Expression Of LL-37 Research Articles

Expression and Characterization of Antimicrobial Peptide LL-37 in Dog Peripheral Blood Endothelial Progenitor Cells In Vitro

This study aimed to construct antimicrobial peptide LL-37 expressed lentiviral vector and explore the expression of LL-37 in endothelial progenitor cells (EPCs) from dog periphery mononuclear cells (PBMCs). The methodology on obtaining EPCs from dog PBMCs was explored and infection approach about the expression of LL-37 in EPCs was optimized. LL-37 was amplified, digested by Age I and constructed into pGC-FU vector. Meanwhile, EPCs were generated from PBMCs using endothelial growth medium. Markers including acetylated low density lipoproteins (Dilac-LDL), Ulex lectin (UEA-1), von Willebrand factor (vWF), VE-cadherin, CD34 and CD133 were used to identify EPCs. Then, LL-37 was introduced into EPCs using lentivirus infection with green fluorescent protein (GFP) as the biomarker. Finally, we used western-blot to examine the expression of LL-37 in EPCs. The pGC-FU-LL-37-GFP was successfully constructed. Viral particles existed in 10−4 μL group with titer of 2 × 108 TU/mL. Both early (round-shaped morphology) and late (pebble-shaped appearance) EPCs were observed. And these EPCs were confirmed by Dil-acLDL uptake and FITC-UEA-1 binding. An approximate 50 % of infection efficacy was confirmed by immunofluorescence. Furthermore, we successfully observed LL-37 expression in EPCs via western blot analysis. In conclusions, LL-37 expressed in the EPCs was successfully achieved. However, the anti-infection and angiogenesis of LL-37 expressed EPCs need further research in future.

Pulmonary tuberculosis patients with a vitamin D deficiency demonstrate low local expression of the antimicrobial peptide LL-37 but enhanced FoxP3+ regulatory T cells and IgG-secreting cells

Control of human tuberculosis (TB) requires induction and maintenance of both macrophage and T cell effector functions. We demonstrate that pulmonary TB patients with a vitamin D deficiency had significantly reduced local levels of the vitamin D-inducible antimicrobial peptide LL-37 in granulomatous lesions compared to distal parenchyma from the infected lung. Instead, TB lesions were abundant in CD3+ T cells and FoxP3+ regulatory T cells as well as IgG-secreting CD20+ B cells, particularly in sputum-smear positive patients with cavitary TB. Mycobacteria-specific serum IgG titers were also elevated in patients with active TB. An up-regulation of the B cell stimulatory cytokine IL-21 correlated with mRNA expression of CD20, total IgG and also IL-10 in the TB lesions. Altogether, vitamin D-deficient TB patients expressed a weak antimicrobial response but an IL-21 associated expansion of IgG-secreting B cells combined with a rise in FoxP3+ regulatory T cells at the local site of infection.

Phenylbutyrate induces cathelicidin expression via the vitamin D receptor: Linkage to inflammatory and growth factor cytokines pathways

Antimicrobial peptides (AMPs) constitute an indispensable arm of innate immunity against infectious microbes in humans. Induction of endogenous AMPs may become an alternative therapy against infections. Our previous studies have demonstrated phenylbutyrate (PBA) as a novel inducer of the AMPs cathelicidin (encoded by the CAMP gene) and human beta-defensin-1 in the human bronchial epithelial cell line VA10. In this work, we have continued by studying molecular mechanisms of PBA mediated induction of LL-37 expression and associated pathways in the human bronchial epithelial cell line VA10. In this study we demonstrate vitamin D receptor (VDR) as a key transcription factor required for PBA mediated up-regulation of the CAMP gene expression. PBA also increases mRNA expression of the vitamin D3 regulated genes CYP24A1 and CD14. The siRNA knockdown of VDR reduced PBA mediated increase in CAMP, CYP24A1 and CD14 expression. Furthermore, we demonstrate that PBA enhances Toll-Like Receptor 5 ligand flagellin regulated mRNA expression of the inflammatory cytokine TNFα and chemokine CXCL8. PBA also up-regulates the expression of the genes encoding the growth factor cytokines transforming growth factor (TGF) α, TGFβ1 and TGFβ2. Our results indicate that TGFβ type I receptor and epidermal growth factor receptor are involved in PBA mediated CAMP regulation. Finally, we show that co-treatment with PBA and vitamin D3 reduces Pseudomonas aeruginosa growth in vitro.

