The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis.

Psoriasis is a common T-cell-mediated skin disease with 2-3% prevalence worldwide. Psoriasis is considered to be an autoimmune disease, but the precise nature of the autoantigens triggering T-cell activation remains poorly understood. Here we find that two-thirds of patients with moderate-to-severe plaque psoriasis harbour CD4(+) and/or CD8(+) T cells specific for LL37, an antimicrobial peptide (AMP) overexpressed in psoriatic skin and reported to trigger activation of innate immune cells. LL37-specific T cells produce IFN-γ, and CD4(+) T cells also produce Th17 cytokines. LL37-specific T cells can infiltrate lesional skin and may be tracked in patients blood by tetramers staining. Presence of circulating LL37-specific T cells correlates significantly with disease activity, suggesting a contribution to disease pathogenesis. Thus, we uncover a role of LL37 as a T-cell autoantigen in psoriasis and provide evidence for a role of AMPs in both innate and adaptive immune cell activation.

Pestiviral E(rns) blocks TLR-3-dependent IFN synthesis by LL37 complexed RNA

The ribonuclease activity of the soluble glycoprotein E(rns) of pestiviruses represents a unique mechanism to circumvent the host's innate immune system by blocking interferon type-I synthesis in response to extracellularly added single- (ss) and double-stranded (ds) RNA. However, the reason why pestiviruses encode a ribonuclease in addition to the abundant serum RNases remained elusive. Here, we show that the 5' UTR and NS5B regions of various strains of the RNA genome of the pestivirus bovine viral diarrhea virus (BVDV) are resistant to serum RNases and are potent TLR-3 agonists. Inhibitory activity of E(rns) was restricted to cleavable RNA products, and did not extend to the synthetic TLR-7/8 agonist R-848. RNA complexed with the antimicrobial peptide LL37 was protected from degradation by E(rns)in vitro but was fully inhibited by E(rns) in its ability to induce IFN in cell cultures, suggesting that the viral protein is mainly active in cleaving RNA in an intracellular compartment. We propose that secreted E(rns) represents a potent IFN antagonist, which degrades viral RNA that is resistant to the ubiquitous host RNases in the extracellular space. Thus, the viral RNase prevents its own pathogen-associated molecular pattern (PAMP) to inadvertently activate the IFN response that might break innate immunotolerance required for persistent pestivirus infections.

Antibacterial Effect of Human Mesenchymal Stem Cells is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/ LL-37.

Cathelicidin LL-37:a defense molecule with a potential role in psoriasis pathogenesis

Epidermal keratinocytes produce and secrete antimicrobial peptides (AMPs) that subsequently form a chemical shield on the skin surface. Cathelicidins are one family of AMPs in skin with various further immune functions. Consequently, dysfunction of these peptides has been implicated in the pathogenesis of inflammatory skin disease. In particular, the cathelicidin LL-37 is overexpressed in inflamed skin in psoriasis, binds to extracellular self-DNA released from dying cells and converts self-DNA in a potent stimulus for plasmacytoid dendritic cells (pDCs). Subsequently, pDCs secrete type I interferons and trigger an auto-inflammatory cascade. Paradoxically, therapies targeting the vitamin D pathway such as vitamin D analogues or UVB phototherapy ameliorate cutaneous inflammation in psoriasis but strongly induce cathelicidin expression in skin at the same time. Current evidence now suggests that self-DNA present in the cytosol of keratinocytes is also pro-inflammatory active and triggers IL-1β secretion in psoriatic lesions through the AIM2 inflammasome. This time, however, binding of LL-37 to self-DNA neutralizes DNA-mediated inflammation. Hence, cathelicidin LL-37 shows contrasting roles in skin inflammation in psoriasis and might serve as a target for novel therapies for this chronic skin disease.

