LL-37 scrambled peptide

Epidermal keratinocytes produce proinflammatory cytokines/chemokines upon stimulation with cytokine milieus and Toll-like receptor ligands, which are considered to reflect epidermal environments in inflamed skin. The human antimicrobial peptide LL-37, besides having microbicidal functions, plays multiple roles as a "host defense peptide" in the immune system. Here, we examined the effect of LL-37 on proinflammatory responses induced by double-stranded RNA (dsRNA) and cytokines in primary human keratinocytes. LL-37 inhibited dsRNA-induced production of thymic stromal lymphopoietin (TSLP), CCL5/RANTES, CXCL10/IP-10, and CXCL8/IL-8, which was attributable to interaction between LL-37 and dsRNA, although LL-37 upregulated CXCL8 expression at an earlier time point (8 h). LL-37 inhibited the increase of CXCL10 and CCL5 induced by TNF-α- and/or IFN-γ but enhanced that of CXCL8. LL-37 and Th17 cytokines (IL-17 and IL-22) synergistically upregulated the expression of CXCL8 and IL-6. LL-37 showed the effects above at a high concentration (25 μg/ml, 5.6 μM). We also examined effects of a peptide with a scrambled LL-37 sequence, which has been frequently used as a negative control, and those of another peptide with the reversed LL-37 sequence, activities of which have not been well investigated. Interestingly, the reversed LL-37 had effects similar to LL-37 but the scrambled LL-37 did not. The modulation by LL-37 of the keratinocyte proinflammatory responses induced by cytokine milieus and dsRNA suggests novel roles for LL-37 in skin inflammation such as the promotion of IL17/IL-22/IL-6-associated psoriasis and suppression of TSLP-associated atopic dermatitis.

Human Cathelicidin antimicrobial peptide LL-37 is known to have antiviral activity against many viruses. In the present study, we investigated the in-vitro effect of LL-37 on dengue virus type 2 (DENV-2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL-37, the virus was pretreated with different concentrations of LL-37 (2.5μM-15μM) or scrambled (Scr) LL-37(5μM-15μM) and used for infection or the cells were first treated with LL-37 and infected. To study the effect of LL-37 post infection (PI), the cells were infected first followed by addition of LL-37 to the culture medium 24h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT-PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10-15μM LL-37 significantly reduced its infectivity as compared to virus control (P0.05). Pretreatment of cells before infection or addition of LL-37 to the culture 24h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL-37 to both the units of DENV envelope (E) protein dimer. Together, the in-vitro experiments and in-silico analyses suggest that LL-37 inhibits DENV-2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in-vivo studies.

The pulmonary mucus of cystic fibrosis (CF) patients displays elevated levels of the cathelicidin antimicrobial peptide LL-37, and the aim of this work was to assess the effect of LL-37 on the growth of Aspergillus fumigatus, a common pathogen of CF patients. Exposure of A. fumigatus to LL-37 and its derived fragment RK-31 (1.95 μg/ml) for 24 h had a positive effect on growth (199.94% ± 6.172% [P < 0.05] and 218.20% ± 4.63% [P < 0.05], respectively), whereas scrambled LL-37 peptide did not (85.12% ± 2.92%). Exposure of mycelium (preformed for 24 h) to 5 μg/ml intact LL-37 for 48 h increased hyphal wet weight (4.37 ± 0.23 g, P < 0.001) compared to the control (2.67 ± 0.05 g) and scrambled LL-37 (2.23 ± 0.09 g) treatments. Gliotoxin secretion from LL-37 exposed hyphae (169.1 ± 6.36 ng/mg hyphae, P < 0.05) was increased at 24 h compared to the results seen with the control treatment (102 ± 18.81 ng/mg hyphae) and the scrambled LL-37 treatment (96.09 ± 15.15 ng/mg hyphae). Shotgun proteomic analysis of 24-h LL-37-treated hyphae revealed an increase in the abundance of proteins associated with growth (eukaryotic translation initiation factor 5A [eIF-5A] [16.3-fold increased]), tissue degradation (aspartic endopeptidase [4.7-fold increased]), and allergic reactions (Asp F13 [10-fold increased]). By 48 h, there was an increase in protein levels indicative of cellular stress (glutathione peroxidase [9-fold increased]), growth (eIF-5A [6-fold increased]), and virulence (RNase mitogillin [3.7-fold increased]). These results indicate that LL-37 stimulates A. fumigatus growth and that this stimulation can result in increased fungal growth and secretion of toxins in the lungs of CF patients.