Vesicular LL-37 contributes to inflammation of the lesional skin of palmoplantar pustulosis.

“Pustulosis palmaris et plantaris”, or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1β in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1β at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.

PORF5 plasmid protein promotes Chlamydia trachomatis infection through inhibition of the antibacterial peptide LL37: a preliminary study

Objective To investigate whether pORF5 plasmid protein promotes Chlamydia trachomatis (Ct)infection through inhibition of the antibacterial peptide LL37,and to explore its potential molecular mechanism.Methods After expression and purification,the glutathione S-transferase (GST)-pORF5 fusion protein was digested with proteases to obtain pORF5 protein without GST tag.Some Hela cells were classified into four groups:Ct group infected with unpretreated Ct,pORF5 group infected with Ct that had been pretreated with pORF5 of 30 μg/ml for 2 hours,LL37 group infected with Ct that had been pretreated with LL37 of 20 μg/ml for 2 hours,combination group infected with Ct that had been pretreated with pORF5 of 30 μg/ml and LL37 of 20 μg/ml for 2 hours.After additional culture,indirect immunofluorescence assay was performed to count the number of Ct inclusion-forming units (IFUs),and real-time,fluorescence-based quantitative PCR and enzyme-linked immunosorbent assay (ELISA) to detect the protein and mRNA expressions of tumor necrosis factor-α (TNF-α) respectively.Some Hela cells were divided into three groups:blank control group remaining untreated,Ct group infected with unpretreated Ct,pORF5 group infected with Ct that had been pretreated with pORF5 of 30 μg/ml for 2 hours.After 6 hours of additional culture,real-time,fluorescence-based quantitative PCR and ELISA were conducted to measure the mRNA and protein expressions of LL37,respectively.Results The concentration of IFUs was significantly higher in the Ct group,pORF5 group and combination group than in the LL37 group (3.8 × 105/ml,3.0 × 105/ml and 3.1 × 105/ml vs.2.0 × 105/ml,all P ＜ 0.05),with no significant differences between the Ct group,pORF5 group and combination group (P ＞ 0.05).The expressions of TNF-α mRNA and protein were significantly increased in the combination group compared with the LL37 group.The mRNA expression of LL37 was reduced by 19％ in the pORF5 group compared with the Ct group.Conclusions pORF5 plasmid protein could promote Ct infection by counteracting the antibacterial peptide LL37,which may be associated with the up-regulation of TNF-α expression and downregulation of LL37 expression in Ct-infected cells. Key words: Chlamydia trachomatis; pORF5 plasmid protein; LL37; Antimicrobial peptides

LL-37 secreted by epithelium promotes fibroblast collagen production: a potential mechanism of small airway remodeling in chronic obstructive pulmonary disease

Emerging evidence suggests that the process of small airway remodeling is mediated by profibrotic growth factors produced by epithelium, which are capable of activating the underlying mesenchymal cells with excessive collagen production. It has been demonstrated that human cathelicidin antimicrobial protein LL-37 is highly expressed in small airway epithelium from COPD patients. However, it is unknown whether the increased levels of LL-37 in epithelium are involved in the pathogenesis of small airway remodeling in COPD. In this study, we examined the expression of LL-37 in small airways from smokers with COPD and controls (non-smokers and smokers without COPD) by immunohistochemistry, and then the association between LL-37 expression in epithelium and the structural changes of small airway remodeling was analyzed. In vitro, the effect of CSE-induced epithelial secretion of LL-37 on collagen production in human lung fibroblasts (HFL-1 cell line) was studied in a co-culture system. Finally, the signaling pathways involved in the effect of LL-37 on fibroblast collagen production were evaluated. The results showed that LL-37 immunoreactivity in airway epithelium was significantly elevated in smokers with COPD compared with controls. In addition, the magnitude of LL-37 expression in epithelium was positively correlated with airway wall thickness and collagen deposition. In vitro, CSE-induced epithelial secretion of LL-37 promoted fibroblast collagen production. Finally, we showed that formyl peptide receptor-like 1 (FPRL1)-dependent extracellular signal-regulated kinase (ERK) signaling pathway was essential for LL-37-induced collagen production in HFL-1 cells. These results suggest that after cigarette smoke exposure, the increased levels of LL-37 in airway epithelium could stimulate collagen production in the underlying lung fibroblasts and may contribute to small airway remodeling in COPD.

