Upregulation of human ?-defensin-3 and cathelicidin LL-37 in Kaposi's sarcoma.
ABSTRACT: Kaposi's sarcoma (KS) is a rare neoplasm of lymphatic endothelial cells. Human herpes virus 8 (HHV-8) is considered to be a necessary, but not sufficient causal agent of KS and additional cofactors remain unknown. In this study we evaluated the expression of human ? defensin (HBD)-3 and LL-37 in cutaneous lesions of KS in comparison to the healthy skin of normal subjects.We performed a quantitative immunohistochemical study of HBD-3 and LL-37 on skin lesions from 18 patients having KS, and on healthy skin from 12 normal controls.HBD-3 and LL-37 were significantly upregulated in epidermal and dermal specimens of all KS patients in comparison to normal skin of healthy controls. The immunostaining score of dermal HBD-3 was significantly higher in nodular lesions (9.6 ± 2.4) versus plaque lesions (4.1 ± 2.2), P = 0.001. Also the immunostaining score of dermal LL-37 was significantly higher in nodular lesions versus plaque lesions (P = 0.001).We have demonstrated for the first time that HBD-3 and LL-37 are significantly upregulated in lesional skin of KS in comparison to the skin of healthy controls. The obtained data suggest a possible involvement of these antimicrobial peptides in the pathogenesis of KS. However, the biological significance of HBD-3 and LL-37 in KS lesions needs further research.
Project description:Decreased levels of antimicrobial peptides (AMPs) in atopic dermatitis (AD) have previously been reported and have been linked to the increased susceptibility to skin infections found in AD patients. This study intents to identify AMPs: hBD-2, hBD-3, RNase7, psoriasin and LL-37 in AD patients and healthy controls, and determine concentrations in consecutive depths of the outer most skin layers. Tape stripping was used on lesional and non-lesional skin. From each skin site, 35 consecutive tape strips were collected and pooled in groups of 5. Commercially available ELISA kits were used to determine AMP concentration in stratum corneum samples. hBD-2, hBD-3, RNase7 and psoriasin were identified in stratum corneum samples. hBD-3-level was markedly higher in AD non-lesional skin compared to healthy controls, and a similar trend was observed for RNase7. Most AMPs were distributed evenly through 35 tape strips, implying a homogeneous distribution of antimicrobial defense in the outer most skin layers. The findings indicate that AD patients may not suffer from a general baseline deficiency in AMPs, and that the innate immune defense is present throughout the stratum corneum, both insights of importance for understanding the role of AMPs in AD.
Project description:Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.
Project description:Treatment of shigellosis in rabbits with butyrate reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Here, we aimed to evaluate whether butyrate can be used as an adjunct to antibiotics in the treatment of shigellosis in patients.A randomized, double-blind, placebo-controlled, parallel-group designed clinical trial was conducted. Eighty adult patients with shigellosis were randomized to either the Intervention group (butyrate, n?=?40) or the Placebo group (normal saline, n?=?40). The Intervention group was given an enema containing sodium butyrate (80 mM), twice daily for 3 days, while the Placebo group received the same dose of normal saline. The primary endpoint of the trial was to assess the efficacy of butyrate in improving clinical, endoscopic and histological features of shigellosis. The secondary endpoint was to study the effect of butyrate on the induction of antimicrobial peptides in the rectum. Clinical outcomes were assessed and concentrations of antimicrobial peptides (LL-37, human beta defensin1 [HBD-1] and human beta defensin 3 [HBD-3]) and pro-inflammatory cytokines (interleukin-1? [IL-1?] and interleukin-8 [IL-8]) were measured in the stool. Sigmoidoscopic and histopathological analyses, and immunostaining of LL-37 in the rectal mucosa were performed in a subgroup of patients.Compared with placebo, butyrate therapy led to the early reduction of macrophages, pus cells, IL-8 and IL-1? in the stool and improvement in rectal histopathology. Butyrate treatment induced LL-37 expression in the rectal epithelia. Stool concentration of LL-37 remained significantly higher in the Intervention group on days 4 and 7.Adjunct therapy with butyrate during shigellosis led to early reduction of inflammation and enhanced LL-37 expression in the rectal epithelia with prolonged release of LL-37 in the stool.ClinicalTrials.gov, NCT00800930.
