Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation
© Lewkowich et al; licensee BioMed Central Ltd. 2011
Received: 16 June 2011
Accepted: 21 September 2011
Published: 1 December 2011
A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear.
Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry.
Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass-stimulated wild type BMDCs were sufficient to induce AHR and allergic airway inflammation, while GC frass-stimulated PAR-2-deficient BMDC had attenuated responses.
Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses.
It is generally accepted that allergic asthma results from an inappropriate Th2-dominated immune response to an otherwise innocuous protein. Allergens are derived from a number of sources, including plants (grasses and trees), arthropods (mite and cockroach), animals (cats, dogs), and fungi. While allergens are from diverse sources, a common characteristic of allergens is that they contain intrinsic protease activity, or are presented in the airways along with particles that contain protease activity. For example, German cockroach (GC) contains serine proteases , while HDM contains both the cysteine protease Der p1 [2, 3] and serine proteases Der p3 and Der p9 . In addition, fungal extracts of Alternaria alternate and Cladosporium herbarum , as well as the cat allergen Felis domesticus (Fel d 1)  contain proteolytic activity. We recently showed that the active serine proteases in GC feces (frass) played a role in regulating airway hyperresponsiveness (AHR) to acetylcholine and mucin production in a mouse model of allergic airway inflammation . In addition, removal of proteases from either A. fumigatus , American cockroach Per a 10 antigen , Epi p1 antigen from the fungus Epicoccum purpurascens or Cur 11 antigen from the mold Curvularia Iunata  decreased airway inflammation and airway hyperresponsiveness in mouse models. To date, the mechanism(s) by which proteases mediate their effects is unclear.
Allergen-associated proteases are thought to regulate biological effects through the activation of protease-activated receptors (PARs). PARs (-1, -2, -3, -4) are a family of proteolytically activated G-coupled receptors which, when activated, initiate a signal transduction pathway leading to transcriptional regulation. Of particular interest is PAR-2, which has been implicated in allergic diseases. To date, only a few studies have investigated the importance of PAR-2 in modulating allergic airway disease. In studies in which systemically-induced (OVA bound to alum administered by intraperitoneal injection) airway responses were compared in wild type and PAR-2-deficient mice, PAR-2-deficient mice had decreased cellular infiltration compared to controls . They also showed that sensitization and challenge of PAR-2 overexpressing mice with OVA resulted in increased AHR compared to wild type mice . Since OVA does not contain protease activity, this study addressed the role of endogenous proteases (i.e. mast cell tryptase) released following an initial inflammatory event. We recently confirmed a role for PAR-2 in mediating allergen-derived allergic airway inflammation ; however the mechanism by which PAR-2 regulated these events is currently unclear.
Dendritic cells (DC) are the most potent antigen presenting cells and are thought to bridge innate and adaptive immunity. Mucosal DCs form a dense network associated with the airway epithelium and can form long extensions into the airway lumen . To date, little is known regarding the role of proteases or PAR-2 in activating DCs. One report showed that serine protease activation of PAR-2 stimulated the development of DCs from bone marrow progenitor cells . Thus it is possible that protease activation of PAR-2 may be important for DC maturation, thus promoting DC's to switch from a sentinel, antigen-capturing mode to a mature antigen-presenting mode. Recent evidence suggests that specific subsets of DCs are critical not only for the initiation of allergic airway responses, but also to drive immunity (myeloid, mDC) or tolerance (plasmacytoid, pDC) [16–18]. To date, it is unclear how these subsets are regulated in the airways. Interestingly, uptake of Alexa Fluor 488-labeled OVA by DCs was enhanced when PAR-2 was activated using a selective PAR-2 agonist . Clearly, protease-PAR-2 may play an important, yet undefined, role in the regulation of DC maturation, function, and activation.
In this report we investigate the role of PAR-2 in mediating the development of allergen-induced allergic airway inflammation through the activation of DCs. We confirmed the importance of functional PAR-2 for allergic airway inflammation and show that the isolated protease from GC frass was sufficient to induce AHR, increased serum IgE and a Th2 skewing phenotype when in the presence of OVA. We found that GC frass upregulated PAR-2 expression on pulmonary mDCs but failed to detect PAR-2 on pDCs. While we failed to find differences in uptake of allergen in the PAR-2-deficient mice, there was a considerable difference in T cell skewing cytokine production in the PAR-2-deficient BMDC. Finally, we confirmed that GC frass activation of wild type BMDC was sufficient to induce AHR and airway inflammation; however this response was partially dependent on a functional PAR-2 on the BMDC. These data suggest the importance of protease-PAR-2 activation of the DC in the regulation of allergic airway responses.