Cathelicidin antimicrobial peptide LL ‐37 augments interferon‐β expression and antiviral activity induced by double‐stranded RNA in keratinocytes

Cathelicidin antimicrobial peptide LL-37 has the capacity to kill a wide range of microbes and to modify host immunity. Recently, our group observed that the activation of keratinocytes by LL-37 and DNA greatly increases interferon (IFN)-β through Toll-like receptor (TLR)9. However, the effect of LL-37 on the induction of IFN-β through TLR3, a sensor of double-stranded (ds) RNA, in keratinocytes is not well known. To investigate whether LL-37 could affect TLR3 signalling and antiviral activity in normal human epidermal keratinocytes (NHEKs). We investigated the production of IFN-β in NHEKs stimulated with a TLR3 ligand, poly (I:C), in the presence of LL-37. To examine the effect of LL-37 and poly (I:C) on antiviral activity, a virus plaque assay using herpes simplex (HS) virus type-1 was carried out. The uptake of poly (I:C) conjugated with fluorescein isothiocyanate (FITC) into the keratinocytes was observed in the presence of LL-37. Immunostaining for TLR3 and LL-37 was performed using skin samples from HS. LL-37 and poly (I:C) synergistically induced the expression of IFN-β in NHEKs. Furthermore, co-stimulation with LL-37 and poly (I:C) significantly decreased the viral plaque numbers compared with poly (I:C) or LL-37 alone. LL-37 enhanced the uptake of FITC-conjugated poly (I:C) into cells. Immunohistochemical analysis demonstrated that the expression of TLR3 and LL-37 is upregulated in HS lesions. Our findings suggest that LL-37 augments the antiviral activity induced by dsRNA in keratinocytes, which may contribute to the innate immune response to cutaneous viral infections such as HS.

Prenatal vitamin D₃ supplementation suppresses LL-37 peptide expression in ex vivo activated neonatal macrophages but not their killing capacity.

Vitamin D has regulatory effects on innate immunity. In the present study, we aimed to assess the effect of prenatal vitamin D₃ (vitD₃) supplementation on neonatal innate immunity in a randomised, placebo-controlled trial by evaluating cathelicidin (LL-37) expression and the killing capacity of macrophages. Healthy pregnant women (n 129) attending a clinic in Dhaka were randomised to receive either a weekly oral dose of 0·875 mg vitD₃ or placebo starting from 26 weeks of gestation up to delivery. Serum, plasma and monocyte-derived macrophages (MDM) were obtained from the cord blood. 25-Hydroxyvitamin D (25(OH)D) concentration was measured in serum. MDM were stimulated with or without Toll-like-receptor 4 ligand (TLR4L). Innate immune function was assessed by measuring LL-37 peptide levels in the culture supernatant of MDM by ELISA, LL-37 transcript levels by quantitative PCR, and ex vivo bactericidal capacity of MDM. VitD₃ supplementation did not increase LL-37 peptide levels in plasma or in the extracellular fluid of macrophages with or without TLR4L induction. However, stimulated intracellular LL-37 expression (ratio of stimulated:unstimulated MDM) was significantly reduced in the vitamin D group v. placebo (P=0·02). Multivariate-adjusted analyses showed that intracellular LL-37 peptide concentration from stimulated MDM was inversely associated with 25(OH)D concentration in serum (P=0·03). TLR4L stimulation increased the bactericidal capacity of MDM compared with the unstimulated ones (P=0·01); however, there was no difference in killing capacity between the two groups. A weekly dose of 0·875 mg vitD₃ to healthy pregnant women suppressed the intracellular LL-37 peptide stores of activated macrophages, but did not significantly affect the ex vivo bactericidal capacity of cord blood MDM.

LL-37 attenuates inflammatory impairment via mTOR signaling-dependent mitochondrial protection

