German cockroach frass proteases modulate the innate immune response via activation of protease-activated receptor-2

Abstract

Allergen exposure can induce an early innate immune response; however, the mechanism by which this occurs has not been addressed. In this report, we demonstrate a role for the active serine proteases in German cockroach (GC) feces (frass) and protease-activated receptor (PAR)-2 in modulating the innate immune response. A single exposure of GC frass induced inflammatory cytokine production and cellular infiltration in the airways of mice. In comparison, exposure to protease-depleted GC frass resulted in diminution of inflammatory cytokine production and airway neutrophilia, but had no effect on macrophage infiltration. Selective activation of PAR-2 confirmed that PAR-2 was sufficient to induce airway inflammation. Exposure of GC frass to PAR-2-deficient mice led to decreased immune responses to GC frass compared to wild-type mice. Using the macrophage as an early marker of the innate immune response, we found that GC frass induced significant release of tumor necrosis factor-alpha from primary alveolar macrophages. This effect was dependent on the intrinsic proteases in GC frass. We confirmed GC frass-induced cytokine expression was mediated by activation of NF-kappaB and ERK in a macrophage cell line. Collectively, these data suggest a central role for GC frass protease-PAR-2 activation in regulating the innate immune response through the activation of alveolar macrophages. Understanding the potential role of protease-PAR-2 activation as a danger signal or adjuvant could yield attractive therapeutic targets.

Fig. 1

GC frass-associated proteases partially regulated TNF α and KC expression in the airways of mice. Balb/c mice were given a single intratracheal inhalation of PBS, aprotinin-treated PBS, GC frass (40 μg/40 μl), or protease-depleted GC frass (40 μg/40 μl); 18 h later, BAL fluid was harvested, clarified, and cytokines were analyzed by ELISA. Means ± SEM (n = 5-7 mice per group). a KC (∗ p < 0.004 vs. control; ∗∗ p = 0.005 vs. GC frass). b TNF α (∗ p < 0.001 vs. control; ∗∗ p < 0.001 vs. GC frass).

Fig. 2

Selective activation of PAR-2 increased KC and TNFα expression in the airways of mice. Naïve wild-type mice were given a single intratracheal inhalation of PBS, PAR-2-AP, or PAR-2-CP; 18 h later, BAL fluid was harvested, clarified, and cytokines were analyzed by ELISA. Means ± SEM (n = 4-6 mice per group). a KC levels (∗ p < 0.001 vs. PBS control). b TNFα levels (∗ p < 0.001 vs. PBS control).

Fig. 3

PAR-2 regulated KC and TNFα expression in vivo. PAR-2-deficient mice were given a single intratracheal inhalation of PBS or GC frass (40 μg/40 μl); 18 h later, BAL fluid was harvested, clarified, and cytokines were analyzed by ELISA. Means ± SEM (n = 4-6 mice per group). a KC (∗ p < 0.001 vs. PBS control; ∗∗ p = 0.001 vs. GC frass). b TNFα (∗ p < 0.001 vs. PBS control; ∗∗ p < 0.001 vs. GC frass).

Fig. 4

GC frass proteases and PAR-2 mediate TNFα release from alveolar macrophages. a BAL fluid from naïve Balb/c mice was cultured and treated with PBS, aprotinin-treated PBS, GC frass (1μ g/ml), or protease-depleted GC frass (1μ g/ml) for 18 h. Cell supernatants were harvested, clarified and analyzed for TNFα by ELISA. * p < 0.001 and * * p = 0.002 vs. control; * * * p = 0.004 vs. GC frass, n = 5 separate experiments. b BAL fluid from Balb/c and PAR-2-deficient mice were cultured and treated with PBS or GC frass (1μ g/ml). * p < 0.001 and * * p = 0.008 vs. PBS; * * * p = 0.004 vs. GC frass, n = 3 separate experiments.

Fig. 5

GC frass induction of TNFα is dependent on NF-κ B activation in macrophages. a MHS cells were pretreated with isohelenin (30 μM), PD98059 (30 μ M), SB202190 (10 μ M), or DMSO for 1 h prior to treatment with GC frass (1 μ g/ml). Means ± SEM for 4 separate experiments (* p < 0.001 vs. PBS; * * p = 0.002 and * * * p = 0.004 vs. GC frass). b MHS cells were treated with GC frass (1 μ g/ml) for different times as indicated (0–90 min). Cell lysates were harvested and analyzed for Iκ B α expression by Western blot. Membrane was stripped and reprobed for β-actin expression. c Lysates from b were analyzed by Western blot for phospho (p)-ERK, and membrane was subsequently stripped and reprobed for ERK. The data shown are representative of 3 separate experiments. d MHS cells were treated with PBS (P), aprotinintreated PBS (PA), GC frass (G), or aprotinin-treated GC frass (GA) for 4 h and nuclear extracts were isolated and analyzed by EMSA. The oligonucleotide probe for the left gel is NF-κ B and AP-1 for the right gel. The data shown are representative of 2 separate experiments