Project description:LL-37 is a human cathelicidin antimicrobial peptide that is released in the skin after injury and acts to defend against infection and modulate the local cellular immune response. We observed in human dermal keloids that fibrosis was inversely related to the expression of cathelicidin and sought to determine how LL-37 influenced expression of types I and III collagen genes in dermal fibroblasts. At nano-molar concentrations, LL-37 inhibited baseline and transforming growth factor-beta-induced collagen expression. At these concentrations, LL-37 also induced phosphorylation of extracellular signal-regulated kinase (ERK) within 30 minutes. Activation of ERK, and the activation of a G-protein-dependent pathway, was essential for inhibition of collagen expression as pertussis toxin or an inhibitor of ERK blocked the inhibitory effects of LL-37. c-Jun N-terminal kinase and p38 mitogen-activated protein kinase inhibitors did not alter the effects of cathelicidin. Silencing of the Ets-1 reversed inhibitory effects of LL-37. Taken together, these findings show that LL-37 can directly act on dermal fibroblasts and may have antifibrotic action during the wound repair process.
Project description:Considering the significance of mast cells (MCs) in the course of various physiological and pathological processes, and the pivotal role of endogenous molecules, i.e., cathelicidins and defensins as multifunctional modulators, the study examines the constitutive and cathelicidin LL-37/defensin hBD-2-induced expression of certain NLRs and RLRs, i.e., NOD1, NOD2, and RIG-I, in fully-mature tissue MCs, and the impact of LL-37 and hBD-2 on MC pro-inflammatory activity. All experiments were carried out in vitro on freshly-isolated peritoneal (P)MCs. qRT-PCR, western blotting, flow cytometry, and confocal microscopy were used to evaluate both constitutive and LL-37/hBD-2-induced expression of NOD1, NOD2, and RIG-I receptors. ROS was determined using H2DCFDA, and Boyden microchamber assay was used to define the migratory response. Standard techniques assessed histamine, cysLT, and chemokine generation. PMCs express NOD1, NOD2, and RIG-I constitutively. LL-37 and hBD-2 enhance the expression and induce translocation of the studied receptors and directly activate the pro-inflammatory and migratory responses of PMCs. Observations demonstrate that LL-37 and hBD-2 might augment MC capability and sensitivity to NLR and RLR ligands and strengthen the role of MCs in inflammation.
Project description:The proinflammatory cytokine interleukin-1? (IL-1?) plays a central role in the pathogenesis and the course of inflammatory skin diseases, including psoriasis. Posttranscriptional activation of IL-1? is mediated by inflammasomes; however, the mechanisms triggering IL-1? processing remain unknown. Recently, cytosolic DNA has been identified as a danger signal that activates inflammasomes containing the DNA sensor AIM2. In this study, we detected abundant cytosolic DNA and increased AIM2 expression in keratinocytes in psoriatic lesions but not in healthy skin. In cultured keratinocytes, interferon-? induced AIM2, and cytosolic DNA triggered the release of IL-1? via the AIM2 inflammasome. Moreover, the antimicrobial cathelicidin peptide LL-37, which can interact with DNA in psoriatic skin, neutralized cytosolic DNA in keratinocytes and blocked AIM2 inflammasome activation. Together, these data suggest that cytosolic DNA is an important disease-associated molecular pattern that can trigger AIM2 inflammasome and IL-1? activation in psoriasis. Furthermore, cathelicidin LL-37 interfered with DNA-sensing inflammasomes, which thereby suggests an anti-inflammatory function for this peptide. Thus, our data reveal a link between the AIM2 inflammasome, cathelicidin LL-37, and autoinflammation in psoriasis, providing new potential targets for the treatment of this chronic skin disease.
Project description:Here we show that keratinocytes in psoriatic lesional skin express increased Toll-like receptor (TLR) 9 that similarly localizes with elevated expression of the cathelicidin antimicrobial peptide LL-37. In culture, normal human keratinocytes exposed to LL-37 increased TLR9 expression. Furthermore, when keratinocytes were exposed to LL-37 and subsequently treated with TLR9 ligands, such as CpG or genomic DNA, they greatly increased production of type I IFNs. This response mimicked observations in the epidermis of psoriatic lesional skin as keratinocytes in psoriatic lesions produce greater amounts of IFN-? than normal skin lacking LL-37. The mechanism for induction of type I IFNs in keratinocytes was dependent on TLR9 expression but not on a DNA-LL-37 complex. These findings suggest that keratinocytes recognize and respond to DNA and can actively participate in contributing to the immunological environment that characterizes psoriasis.