Materials and methods
German cockroach frass
The fecal remnants (frass) from one cage of German cockroaches were transferred to a sterile container and stored at 4°C. Frass were resuspended in endotoxin-free double-distilled water (2 h at 4°C while rocking). Extracts were centrifuged to remove debris (10,000 g for 10 min at 4°C), supernatants harvested, and total protein was measured using the Bio-Rad Protein Assay Dye (Bio-Rad, Hercules, CA). GC frass was frozen in aliquots for use throughout the entire study. AlexaFluor-405 (Invitrogen, Carlsbad, CA) labeled GC frass (AF405-GC frass) was made according to manufacturers' specifications.
Protease-enhancement of GC frass
GC frass was run through a size exclusion column (Sephadex G75 superfine, Amersham Pharmacia, Piscataway, NJ) and the fractions in the protease-containing peak were combined and run through a prepacked HiTrap Benzamidine FF affinity column (GE Healthcare, Piscataway, NJ). Serine proteases bind this column and are eluted using a buffer containing 20 mM para-aminobenzamidine (Spectrum Chemical Corp, Gardenia, CA). The fractions containing protease activity were combined, dialyzed against ddH2O and measured for protein concentration and protease activity as previously described . Information regarding the amount of protein, enzymatic activity and endotoxin in the starting material GC frass and in the final column-purified protease sample is shown elsewhere . The protease-enhanced GC frass was frozen in aliquots and used for the remainder of the studies.
Animals and GC frass exposure
BALB/c and PAR-2-deficient mice were obtained from Jackson Laboratory (Bar Harbor, ME). PAR-2-C57Bl/6 mice were backcrossed for 10 generations onto the BALB/c background. For sensitization and challenge experiments, mice (6-8 weeks old) were anesthetized with ketamine (45 mg/kg)/xylazine (8 mg/kg) prior to inhalation of PBS (40 μl), LPS-free ovalbumin (OVA; 100 μg/mouse; Worthington Biochem Corp, Lakewood NJ), OVA (100 μg) plus enriched protease (0.5 units), or OVA (100 μg) plus LPS (0.1 μg/mouse; Sigma Chemical Corp, St. Louis MO #055:B5) on day 0, 14, and 21 . Mice were harvested on Day 24. For the adoptive transfer of bone marrow-derived DCs (BMDC), 40 μl of BMDC (1 × 106 cells) suspension was administered via instillation into the airways of anesthetized mice. 14 d later, mice were exposed to a single intratracheal inhalation of either PBS (40 μl) or GC frass (40 μg/40 μl). In all cases, 72 h following the final inhalation, airway responses were measured. For flow cytometry studies, a single exposure to PBS or GC frass was followed by a lethal dose of sodium pentobarbital 20 h later. These studies were approved by the Cincinnati Children's Hospital Medical Center Institutional Animal Care and Use Committee.
Airway hyperresponsiveness measurements
Allergen-induced airway hyperresponsiveness (AHR) was determined as we have previously described . Briefly, mice were anesthetized 72 h after the last GC frass exposure, intubated and ventilated at a rate of 120 breaths per minute with a constant tidal volume of air (0.2 ml), and paralyzed with decamethonium bromide (25 mg/kg). After establishment of a stable airway pressure, 25 μg/kg weight of acetylcholine was injected i.v. and dynamic airway pressure (airway pressure time index [APTI] in cm-H2O × sec-1) was followed for 5 min.
Assessment of airway inflammation
Lungs were lavaged with 1 ml of Hanks balanced salt solution without calcium or magnesium. The lavage fluid was centrifuged (1,800 rpm for 10 min), the supernatant was removed for cytokine analysis and immediately stored at -80°C. Total cell numbers were counted on a hemocytometer. Smears of BAL cells prepared with a Cytospin II (Shandon Thermo, Waltham, MA) were stained with Diff-Quick (Thermo Electron Corporation, Pittsburg, PA) solution for differential cell counting.
Animals were bled and serum isolated for total IgE levels using antibodies from BD Biosciences (San Diego, CA).
Liberase/DNase I digests of the lung were prepared to obtain single lung cell suspensions. Single cell suspensions (2.5 × 105) were incubated for 72 hours in culture medium (RPMI) or in RPMI treated with GC frass (1 μg/ml) or ConA (10 μg/ml) and supernatants were analyzed by ELISA for Th2 cytokine expression as previously described .