The human cationic antimicrobial protein LL-37 is a multifunctional host defense peptide with a wide range of immunomodulatory activities. Previous work has shown that LL-37 exerts both pro- and anti-inflammatory effects. The role of mitochondria in the skin inflammatory effects of LL-37 has not been well studied. Therefore, our aim was to investigate the immunomodulatory effect of LL-37 in HaCaT cells and to delineate the underlying mechanisms related to mitochondrial function. Immunohistochemistry results from tissue microarrays showed strong cytoplasmic LL-37 staining in inflammatory cells in chronic dermatic inflammation. Using exogenous LL-37 stimulation and LL-37 knockdown and overexpression, LL-37 was demonstrated to dramatically reduce the mRNA levels and protein secretion of inflammatory cytokines including IL-6, IL-8, IL-1α and tumor necrosis factor-α (TNF-α), which are induced by lipopolysaccharides (LPS). The anti-inflammatory effects of LL-37 are dependent upon its ability to increase mitochondrial biogenesis and to maintain mitochondrial homeostasis. Furthermore, we observed that LL-37 enhances the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and mammalian target of rapamycin (mTOR). The mTOR inhibitor rapamycin can neutralize the protective effects of LL-37 on mitochondria. In conclusion, these results suggest that high LL-37 expression levels correlate with chronic skin inflammation; mitochondrial dysfunction occurs in HaCaT cells during inflammation; and LL-37 attenuates inflammatory impairment by stimulating mitochondrial biogenesis and protecting mitochondrial function, which are dependent upon mTOR signaling. These findings provide new insights into targeting mitochondria with LL-37 to prevent skin inflammatory reactions.

LEAP-2, LL-37 and RNase7 in tonsillar tissue: downregulated expression in seasonal allergic rhinitis.

In the upper airway, the production of antimicrobial peptides (AMPs) protects against bacteria, viruses and fungi. Previous investigations have revealed downregulated expression of AMPs in different manifestations of allergic disease. In this study, we examined the expression of LL-37, Ribonuclease7 (RNase7) and Liver-expressed antimicrobial peptide 2 (LEAP-2) in tonsillar tissue and studied a possible relation to seasonal allergic rhinitis (SAR). Tonsils, obtained from patients with SAR and nonallergic controls, were examined for the occurrence of LL-37, RNase7 and LEAP-2 with real-time RT-PCR and immunohistochemistry. Tonsillar mononuclear cells were cultured in presence or absence of LEAP-2 or LL-37 and analyzed for cytokine levels using ELISA. mRNA and protein for LL-37, RNase 7 and LEAP-2 were found in all tonsils. Immunohistochemistry revealed prominent staining for LL-37 and RNase7 in the tonsillar epithelium, whereas a moderate staining was seen with LEAP-2. Real-time RT-PCR showed a downregulation of RNase7 and LEAP-2 in the allergic as compared to the nonallergic group. Mononuclear cells cultured in presence of LEAP-2 or LL-37 demonstrated reduced levels of IL-10. The present study demonstrates the presence and function of LEAP-2, LL-37 and RNase7 in tonsils. Moreover, a downregulation of LEAP-2 and RNase7 is seen in SAR patients, indicating that allergic individuals may be more susceptible to respiratory tract infections due to an impaired antimicrobial defense.

Skin Electroporation of a Plasmid Encoding hCAP-18/LL-37 Host Defense Peptide Promotes Wound Healing

Host defense peptides, in particular LL-37, are emerging as potential therapeutics for promoting wound healing and inhibiting bacterial growth. However, effective delivery of the LL-37 peptide remains limiting. We hypothesized that skin-targeted electroporation of a plasmid encoding hCAP-18/LL-37 would promote the healing of wounds. The plasmid was efficiently delivered to full-thickness skin wounds by electroporation and it induced expression of LL-37 in the epithelium. It significantly accelerated reepithelialization of nondiabetic and diabetic wounds and caused a significant VEGFa and interleukin (IL)-6 induction. IL-6 was involved in LL-37-mediated keratinocyte migration in vitro and IL-6 neutralizing antibodies delivered to mice were able to suppress the wound healing activity of the hCAP-18/LL-37 plasmid. In a hindlimb ischemia model, electroporation of the hCAP-18/LL-37 plasmid increased blood perfusion, reduced muscular atrophy, and upregulated the angiogenic chemokines VEGFa and SDF-1a, and their receptors VEGF-R and CXCR-4. These findings demonstrate that a localized gene therapy with LL-37 is a promising approach for the treatment of wounds.