The Host Defence Peptide LL-37 is Susceptible to Proteolytic Degradation by Wound Fluid Isolated from Foot Ulcers of Diabetic Patients

The pleiotropic effects of host defence peptides (HDPs), including the ability to kill microorganisms, enhance re-epithelialisation and increase angiogenesis, indicates a role for these important peptides as potential therapeutic agents in the treatment of chronic, non-healing wounds. However, the maintenance of peptide integrity, through resistance to degradation by the array of proteinases present at the wound site, is a prerequisite for clinical success. In this study we explored the degradation of exogenous LL-37, one such HDP, by wound fluid from diabetic foot ulcers to determine its susceptibility to proteolytic degradation. Our results suggest that LL-37 is unstable in the diabetic foot ulcer microenvironment. Following overnight treatment with wound fluid, LL-37 was completely degraded. Analysis of cleavage sites suggested potential involvement of both host- and bacterial-derived proteinases. The degradation products were shown to retain some antibacterial activity against Pseudomonas aeruginosa but were inactive against Staphylococcus aureus. In conclusion, our data suggest that stabilising selected peptide bonds within the sequence of LL-37 would represent an avenue for future research prior to clinical studies to address its potential as an exogenously-applied therapeutic in diabetic wounds. 

Effects of LL-37 on Human Gingival Fibroblast Function

Background and Objectives: Gingival fibroblasts play a significant role in the innate immune response of the periodontium to bacterial stimulation. A number of microorganisms and their by-products induce a host response that commonly leads to tissue destruction and periodontal disease progression. LL-37 is an antimicrobial peptide which has multiple roles in host defence including immunomodulation and wound-healing. We have investigated the role of LL-37 on the responsiveness of human gingival fibroblasts to microbial challenge from E. coli lipopolysaccharide (LPS) and P. gingivalis LPS, as well as exploring the direct effects of LL-37 on human gingival fibroblasts. Methods: The effect of LL-37 on bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. The influence of LL-37 on bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot. The direct effects of LL-37 on modulating gingival fibroblasts gene expression were initially determined by DNA microarray analysis and subsequently confirmed by quantitative polymerase chain reaction (Q-PCR) and ELISA analysis of 9 selected genes. Results: Bacterial LPS-induced IL-8 and IL-6 production by human gingival fibroblasts were significantly reduced in the presence of LL-37 at concentrations in the range of 1-10 µg/ml (p<0.05). The presence of LL-37 at a concentration of 5 µg/ml led to a reduction in LPS-induced IκBα degradation by E. coli LPS (100 ng/ml) and P. gingivalis LPS (10 µg/ml). LL-37 (50 µg/ml) significantly altered the gene expression of 367 genes in human gingival fibroblasts by at least 2-fold. CXCL1, CXCL2, CXCL3, IL-24, IL-8, CCL2, and SOCS3 mRNA were significantly upregulated by LL-37 (p<0.05). LL-37 also significantly stimulated expression of IL-8, hepatocyte growth factor (HGF) and CXCL1 (p<0.05) at the protein level. Discussion: LL-37 plays an important role in the innate immune response due to its broad spectrum antimicrobial and immunomodulatory activity. The ability of LL-37 to directly regulate expression of a range of genes, central to the pathogenesis of periodontitis, identifies multiple roles for the peptide in host homeostasis.

The role of antimicrobial peptides in periodontal disease.

Objectives Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

The role of the cathelicidin LL-37 in periodontal disease.

Abstract Background Fibroblasts respond to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. As such fibroblasts play a significant role as regulators of the host response in periodontal disease. LL-37, an antimicrobial peptide, found in saliva and GCF, inhibits LPS-induced cytokine signalling in macrophages, suggesting a role in host defence in periodontal disease. This study investigated the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and also its effect on fibroblast response to LPS activation. Methods Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (0.01µg/ml) along with LL-37 (0-50µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Results At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL37 induced IL-8 production independent of LPS. Microarray analysis revealed that LL-37 upregulated a significant number of cytokines and chemokines by > 5 fold. The stimulatory effect on IL-8 mRNA expression was confirmed by Q-PCR. Conclusion LL-37 appears to have pleiotrophic effects in innate immunity. Its ability, at low concentrations, to reduce bacterial LPS-induced cytokine production in gingival fibroblasts suggests a potential therapeutic role in the management of periodontal disease.