Project description:AIMS:Antimicrobial peptides (AMPs) have been implicated in the pathogenesis of several cancers, although there is also evidence suggesting potential for novel, AMP-based antitumor therapies. Discerning potential roles of AMPs in tumor pathogenesis may provide valuable insight into the mechanisms of novel AMP-based antitumor therapy. METHODS:mRNA expression of the AMPs ? defensin (HNP-1); cathelicidin (LL-37); and ? defensins (hBD-1, hBD-2, hBD-3, hBD-4) in human uveal and cutaneous melanoma cell lines, primary human uveal melanocytes, and primary human uveal melanoma cells was determined by reverse transcriptase polymerase chain reaction. An in vitro scratch assay and custom Matlab analysis were used to determine the AMP effects on melanoma cell migration. Last, the effect of specific AMPs on vasculogenic mimicry was determined by three-dimensional (3D) culture and light and fluorescence microscopy. RESULTS:Low-to-moderate AMP transcript levels were detected, and these varied across the cells tested. Overall, LL-37 expression was increased while hBD-4 was decreased in most melanoma cell lines, compared to primary cultured uveal melanocytes. There was no observable influence of HNP-1 and LL-37 on tumor cell migration. Additionally, aggressive cutaneous melanoma cells grown in 3D cultures exhibited vasculogenic mimicry, although AMP exposure did not alter this process. CONCLUSIONS:Collectively, our data show that although AMP mRNA expression is variable between uveal and cutaneous melanoma cells, these peptides have little influence on major characteristics that contribute to tumor aggressiveness and progression.
Project description:The pulmonary airways are continuously exposed to bacteria. As a first line of defense against infection, the airway surface liquid (ASL) contains a complex mixture of antimicrobial factors that kill inhaled and aspirated bacteria. The composition of ASL is critical for antimicrobial effectiveness. For example, in cystic fibrosis an abnormally acidic ASL inhibits antimicrobial activity. Here, we tested the effect of pH on the activity of an ASL defensin, human ?-defensin-3 (hBD-3), and the cathelicidin-related peptide, LL-37. We found that reducing pH from 8.0 to 6.8 reduced the ability of both peptides to kill Staphylococcus aureus. An acidic pH also attenuated LL-37 killing of Pseudomonas aeruginosa. In addition, we discovered synergism between hBD-3 and LL-37 in killing S. aureus. LL-37 and lysozyme were also synergistic. Importantly, an acidic pH reduced the synergistic effects of combinations of ASL antibacterials. These results indicate that an acidic pH reduces the activity of individual ASL antimicrobials, impairs synergism between them, and thus may disrupt an important airway host defense mechanism.
Project description:Various sebum free fatty acids (FFAs) have shown antibacterial activity against a broad range of gram-positive bacteria, resulting in the suggestion that they are accountable, at least partially, for the direct antimicrobial activity of the skin surface. In this study, we examined the effects of sebum FFAs on the antimicrobial peptide (AMP)-mediated innate immune defense of human sebocytes. Incubation of lauric acid, palmitic acid, or oleic acid (OA) with human sebocytes dramatically enhanced their expression of human beta-defensin (hBD)-2, one of the predominant AMPs found in the skin, whereas remarkable increases in hBD-1, hBD-3, and human cathelicidin LL-37 were not observed. Secreted hBD-2 was detectable by western blotting in the supernatant of sebocyte culture incubated with each FFA, but not with a vehicle control. The supernatant of FFA-incubated sebocyte culture showed antimicrobial activity against Propionibacterium acnes, whereas the enhanced antimicrobial activity of human sebocytes was neutralized by anti-hBD-2 IgG. In addition, the FFA-induced hBD-2 expression was suppressed by blocking the cluster of differentiation (CD)36 fatty acid translocase on the surface of sebocytes with anti-human CD36 IgG or blocking the NF-kappaB signaling pathway with BMS-345541, a highly selective inhibitor of inhibitory kappaB kinase. These data suggest that sebum FFAs upregulate the expression of hBD-2 in human sebocytes, which may enhance the disinfecting activity of the human sebaceous gland. The FFA-induced upregulation of hBD-2 is facilitated by CD36-mediated FFA uptake and NF-kappaB-mediated transactivation. The upregulation of mouse beta-defensin 4, a mouse ortholog for hBD-2, was also observed in the hair follicle sebaceous glands of mouse ear skin after an epicutaneous application of OA, the most hBD-2-inducible FFA tested. This report highlights the potential of using FFAs as a multifunctional antimicrobial therapy agent for acne vulgaris treatment; FFAs may provide direct antibacterial activities against P. acnes and enhance the skin's innate antibacterial defense by inducing the expression of hBD-2 in sebocytes as well.