Cathelicidin Antimicrobial Peptide LL‐37 in Cholesteatoma Enables Keratinocyte Reactivity with Cytosolic DNA

The purpose of this study was to determine whether self-DNA can trigger the inflammatory response in cholesteatoma. Specimens were collected from nine patients with invasive cholesteatoma, nine patients with attic-type cholesteatoma (pars flaccida was perforated in five patients and intact in four) and four healthy skins. Expression and localization of LL-37 and interferon-alpha were detected by immunofluorescence and immunoblot analysis. Cultures of human cholesteatomatous keratinocytes were exposed to CpG DNA, LL-37 or CpG DNA complexed to LL-37 for 24h. Expression of interferon-alpha was detected by RT-PCR. We detected abundant cytosolic DNA, increased LL-37 and interferon-alpha in keratinocytes in invasive cholesteatoma and attic-type cholesteatoma with pars flaccida perforation, but not in attic-type cholesteatoma with pars flaccida intact and normal skin. In cultured keratinocytes, LL-37-DNA complexes induced IFN-α expression. These data suggest that cytosolic DNA is an important disease-associated molecular pattern that triggers the inflammation response in cholesteatoma. Furthermore, LL-37 played an important role in DNA-triggered inflammation. Thus, we have identified a link between cytosolic DNA, LL-37 and autoinflammation in cholesteatoma, providing new potential targets for the treatment of this disease.

Antibacterial responses of retinal Müller glia: production of antimicrobial peptides, oxidative burst and phagocytosis

BackgroundWe have previously shown that, in response to microbial infection, activated Müller glia secrete inflammatory cytokines/chemokines and exhibit antimicrobial properties. The aim of this study is to understand the mechanisms and the key components involved in this response.MethodsImmortalized human retinal Müller glia (MIO-M1 cells) were challenged with Staphylococcus (S) aureus, the leading cause of severe intraocular infection followed by RT2 profile PCR array analysis. The expression of human β-defensin 1 (HBD1), 2 (HBD2), 3 (HBD3), hepcidine and cathelicidin LL37 was checked by RT-PCR and quantified by Taqman® qPCR. The expression of AMPs was confirmed at protein level by dot-blot analysis. The production of ROS was measured by dicholoro-dihydro-fluorescein diacetate (DCFH-DA) staining by flow cytometry as well as fluorescence microscopy. The level of nitric oxide (NO) was measured by measuring a stable metabolite, nitrite using the Griess reagent. In vitro killing assay was performed by Live/Dead® BacLight™ staining as well as by dilution plating in suspension and adherent conditions following S. aureus infection. Phagocytosis was measured by CFU enumeration following infection.ResultsPCR array data showed that, in comparison to uninfected control cells, bacterial challenge significantly (> two-fold) induced the expression of 26 genes involved in cytokine/chemokine, antimicrobials, Toll-like receptor, apoptotic, and NF-κB signaling. RT-PCR analysis showed time-dependent increased expression of HBD1, HBD2, HBD3, LL-37, and hepcidin mRNA in bacteria-challenged Müller glia. The expression of these antimicrobial molecules was also increased at the protein level in the culture supernatant, as detected by dot-blot analysis. Additionally, the bacteria-stimulated Müller glia were found to produce reactive oxygen (ROS) and reactive nitrogen (RNS) species. In vitro, killing assays revealed that Müller glia exhibited bactericidal activity against S. aureus in both adherent and suspension cultures. Furthermore, our data demonstrated that Müller glia can phagocytize and kill the bacteria in a time-dependent manner.ConclusionsThese data suggest that retinal Müller glia behave like classical innate immune cells by producing a variety of antimicrobial molecules in response to bacterial challenge, suggesting their pivotal role in retinal innate defense.

Effect of vitamin D supplementation on cathelicidin, IFN-γ, IL-4 and Th1/Th2 transcription factors in young healthy females

We assessed the effect of cholecalciferol and calcium supplementation on mRNA expression of cathelicidin (LL-37), Th1 and Th2 cytokines and their transcription factors in the peripheral blood mononuclear cells (PBMCs) in healthy females with vitamin D deficiency (VDD). Subjects included 131 females with biochemical VDD randomized to receive (a) oral cholecalciferol (60,000 IU/week for 8 weeks followed by 60,000 IU/fortnight (b) calcium (elemental calcium 500 mg twice/day) (c), dual supplementation and (d) placebo for 6 months. The mRNA expression of cathelicidin, Th1 (IFN-γ) and Th2 (IL-4 and its antagonist-IL-4δ2) cytokines and their transcription factors (T-bet, STAT4, GATA-3, STAT6) were measured in the PBMC by real-time PCR before and after intervention. Cholecalciferol-supplemented groups showed significant rise of mean serum 25(OH)D (30.6 ± 7.51 and 28.6 ± 8.41 ng/ml). The expression of LL-37, IFN-γ, IL-4, IL-4δ2 and transcription factors were comparable in the four groups at baseline. Despite significant increase in mean serum 25(OH)D in the cholecalciferol-supplemented groups, their mean mRNA transcripts of LL-37, IFN-γ, IL-4, transcription factors and their IFN-γ/IL-4 and T-bet/GATA-3 ratios were similar to that of calcium and placebo groups. Six months of cholecalciferol/calcium supplementation in young females with VDD do not lead to significant alteration in mRNA expression of LL-37, Th1/Th2 cytokines and their transcription factors.

