation through STAT6 signaling to impair TLR2 expression and subsequent production of IL-6, and consequently dampen the host defense against Mp infection in allergic lungs (illustrated by blue arrow).

 

   Acknowledgments
 
The authors thank Richard A. Willis, Philippa C. Marrack, Rachel Breed, Taylor Moss, Xiaona Ge, Jyoti Thaikoottathil, David Bosch, Maisha Minor, and Sandy Ho for their technical assistance.


   FOOTNOTES
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200709-1387OC on January 17, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 18, 2007; accepted in final form January 11, 2008


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 RESULTS
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