Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein Inhibits Mite Allergen Der p 2 Induced Inflammasome Activation

<div><p>Newly discovered cell penetration peptides derived from human eosinophil cationic proteins (CPPecp) have the characteristic of cell internalization, but the effect of CPPecp on immunomodulation has not been clarified. House dust mite (HDM) major allergen, Der p 2, can induce proinflammatory cytokine production which contributes to airway inflammation and allergic asthma. However, the mechanism of Der p 2 on NLRP3 inflammasome activation remains unclear. The aim of this study was to investigate the immunomodulatory effect of CPPecp on inhibition of Der p 2 induced inflammasome activation. We showed that proinflammatory cytokines IL-1β, IL-6 and IL-8 were significantly upregulated in peripheral blood mononuclear cells (PBMCs) derived from HDM allergic patients after Der p 2 stimulation. Expression of NLRP3, ASC, Caspase-1, IL-1β and Caspase-1 activity was upregulated in THP-1 cells after Der p 2 stimulation. Proinflammatory cytokine production, NLRP3 inflammasome activation and caspase-1 activity were downregulated in THP-1 cells and CD14+ cells co-cultured with Der p 2 and CPPecp. The immunomodulatory effect of CPPecp was through upregulation of IFN-α production but not induction of autophagy. These results suggested Der p 2 plays an important role in NLRP3 inflammasome activation and CPPecp has the potential to be a novel anti-inflammatory agent for allergic inflammation treatment in the future.</p></div

Inflammasome activation can be induced by Der p 2 stimulation.

<p>(A) THP-1 cells were treated with Der p 2 (1.5ug/ml) at different time points. Cells treated with LPS (500ng/ml) for six hours were used as positive control. After stimulation, cell lysates were collected and separated with SDS-PAGE. Expressions of NLRP3, ASC and caspase-1 were detected by Western blot. Caspase-1 enzymatic activity was assayed in cell lysates of THP-1 cells stimulated with Der p 2 (1.5ug/ml) at different time points. Caspase-1activities were expressed as percent of control with *p-value <0.05 compared to control (B). IL-1β production from (C) THP-1 cells and (D) CD14⁺ cells derived from HDM allergic subjects (n = 5) were measured by ELISA. Bars and error bars indicate mean and standard error of the mean (SEM), respectively. * p<0.05, compared to control. Results shown are representative of three independent experiments.</p

CPPecp inhibits inflammasome activation and downregulates proinflammatory cytokine production.

<p>(A) THP-1 cells were co-cultured with Der p 2 (1.5ug/ml) and CPPecp (10 to 100 uM) for 6 hours; LPS (500ng/ml) was used as control. Protein lysates were collected and expressions of NLRP3, ASC, caspase-1 was detected by Western blot. Caspase-1 enzymatic activity was assayed in cell lysates of THP-1 cells stimulated with Der p 2 (1.5ug/ml) co-cultured with CPPecp (10 to 100 uM) for 6 hours; LPS (500ng/ml) was used as control. Caspase-1activities were expressed as percent of control with *p-value <0.05 compared to treated with Der p 2 treatment group (B). THP-1 cells (C) and CD14⁺(D) cells co-cultured with Der p 2 (1.5ug/ml) and CPPecp (10 to 100 uM) for 6 hours, mRNA expression of IL-1β, IL-6, IL-8 and GAPDH were detected by RT-PCR. THP-1 cells (E) were co-cultured with CPPecp (10 to 100 uM) and PBMCs (F) derived from HDM allergic patients (n = 10) were co-cultured with Der p 2 1.5ug/ml and CPPecp 100uM for six hours. Culture supernatant was collected and IL-1β concentration was measured by ELISA. Bars and error bars indicate mean and standard error of the mean (SEM), respectively. * p<0.05 compared to treated with Der p 2 treatment group. THP-1 cells (G) were treated with 10 and 100uM CPPecp for different incubation periods. After treatment, cell viability was measured by trypan blue exclusion. Tamaxifen (10uM) was used as control. Results shown are representative of three independent experiments.</p