Effect of Narrowband Ultraviolet B Therapy on Serum Vitamin D and Cathelicidin (LL-37) in Patients with Chronic Plaque Psoriasis

Narrowband ultraviolet B (NB-UVB) has been widely used in the treatment of psoriasis. It has been shown that vitamin D is a major regulator of the expression of human antimicrobial peptide cathelicidin LL-37, which has a critical role in inflammatory cascade in psoriasis. To evaluate the effect of NB-UVB therapy on serum levels of cathelicidin LL-37 and 25-hydroxyvitamin D [25(OH)D] in psoriasis patients. Ninety-three psoriasis patients and 50 controls were included in the study. For psoriasis patients, serum levels of 25(OH)D and cathelicidin LL-37 were estimated before and after NB-UVB therapy. Before treatment, serum 25(OH)D levels were significantly lower in psoriasis patients (31.5 ± 14.41 nmol/L) compared to controls (53.5 ± 19.6 nmol/L), p = .015. In contrast, serum LL-37 was significantly higher in psoriasis patients (13.24 ± 3.2 ng/mL) than in controls (7.92 ± 5.33 ng/mL), p < .001. After NB-UVB treatment, there was a highly significant elevation of serum 25(OH)D to reach 56.85 ± 5.2 nmol/L (p < .001) and further elevation of serum LL-37 to reach 29.4 ± 4.2 (p = .02). The elevation of serum 25(OH)D and cathelicidin LL-37 could be an additional possible mechanism of action of NB-UVB therapy in the treatment of psoriasis.

Calcitriol May Down-Regulate mRNA Over-Expression of Toll-Like Receptor-2 and -4,LL-37and Proinflammatory Cytokines in Cultured Human Keratinocytes

BackgroundAlthough vitamin D analogs have been used in the topical treatment of psoriasis, their mechanisms of action are not well understand. Calcitriol, the hormonally active vitamin D3 metabolite, has been demonstrated to exert immunomodulatory effects in the skin by down-regulating the expression of Toll-like receptors (TLRs) and proinflammatory cytokines.ObjectiveWe investigated the effects of calcitriol on the expression of TLR2, TLR4, antimicrobial peptide LL-37, and proinflammatory cytokines in cultured human keratinocytes.MethodsThe mRNA expression levels of TLR2, TLR4, tumor necrosis factor α (TNF-α), interleukin (IL)-1β and LL-37 in cultured human keratinocytes were measured by real-time polymerase chain reaction (PCR) and reverse transcription (RT). Furthermore, we measured supernatant TNF-α levels by an enzyme-linked immunosorbent assay (ELISA) to confirm the effects of calcitriol on TLR2 and TLR4.ResultsAs measured by RT-PCR and real-time PCR, calcitriol was found to suppress the lipopolysaccharide- and ultraviolet B radiation-mediated induction of expression of TLRs, LL-37 and proinflammatory cytokines such as TNF-α and IL-1β in normal human keratinocytes. The supernatant TNF-α levels measured by ELISA were also suppressed after treatment with calcitriol.ConclusionCalcitriol may down-regulate inflammatory stated over-expression of LL-37 and proinflammatory cytokines.

Human Corneal Epithelial Cells Produce Antimicrobial Peptides LL-37 and β-Defensins in Response to Heat-Killed Candida albicans