Host Defence Peptide LL-37; Effects of truncation on antimicrobial activity

Background: The oral cavity is an ideal environment for colonisation by micro-organisms. A first line of defence against microbial infection is the secretion of broad spectrum host defence peptides (HDPs). In the current climate of antibiotic resistance, exploiting naturally occurring HDPs or synthetic derivatives (mimetics) to combat infection is particularly appealing. The human cathelicidin, LL-37 is one such HDP expressed ubiquitously by epithelial cells and neutrophils. LL-37 exhibits the ability to bind lipopolysaccharide (LPS) and displays broad spectrum activity against a wide range of bacteria. The current study focuses on truncation of LL-37 and defining the antimicrobial and LPS binding activity of the resultant mimetics. Objectives: To assess the antimicrobial and LPS binding activity of LL-37 and three truncated mimetics (KE-18, EF-14 and KR-12). Methods: Peptides were synthesised in-house by Fmoc solid phase peptide synthesis or obtained commercially. Antimicrobial activity was determined using a radial diffusion assay and ability to bind LPS was determined by indirect ELISA. Results: LL-37 and mimetics displayed antimicrobial activity against Streptococcus mutans and Enterococcus Faecalis. KE-18 and KR-12 were shown to possess antimicrobial activity against both pathogens whereas EF-14 was the least antimicrobial. In terms of LPS binding, KE-18 and KR-12 were both effective whereas EF-14 showed the least activity of the three mimetics. Conclusion: Truncation of LL-37 can yield peptides which retain antimicrobial activities and have the ability to bind LPS. Interestingly in some cases the truncation of LL-37 produced mimetics with greater potency than the parent molecule in terms of antimicrobial activity and LPS binding. This work was funded by DEL and the Diabetes Wellness Foundation.

LL-37 quantification in gingival crevicular fluid (GCF) from periodontitis patients

Background: LL-37, an anti-microbial peptide belonging to the cathelicidin family, derives its name from two N-terminal leucine residues and the 37 amino acids comprising the peptide. LL-37 is the only known cathelicidin to exist in humans. It exhibits both anti-bacterial and immunomodulatory properties. Objectives: In the current study, LL-37 was quantified in GCF from periodontitis patients. Previous studies have relied on qualitative results from Western blotting to detect LL-37 in GCF. This study aims to quantitatively determine LL-37 levels in GCF. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 sites in 12 patients presenting with advanced periodontitis. Plaque samples were analysed by QPCR for the presence or absence of the periodontopathic bacterium Porphyromonas gingivalis (P. gingivalis). The concentrations of LL-37 in patient samples pre- and post-treatment were deduced by indirect enzyme linked immunosorbent assay (ELISA). Results: Concentrations of LL-37 in samples varied between a minimum and maximum of 1 and 40 ng/ml. LL-37 levels were shown, pre-treatment, to be higher in deep pockets (6-9 mm) compared with shallower pockets (3-5 mm) and highest in those sites which were positive for P. gingivalis. Non-surgical therapy resulted in a significant improvement in clinical indices while expression levels of P. gingivalis were reduced. Following treatment, LL-37 levels in GCF decreased from an average of 6.5 ± 1 - 5.8 ± 1.2 ng/ml. The most interesting observation however was the reduction in LL-37 levels, from an average of 7 ± 1.3 – 2.5 ± 1.1 ng/ml in those sites where P. gingivalis infection was eradicated post-treatment. Conclusions: LL-37 levels are increased at sites showing advanced periodontal disease, reduce following treatment and appear to be linked to the presence of P. gingivalis. This study will further our knowledge of host defence in chronic diseases such as periodontitis.