All ELISAs were from R&D Systems (Minneapolis, MN) and run according to manufacturer's specifications.
Whole lungs were removed and formalin fixed. Lungs were embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H&E) and Periodic Acid Schiff (PAS).
Whole lungs were isolated from mice 20 h following exposure, minced and placed in RPMI 1640 containing Liberase CI (0.5 mg/ml; Roche Diagnostics, Indianapolis, IN) and DNase I (0.5 mg/ml; Sigma, St. Louis MO) at 37°C for 45 minutes. The tissue was forced through a 70-micron cell strainer, and red blood cells were lysed with ACK lysis buffer (Invitrogen, Carlsbad, CA). Cells were washed with RPMI containing 10% FBS, counted and plated at 500,000 cells per well in a 96 well plate. Staining reactions were performed at 4°C following incubation with Fc block (mAb 2.4G2) for 30 min. Myeloid DCs (CD11c+, CD11b+, Gr1neg, CD317neg) and plasmacytoid DCs (CD11c+, CD11bneg, Gr1low, CD317+) were quantified using anti-CD11c-APC (HL3), anti-CD11b-PE-Cy7 (M1/70), and anti GR-1-APC-Cy7 (RB6-8C5). PAR-2 expression was examined using a PE-conjugated mAb to PAR-2 (Santa Cruz, Santa Cruz, CA). Co-stimulatory molecule expression was examined using PE-conjugated mAbs to CD86 (GL1) or CD80 (16-10A1). Dead cells were excluded using 7-AAD. All antibodies (with the exception of PAR-2) were purchased from eBioscience (San Diego, CA). Data were acquired with an LSRII flow cytometer (BD Biosciences, San Jose, CA). Spectral overlap was compensated using the FACSDiVa software (BD Biosciences) and analyzed using FlowJo software (Treestar Inc, Ashland, OR).
Isolation and development of mature, GC frass-pulsed bone marrow-derived myeloid DCs
Mice were given a lethal dose of sodium pentobarbital prior to removal of tibias and femurs. Bone marrow cells (1.5 × 107 cells per ml) were cultured on complete RPMI supplemented with GM-CSF (10 ng/ml, Peprotech, Rocky Hills, NJ). Fresh media was added along with GM-CSF (10 ng/ml) on day 3. On day 6, cells were treated with endotoxin-free PBS or GC frass (1 μg/ml) for 24 h. Cells were washed, counted and resuspended at 2.5 × 106 cells/ml.
Quantitative real time PCR
RNA was extracted using a standard TRIzol method of phenol extraction. Total RNA is converted to cDNA by reverse transcription using the Superscript First Strand Synthesis System kit (Invitrogen, Carlsbad, CA). The PAR-2 primers are 5'-CTTAGCCTTCTTGCCAGGTG-3' and 5'-TCTCTGCACCAATCACAAGC-3' and the β-actin primers are 5'-TGTTACCAACTGGGACGACA-3'and 5'-GGGGTGTTGAAGGTCTCAAA-3'. Amplification was performed by PCR using SYBR Green on the iCycler (BioRad Laboratories) as follows: 1 cycle 95°C for 3 min, followed by 40 cycles of [95°C for 5 sec, 57°C for β-actin and 60°C for PAR-2 for 5 sec, 72°C for 10 sec], 95°C for 1 min, 55°C for 1 min and then a hold of 25°C. The target gene is normalized to the reference gene using the Pfaffl method .
When applicable, statistical significance was assessed by Students t-test or one-way analysis of variance (ANOVA). Differences identified by ANOVA were pinpointed by Student-Newman-Keuls' multiple range test using SigmaStat software.
PAR-2 promotes the development of GC frass-induced asthma
Differential cell count in BAL fluid of wild type and PAR-2-deficient mice.
1.4 ± 0.2
1.7 ± 0.4
0.04 ± 0.02
0.09 ± 0.05
1.6 ± 0.3
3.5 ± 1.6
0.02 ± 0.02
0.07 ± 0.03
8.6 ± 3.6
5.2 ± 1.6
1.7 ± 0.5
4.4 ± 0.7 *
1.6 ± 0.04
3.4 ± 1.1
4.0 ± 1.5
0.6 ± 0.3 †
1.2 ± 0.02 †
1.5 ± 0.8
GC frass protease strongly promotes the development of Th2 immunity
PAR-2 is expressed on pulmonary mDCs and is regulated by allergen exposure
One possibility for the decreased allergic airway inflammation in PAR-2 could be differential uptake of allergen in DCs between wild type and PAR-2-deficient mice. To address this, wild type or PAR-2-deficient mice were exposed to a single intratracheal inhalation of PBS or Alexa405-labeled (AF405) GC frass. Twenty-four hours later, whole lungs were harvested, dissociated and stained for flow cytometry. We found no difference in the percentage (Figure 3C) of AF405-GC frass positive mDCs or in the MFI (Figure 3D) between wild type and PAR-2-deficient mice exposed to the allergen. Taken together these data demonstrate that while PAR-2 expression is enhanced on pulmonary mDCs after GC frass exposure, it does not mediate its Th2-enhancing properties by enhancing antigen uptake by pulmonary mDC.