Aims: To explore the innate response of human corneal epithelial cells (HCECs) exposed to fungus by producing antimicrobial peptides LL-37 and β-defensins. Methods: Primary HCECs were treated with heat-killed Candida albicans (HKCA) at different doses (10³-10⁶ cells/ml) for 2-48 h. The cells were subjected to total RNA extraction, reverse transcription and quantitative real-time PCR for mRNA expression. Cells treated for 48 h were used for immunofluorescent staining and ELISA. Results: Human LL-37 and β-defensins (hBDs) 1-4 were detected in normal HCECs. The mRNA expression of LL-37, hBD2, and hBD3 was dose-dependently induced by HKCA with their peak levels at 4 h. HKCA (10⁶ cells/ml) stimulated the mRNA of LL-37, hBD2, and hBD3 4.33 ± 1.81, 3.75 ± 1.31, and 4.91 ± 1.09 fold, respectively, in HCECs. The stimulated production of LL-37, hBD2, and hBD3 by HKCA was confirmed at protein levels by immunofluorescent staining and ELISA. The protein production of LL-37, hBD2, and hBD3 significantly increased to 109.1 ± 18.2 pg/ml, 4.33 ± 1.67 ng/ml, and 296.9 ± 81.8 pg/ml, respectively, in culture medium of HCECs exposed to HKCA (10⁶ cells/ml) compared to untreated HCECs. Conclusions: HCECs produce antimicrobial peptides, LL-37, hBD2 and hBD3, in response to stimulation of HKCA, which suggests a novel innate immune mechanism of the ocular surface in defense against fungal invasion.

Expression of antimicrobial peptides and proteins in epidermis equivalents exposed to salt water and narrowband ultraviolet B radiation.

Expression of antimicrobial peptides and proteins in epidermis equivalents exposed to salt water and narrowband ultraviolet B radiation.

Label-Free Quantitative Mass Spectrometry Reveals Novel Pathways Involved in LL-37 Expression

Antimicrobial peptides are important for a healthy host-microbe homeostasis. In infections characterized by low levels of the human cathelicidin, LL-37, induction of its expression increases clearance of pathogens. Our aim was to discover signaling pathways and compounds capable of affecting the expression of LL-37. We recently observed a synergistic induction of LL-37 expression by stimulating the colonic epithelial cell-line HT-29 with lactose and phenylbutyrate (PBA). Here, we studied regulatory circuits mediating this synergism in HT-29 cells stimulated with lactose (60 g/l) and PBA (2 m<smlcap>M</smlcap>) for 24 h by using mass spectrometry and pathway analyses. Selected pathways were evaluated for their involvement in LL-37 regulation in a CAMP gene-luciferase reporter system. Three pathways were examined in detail: thyroid hormone receptor and retinoid X receptor (TR/RXR) activation, eicosanoid signaling and steroid biosynthesis. Induced expression of LL-37 was observed upon stimulation with triiodothyronine (T3, 2.5 n<smlcap>M</smlcap>-1 µ<smlcap>M</smlcap> for 3-30 h) and thyroxine (T4, 2.5-10 n<smlcap>M</smlcap> for 24 h). Furthermore, the synergism of lactose and PBA was reduced in cells coincubated with inhibitors of phospholipase A2, cyclooxygenase 2 or HMG-CoA reductase. Based on these results, we conclude that proteomics and pathway analyses are valuable tools for dissecting the regulatory networks involved in LL-37 expression.

Differential regulation of human cathelicidin LL-37 by free fatty acids and their analogs

LL-37 is the single cathelicidin host defense peptide in humans with direct antimicrobial and immunomodulatory activities. Specific regulation of LL-37 synthesis has emerged as a novel non-antibiotic approach to disease control and prevention. Short-chain fatty acids, and butyrate in particular, were found recently to be strong inducers of LL-37 gene expression without causing inflammation. Here, we further evaluated the LL-37-inducing efficiency of a broad range of saturated free fatty acids and their derivatives in human HT-29 colonic epithelial cells and U-937 monocytic cells by real-time RT-PCR. Surprisingly, we revealed that valerate, hexanoate, and heptanoate with 5–7 carbons are more potent than 4-carbon butyrate in promoting LL-37 gene expression in both cell types. Free fatty acids with longer than 7 or shorter than 4 carbons showed only a marginal effect on LL-37 expression. Studies with a series of fatty acid derivatives with modifications in the aliphatic chain or carboxylic acid group yielded several analogs such as benzyl butyrate, trans-cinnamyl butyrate, glyceryl tributyrate, and phenethyl butyrate with a comparable LL-37-inducing activity to sodium butyrate. On the other hand, although reactive, the anhydride derivatives of short- and medium-chain fatty acids are as potent as their corresponding free acid forms in LL-37 induction. Thus, these newly identified free fatty acids and their analogs with a strong capacity to augment LL-37 synthesis may hold promise as immune boosting dietary supplements for antimicrobial therapy.