Activation of human mast cells by retrocyclin and protegrin highlight their immunomodulatory and antimicrobial properties

Abstract

Preclinical evaluation of Retrocyclins (RC-100, RC-101) and Protegrin-1 (PG-1) antimicrobial peptides (AMPs) is important because of their therapeutic potential against bacterial, fungal and viral infections. Human mast cells (HMCs) play important roles in host defense and wound healing but the abilities of retrocyclins and protegrin-1 to harness these functions have not been investigated. Here, we report that chemically synthesized RC-100 and PG-1 caused calcium mobilization and degranulation in HMCs but these responses were not blocked by an inhibitor of formyl peptide receptor-like 1 (FPRL1), a known receptor for AMPs. However, RC-100 and PG-1 induced degranulation in rat basophilic leukemia (RBL-2H3) cells stably expressing Mas related G protein coupled receptor X2 (MrgX2). Chemical synthesis of these AMPs is prohibitively expensive and post-synthesis modifications (cyclization, disulfide bonds, folding) are inadequate for optimal antimicrobial activity. Indeed, we found that synthetic RC-100, which caused mast cell degranulation via MrgX2, did not display any antimicrobial activity. Green-fluorescent protein (GFP)-tagged RC-101 (analog of RC-100) and GFP-tagged PG-1 purified from transgenic plant chloroplasts killed bacteria and induced mast cell degranulation. Furthermore, GFP-PG1 bound specifically to RBL-2H3 cells expressing MrgX2. These findings suggest that retrocyclins and protegrins activate HMCs independently of FPRL1 but via MrgX2. Harnessing this novel feature of AMPs to activate mast cell's host defense/wound healing properties in addition to their antimicrobial activities expands their clinical potential. Low cost production of AMPs in plants should facilitate their advancement to the clinic overcoming major hurdles in current production systems.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; MrgX2; antimicrobial peptides; chloroplast; mast cells; protegrin; retrocyclin.

Figure 1. PG-1 and RC-100 induce degranulation and Ca²⁺ mobilization in LAD2 human mast cells; however have differential antimicrobial activities

LAD2 mast cells were stimulated with different concentrations of A. PG-1 or D. RC-100 and percent degranulation (β-hexosaminidase release) was determined. Data are mean ± SEM of three experiments. LAD2 cells were loaded with Indo-1AM and Ca²⁺ mobilization in response B. PG-1 or E. RC-100 (both 3 μg/ml) was determined. To test the biological activity of the commercial peptides a starting inoculum of 4 × 10⁵ CFU/ml of E. coli was incubated in the presence of C. PG-1 and F. RC-100 at the indicated concentrations for a period of 8 hours, after which the cultures were plated on LB Agar and incubated overnight. The colony forming units were counted the next day to identify the survival rate of E. coli. Data shown are representative of 3 similar experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. ** indicates p < 0.001.

Figure 2. Effects of Pertussis toxin on C3a, PG-1 and RC-100-induced Ca²⁺ mobilization and degranulation in human mast cells

A., D. Indo-1 loaded LAD2 cells were exposed to C3a (1 nM), followed by PG-1 (3 μg/ml) or RC-100 (3 μg/ml) and intracellular Ca²⁺ mobilization was determined. B., E. Cells were treated with pertussis toxin (PTx; 100 ng/ml, 16 h) and effects of C3a, PG-1 or RC-100 on Ca²⁺ mobilization was determined. C., F. Cells were exposed to Pertussis toxin (PTx; 100 ng/ml, 16h) and C3a, PG-1 and RC-100-induced degranulation was determined. Data are mean ± SEM of three experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. and ** indicates p < 0.001.

Figure 3. PG-1 and RC-100 induced Ca²⁺ mobilization and degranulation are independent of FPRL1

Indo-1 loaded LAD2 cells were preincubated with buffer (Control) or WRW4 (10 μM) for 30 min and Ca²⁺ mobilization was determined in response to A., B. PG-1 (3 μg/ml) or C., D. RC-100 (3 μg/ml). E. LAD2 cells were pretreated with buffer (Control) or WRW4 (10 μM) and stimulated with 3 μg/ml concentration of PG-1 and RC-100 and percent degranulation was determined. Data are represented as mean ± SEM of three experiments.

Figure 4. PG-1 and RC-100 induce degranulation in RBL-2H3 cells expressing MrgX2

A., B. RBL-2H3 cells stably expressing MrgX2 were exposed to buffer (Control), PG-1 or RC-100 (3 μg/ml, 5 μg/ml) for 30 min and β-hexosaminidase release was measured. Data are represented as mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p < 0.01 and ** indicates p < 0.001.

Figure 5. Quantification and evaluation of GFP-RC101 and GFP-PG1 expressed in transgenic plants

A. Western blot analysis of GFP-RC101 and GFP-PG1 protein extracts from the transgenic tobacco leaves. Total Soluble Protein was loaded at indicated dilutions. B. Western blot quantification of GFP- RC101 purified from transgenic tobacco leaves. Purified protein was loaded at the indicated volumes of 100X and 200X dilutions. C. Fluorescence of GFP-RC101 protein is maintained after column purification as seen in non denaturing native PAGE (12%), observed under UV light.

Figure 6. Antimicrobial and immunomodulatory effects of AMPs produced in plants

A. Antimicrobial activity assay of AMPs purified from transplastomic plants against E. coli. The inhibitory effect of the AMPs on the growth of E. coli (starting inoculum 4 × 10⁵ CFU/ml) was measured by monitoring OD₆₀₀ for 8 h in the presence of vehicle control (GFP), GFP-RC101 (0.8 μg/ml) or GFP-PG1 (0.5 μg/ml). B. LAD2 cells were exposed to vehicle control (GFP), GFP-RC101 (1.8 μg/ml) or GFP-PG1 (2.4 μg/ml) and percent degranulation (β-hexosaminidase release) was determined 30 min after stimulation. Data are represented as mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p < 0.01 and ** indicates p < 0.001.

Figure 7. GFP-PG1 associate with the plasma membrane of MrgX2-expressing RBL-2H3 cells

A. Mock transfected RBL-2H3 cells were exposed to GFP-PG1 (4 μg/ml, 30 min) and B. RBL-2H3 cells transfected with MrgX2 were exposed to either GFP or GFP-PG1 (4 μg/ml, 30 min). Cells were washed with ice cold PBS, fixed in 4% paraformaldehyde, nuclei were counter-stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed using laser scanning confocal microscope (Nikon A1R) with a 60x water objective and 5.6X digital zoom. Images were analyzed using Nikons Elements 4.1 software and representative images from 25 stored images for each condition are shown.