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

Spleen mRNA expression.

<p>Spleen mRNA expression in Foxp3, IL-10, IFN-γ, GATA-3, and RORγt were decreased in hIN and lmwhIN groups. #p = 0.05.</p

The animal protocol of this study.

<p>MIT group, heparin IN (hIN) group and low-molecular-weight heparin IN (lmwhIN) group were sensitized twice with Der p allergen subcutaneously on day 1 and day 8. Der p allergen was administered intratracheally on day 15. The hIN and lmwhIN groups were treated with heparin or lmw heparin intranasally from days 1 to 22. Splenocytes from sacrificed mice stimulated with Der p 16 ug/ml were cultured for 72 hours.</p

Serum Der p-specific IgE levels.

<p>Serum Der p-specific IgE levels were significantly decreased in hIN and lmwhIN groups. Control group, mIT group, hIN group, and lmwhIN group were analyzed. ^★p<0.00000001, p<0.000001.</p

Cytokine levels in supernatants from splenocyte culture stimulated with mite.

<p>Cytokine levels in splenocyte supernatants are shown in this figure. Due to the large differences between cytokines level, we showed each cytokine level as a percentage of the corresponding level in the mIT group. The levels of mIT group are as follows: TGF-beta 2448.25±136.31 pg/mL, IL-10 5.93±0.98 pg/mL, IFN-γ 16.27±8.80 ng/mL, IL-13 2102.10±269.55 pg/mL, IL-4 159.40±20.51 pg/mL, IL-17A/F 708.6±374.52 pg/mL. ^⊚p = 0.012, *p = 0.005, **p = 0.013, ^#p = 0.034, ^@p = 0.0038, ^&p = 0.037,^$p = 0.033, p = 0.001, ^★p = 0.001, ^⊙p = 0.005, ^▴p = 0.034.</p

The risk of tuberculosis disease in rheumatoid arthritis patients on biologics and targeted therapy: A 15-year real world experience in Taiwan

<div><p>The objective of this study is to determine the risk of tuberculosis (TB) disease in biologics users among rheumatoid arthritis (RA) patients in Taiwan from 2000 to 2015. This retrospective cohort study enrolled adult RA patients initiated on first biologics at Taichung Veterans General Hospital. TB risks were determined as hazard ratio (HR) with 95% confidence interval (CI) using cox regression. A total of 951 patients were recruited; etanercept (n = 443), adalimumab (n = 332), abatacept (n = 74), golimumab (n = 60), tocilizumab (n = 31) and tofacitinib (n = 11). Twenty-four TB cases were identified; 13 in etanercept and 11 in adalimumab group with the TB incidence rate of 889.3/ 100,000 and 1055.6/ 100,000 patient-years respectively. There was no significant difference in TB risk between adalimumab and etanercept users with an incidence rate ratio of 1.27 (<i>p</i> = 0.556 by Poisson model). Significant 2-year TB risk factors included elderly patient >65 year-old (HR: 2.72, 95% CI: 1.06–6.99, <i>p</i> = 0.037), history of TB (HR: 6.24, 95% CI: 1.77–22.00, <i>p</i> = 0.004) and daily glucocorticoid use ≥5mg (HR:5.01, 95% CI: 1.46–17.21, <i>p</i> = 0.010). Sulfasalazine treatment appeared to be protective (HR: 0.32, 95% CI: 0.11–0.97, <i>p</i> = 0.043). Risk management plan (RMP) for TB before initiation of biologics commenced in 2012. The 2-year TB risks after RMP was compared with that before 2012 (HR:0.67, 95% CI: 0.30–1.49, <i>p</i> = 0.323). Elderly RA patients with a history of previous TB infection and concomitant moderate dose glucocorticoid were at higher risk of TB disease. Concurrent sulfasalazine treatment appeared to be a protective factor against TB disease.</p></div

Incidence of TB according to bDMARDs and stratified before and after 2012.

<p>Incidence of TB according to bDMARDs and stratified before and after 2012.</p

Demographic data and clinical characteristics among RA patients initiating bDMARDs and tDMARDs.

<p>Demographic data and clinical characteristics among RA patients initiating bDMARDs and tDMARDs.</p