Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

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.

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 [20]. 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 [20]. The protease-enhanced GC frass was frozen in aliquots and used for the remainder of the studies.

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 [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.

Allergen-induced airway hyperresponsiveness (AHR) was determined as we have previously described [22]. 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.

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 [21].

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⁺, Gr1^neg, CD317^neg) and plasmacytoid DCs (CD11c⁺, CD11b^neg, Gr1^low, 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).

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.

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 [23].

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.

To determine the role of PAR-2 in mediating allergen-driven AHR, wild type and PAR-2-deficient mice were exposed to GC frass by intratracheal instillation on day 0 and 14, and three days later airway responses were measured. Exposure of wild type mice to GC frass resulted in increased AHR which was significantly attenuated in PAR-2-deficient mice (Figure 1A). Next we cultured cells from whole lungs and re-stimulated the cells with GC frass to assess the ability of GC frass to induce cytokine production. GC frass induced production of the Th2 cytokines IL-5 (Figure 1B), IL-13 (Figure 1C) and IL-4 (data not shown) and the Th17 cytokine IL-17A (Figure 1D). Importantly, PAR-2-deficient mice had significantly reduced production of the Th2 cytokines and IL-17A. GC frass exposure also induced a significant increase in eosinophilia and neutrophilia into the BAL fluid of wild type mice, and this was also significantly reduced in the PAR-2-deficient mice (Table 1). Together these data suggest an important role for PAR-2 in regulating allergic airway inflammation.

GC frass is a complex mixture containing both active serine proteases, and TLR4 agonists, such as LPS. Both active protease from Aspergillus fumigatus [8] and LPS (0.1 μg) [24] have been shown to enhance Th2 sensitization to a normally tolerogenic antigen (OVA). As such, we wished to determine the relative importance of GC frass-derived serine proteases and LPS in driving Th2 response observed following GC-frass sensitization and challenge. We have previously reported the enrichment of the active serine protease from GC frass which significantly increased the amount of protease and almost completely removed endotoxin (~1 pg/μg protein) [20]. Thus, to test whether GC frass-derived protease acted as an adjuvant for specific Th2 sensitization in the induction of allergic airway responses, we sensitized and challenged mice with OVA, OVA in the presence of GC-frass derived protease, or OVA in the presence of LPS (0.1 μg) on Days 0, 14 and 21. Importantly, while OVA itself failed to induce significant AHR, Th2, or Th17 cytokine production, OVA containing protease was sufficient to induce airway responsiveness to cholinergic agents (Figure 2A), increase serum IgE levels (Figure 2B), and induce the whole lung production of theTh2 cytokines IL-5 (Figure 2C), IL-13 (Figure 2D) and IL-4 (data not shown) alone or in the presence of ConA. IL-17A (Figure 2E) and IFNγ (Figure 2F) levels were unaltered by the protease. Thus, the principal effect of the protease is to enhance the development of Th2 immunity. In contrast, while the presence of LPS moderately enhanced AHR (Figure 2A), serum IgE levels (Figure 2B) and Th2 cytokine production (Figure 2C, D), it markedly enhanced production of IL-17A (Figure 2E) and IFNγ (Figure 2F), suggesting that LPS is capable of enhancing Th2, Th7 and Th1 responses. Together, these data support the observation that the active serine proteases in GC frass are sufficient to induce robust Th2 responses following mucosal allergen exposure in mice.

As uptake of allergen by pulmonary DCs is required for optimal stimulation of T cell responses leading to asthma, and both PAR-2 and active serine proteases are effective at enhancing Th2 responses in vivo, we wished to determine if PAR-2 could be detected on the surface of pulmonary mDCs. To this end, wild type mice were exposed to a single intratracheal inhalation of PBS or GC frass. Twenty hours later, whole lungs were harvested, dissociated and stained for flow cytometric analysis of PAR-2 expression on pulmonary DCs. PAR-2 expression was apparent on neutrophils (data not shown); however PAR-2 expression on pulmonary mDCs and pDCs could not be reliably detected in naïve mice. Importantly, inhalation of GC frass induced the expression of PAR-2 on the pulmonary mDCs (Figure 3A, B) but not on pDCs (data not shown). This suggests the upregulation of PAR-2 on mDCs but not pDCs following 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.