Differential cytokine production from PAR-2-deficient BMDCs
Regulation of co-stimulatory molecule expression on mDC
Adoptive transfer of PAR-2-deficient BMDC into wild type mice fails to induce allergic airway inflammation
Differential cell count in BAL fluid from wild type and PAR2-deficient mice adoptively transferred with mDCs pulsed with PBS or GC frass.
BALB/c DC PBS
2.2 ± 0.8
13.0 ± 3.5
0.1 ± 0.06
0.3 ± 0.2
PAR2-/- DC PBS
3.5 ± 1.3
14.3 ± 2.8
0.1 ± 0.03
0.2 ± 0.05
BALB/c DC frass
5.2 ± 2.1
9.8 ± 2.1
1.6 ± 0.4
9.5 ± 2.1 *
3.4 ± 0.5
PAR2-/- DC frass
3.8 ± 1.4
12.8 ± 3.5
0.7 ± 0.2 †
7.5 ± 1.6 *
3.0 ± 0.4
Herein we describe a mechanism whereby serine proteases promote the development of allergen-induced AHR through activation of PAR-2. Confirming our previous work , and that of others  we again demonstrate that mucosal sensitization to GC frass is reduced in mice lacking PAR-2, resulting in reduced AHR and airway inflammation. Previous studies have focused on the role for PAR-2 in direct activation of bronchial epithelial cells [20, 30–32] triggering the development of innate immunity by causing the release of chemotactic factors specific for the growth and recruitment of pulmonary mDCs (i.e. CCL20, GM-CSF). We demonstrate here that in vivo PAR-2-deficient mice display reduced production of both Th2 and Th17-associated cytokines, suggesting that PAR-2 differentially influences multiple T cell responses. As mucosal exposure to GC frass induces upregulation of PAR-2 on pulmonary mDCs, but not pDCs, we sensitized naïve mice with GC-frass pulsed wild type BMDCs, or BMDCs from PAR-2-deficient mice. As previously observed , the adoptive transfer of allergen-pulsed mDCs yields robust production of IFNγ and IL-17A and airway neutrophilia along with the typical Th2 cytokines and eosinophilia, a response which more closely resembles the mixed Th1/Th2/Th17 response observed in severe asthmatics. However, in this context, the lack of PAR-2 on sensitizing DCs markedly impacts Th2 cytokine production, providing evidence that PAR-2 on mDCs is involved in promoting Th2 immune responses. In contrast, the Th17 response is only slightly diminished in mice sensitized with PAR-2-deficient DCs suggesting that PAR-2 expression on other cell types controls Th17 cytokine production. Thus, the present study suggests that specifically targeting PAR-2 activation of pulmonary mDCs may allow one to limit the development of Th2 responses at mucosal sites.
The observation that the induction of Th2 responses by GC frass is reduced in both PAR-2 deficient mice and mice sensitized with PAR-2 deficient DCs strongly implicates PAR-2 expression on mDCs on the ability of GC frass to induce a Th2-polarized immune response at mucosal surfaces. However, the mechanism whereby PAR-2 promotes the development of Th2 responses is unclear. While we observed that PAR-2 deficient BMDCs produce significantly reduced levels of all cytokines examined (IL-6, IL-23, TNFα) we have previously reported that GC frass-induced production of both IL-6 and IL-23 is completely abrogated in MyD88 -/- BMDC , suggesting that PAR-2 expression may amplify TLR-triggered cytokine production. However, as LPS-depleted serine proteases still demonstrated marked Th2-skewing capacity in vivo, it seems unlikely that the reduced Th2-skewing capacity of PAR-2 DCs is the direct result of reduced cytokine production. In contrast, PAR-2 activation has been shown to enhance maturation of BMDCs (as evidenced by increased MHC Class II and CD86 expression) , suggesting that reduced co-stimulatory molecule expression may be involved. In support of this possibility, we observe decreased expression of the co-stimulatory molecule CD80 and CD86 on pulmonary mDCs from PAR-2 -/- mice. Collectively, these data suggest that PAR-2 expression on pulmonary mDCs is required for optimal induction of Th2 immunity following exposure to GC frass.