To better delineate the importance of PAR-2 expression on the activity of mDCs, we cultured BMDCs from WT or PAR-2-deficient mice with GC frass for 18 hours and cytokine expression was measured by ELISA. BMDCs were > 90% mDCs as characterized by flow cytometry (CD11b⁺ CD11c⁺ Gr1^neg CD317^neg; data not shown). As observed on pulmonary mDCs from in vivo GC frass treated mice, we also observed that PAR-2 expression was induced by GC frass exposure in BMDCs (Figure 4A). GC frass increased cytokine expression from wild type and PAR-2-deficient mDC; however the levels of IL-6 (Figure 4B), IL-23 (Figure 4C), and TNFα (Figure 4D) in PAR-2-deficient mDCs were significantly lower than in wild type mDCs. The levels of IL-12 and IL-10 were not above the level of detection of the assay (data not shown). To confirm that PAR-2-deficient mDCs are capable of producing high levels of cytokines, we treated cells with the TLR2 agonist, Pam3Cys. There was no difference between the wild type and PAR-2-deficient mDC in their response to a TLR2 agonist. These data suggest that activation of PAR-2 plays a role in enhancing the ability of mDCs to produce T cell skewing cytokines following exposure to protease-containing allergens.

To further explore alterations in PAR-2-deficient mDC, we investigated the regulation of the co-stimulatory molecules CD80 and CD86 on the lung mDC population following GC frass inhalation. BALB/c and PAR-2-deficient mice were exposed to PBS or GC frass and 20 h later, whole lungs were harvested, dissociated and stained for flow cytometric analysis of CD80 and CD86 expression. GC frass increased expression of both CD80 and CD86 on mDC following a single exposure (Figure 5). There was no difference in the levels of CD80 or CD86 staining on mDCs from PBS-treated PAR-2-deficient mice compared to wild type mice, suggesting that PAR-2-deficient mice do not have inherent alterations in DC activation. Interestingly, following GC frass exposure, the number of CD86 and CD80 molecules (as indicated by MFI) on the cell surface of mDCs in PAR-2-deficient mice was less compared to wild type mice (Figure 5A and 5C). There was a significant difference in percentage of mDC expressing CD80 in the PAR-2-deficient mice compared to wild type (Figure 5B), however there was no difference in the percentage of mDC expressing CD86 between the two strains of mice (Figure 5D). This data demonstrates an overall decrease in the levels of CD80 and CD86 on mDCs in PAR-2-deficient mice and suggests that pulmonary mDCs from PAR-2-deficient mice have reduced activation and capacity for T cell stimulation following allergen sensitization.

To further explore the role of PAR-2 on mDCs in promoting the development of allergic airway inflammation, we performed an adoptive transfer. To this end, BALB/c BMDCs were treated with PBS or GC frass in vitro for 18 hours and then 1 × 10⁶ cells were adoptively transferred into the lungs of naïve BALB/c mice. Fourteen days later, mice were challenged with intratracheal inhalation of PBS or GC frass and airway responses were measured 72 h post challenge. Adoptive transfer of GC frass-pulsed wild type BMDCs induced AHR to acetylcholine challenge (Figure 6A) and increased IL-5 (Figure 6B), IL-13 (Figure 6C), IL-17A (Figure 6D), and IFNγ (Figure 6E) compared to PBS-pulsed wild type BMDCs. Thus, sensitization with GC-frass pulsed DCs induced a mixed Th1/Th2/Th17 profile similar to that observed in mice sensitized with HDM-pulsed DCs [18], and reminiscent of the profile observed in humans with more severe disease [25‐28]. Importantly, adoptive transfer of GC frass-pulsed PAR-2-deficient BMDCs resulted in significantly reduced levels of AHR (Figure 6A). In addition, the restimulation of the lungs with GC frass in vitro resulted in up to 50% reduction in levels of Th2 cytokines (Figure 6B, C). While Th17 cytokine production was also reduced in mice sensitized with PAR-2-deficient BMDCs compared to wild type GC frass-pulsed BMDCs, the effect was comparatively mild (~10% reduction). There was no effect of adoptive transfer of allergen-pulsed PAR-2-deficient BMDC on IFNγ compared to allergen-pulsed wild type BMDC, suggesting that PAR-2 on mDCs is important in driving Th2 responses, but has limited impact on Th1 or Th17 responses. Moreover, while adoptive transfer of GC frass-pulsed mDCs from both wild type and PAR-2-deficient BMDC increased eosinophil, neutrophil, and lymphocyte infiltration in to the airways compared to PBS-pulsed DCs (Table 2), GC frass-pulsed PAR-2-deficient BMDC induced significantly less infiltration of eosinophils compared to GC-frass-pulsed wild-type BMDC. Mucin production was increased in the lungs of mice administered GC frass-pulsed wild type BMDC compared to mice that received GC frass-pulsed PAR-2-deficient BMDC (Figure 7). Administration of GC frass to either completely naïve animals or mice sensitized with PBS-pulsed BMDCs failed to initiate AHR or Th2 cytokine skewing (data not shown), suggesting the importance of the DC in the initiation of the allergic airway response, and suggesting repetition of allergen exposure is important. These data suggest that protease activation of PAR-2 on BMDC can modulate the immune response and lead to the development of allergic airway inflammation.