While our data suggests that DCs lacking PAR-2 demonstrate reduced Th2 skewing capacity we cannot completely rule out a role for the epithelium in the ability of the GC frass to induce a Th2 response. Epithelial cells treated with allergens with active proteases (Aspergillus extract, Derp1) induce IL-25, a strong inducer of Th2 immunity  in an ERK/p38 dependent manner . As we have shown that GC protease mediated induction of IL-8 from epithelial cells is dependent upon ERK activation , it is likely that IL-25 may also be produced. Moreover, Alternaria mediated PAR-2 cleavage has also been shown to induce TSLP production from bronchial epithelial cells . TSLP in turn directly enhances the ability of DCs to induce a Th2 response [36, 37], suggesting an additional mechanism whereby PAR-2 may amplify Th2 immunity. However, additional studies making use of mice lacking PAR-2 specifically in DC populations or pulmonary epithelial cells will be required to conclusively determine the relative contributions of PAR-2 on DCs and epithelial cells.
It is interesting to note that while there is a substantial (~75%) decrease in the number of neutrophils in the BAL in PAR-2 -/- mice, a dramatic impact on neutrophil recruitment was not observed in mice sensitized with GC frass-pulsed PAR-2 -/- BMDCs (~20% decrease). This is especially striking given that in both PAR-2 -/- and mice sensitized with GC frass-pulsed PAR-2 -/- BMDCs levels of IL-17A are only partially affected. As IL-17A is a strong promoter of neutrophilia, this is somewhat surprising. However, a recent report by Fei et al suggests that elevated IL-17A is not sufficient to drive neutrophilia in a model of allergic bronchopulmonary aspergilliosis . Rather, a combination of both TNFα and IL-17A are required to get maximal neutrophil recruitment, whereas the production of IL-17A alone was associated with a more pronounced eosinophilia . In this report we also demonstrate that PAR-2 expression on mDCs is required for maximal GC frass-induced production of TNFα, suggesting that differential TNFα production may explain differences in neutrophilia observed PAR-2 -/- versus mice sensitized with GC frass-pulsed PAR-2 -/- BMDCs. Indeed, in PAR-2 -/- mice, GC frass sensitization results in limited IL-5, but robust IL-17A production. Moreover, at challenge, PAR-2 -/- mice also lack DC-derived, GC frass-stimulated TNFα production resulting in a milieu with low IL-5 and TNFα, but high IL-17A levels that is not conducive to strong recruitment of eosinophils or neutrophils. In contrast, while sensitization of mice with GC frass-pulsed BMDCs also results in a low IL-5 and high IL-17A levels, GC frass challenge of these mice induces high levels of TNFα as endogenous DCs can respond to serine protease in the GC frass. This resulted in a low IL-5, high TNFα/IL-17A milieu which permits neutrophil recruitment, but resulted in limited recruitment of eosinophils.
The data presented here suggest that the initial response to inhaled GC frass is complex, and that synergy between different components is likely required for maximal allergenic capacity. However, these studies do highlight the importance of protease activity and activation of PAR-2 receptors on pulmonary mDCs in inducing the development of Th2 responses at mucosal surfaces. Taken together with a number of other studies showing the ability of proteases to induce the development of Th2 responses [30, 32, 39], these studies suggest that activity of proteases at mucosal surfaces may be a key factor regulating the development of Th2 immune responses. It is important to note that while there is good evidence to support that cockroach proteases and PAR-2 are involved in the Th2 responses in the airways, this study does not demonstrate the development of antigen-specific T cell responses by cockroach proteases and PAR-2. A greater understanding of the processes that lead to the development of Th2 responses may prove invaluable in therapeutic regulation of Th2 responses that are both undesirable (i.e. allergy, asthma), or desirable (parasitic clearance).
The present study highlights the importance of functional PAR-2 on mDCs as critical for the development of Th2-biased immune response and indicates the mechanism by which allergen-derived proteases initiate allergic airway responses.
List of abbreviations
bone marrow-derived DC
mean fluorescent intensity
Protease activated receptor.
This work was supported by the National Institutes of Health Grant HL075568 (KP), American Medical Association Seed Research Grant (SD), a Parker B. Francis Fellowship (IPL) and partially supported by NIH grant AR47363 for the flow cytometry core at CCHMC.
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