Bile acid metabolites enhance expression of cathelicidin antimicrobial peptide in airway epithelium through activation of the TGR5-ERK1/2 pathway.

Signals for the maintenance of epithelial homeostasis are provided in part by commensal bacteria metabolites, that promote tissue homeostasis in the gut and remote organs as microbiota metabolites enter the bloodstream. In our study, we investigated the effects of bile acid metabolites, 3-oxolithocholic acid (3-oxoLCA), alloisolithocholic acid (AILCA) and isolithocholic acid (ILCA) produced from lithocholic acid (LCA) by microbiota, on the regulation of innate immune responses connected to the expression of host defense peptide cathelicidin in lung epithelial cells. The bile acid metabolites enhanced expression of cathelicidin at low concentrations in human bronchial epithelial cell line BCi-NS1.1 and primary bronchial/tracheal cells (HBEpC), indicating physiological relevance for modulation of innate immunity in airway epithelium by bile acid metabolites. Our study concentrated on deciphering signaling pathways regulating expression of human cathelicidin, revealing that LCA and 3-oxoLCA activate the surface G protein-coupled bile acid receptor 1 (TGR5, Takeda-G-protein-receptor-5)-extracellular signal-regulated kinase (ERK1/2) cascade, rather than the nuclear receptors, aryl hydrocarbon receptor, farnesoid X receptor and vitamin D3 receptor in bronchial epithelium. Overall, our study provides new insights into the modulation of innate immune responses by microbiota bile acid metabolites in the gut-lung axis, highlighting the differences in epithelial responses between different tissues.

Potential role of salivary vitamin D antimicrobial peptide LL-37 and interleukins in severity of dental caries: an exvivo study.

INTRODUCTION: Vitamin D performs various functions as a hormone by promoting calcium absorption but plays a major role in innate immunity,cell differentiation, cell maturation through its genomic effects via vitamin D receptor. The immune response also plays a major role in tooth surface and supporting structure destruction and playing a major factor in high caries formation. The inflammatory cytokines are released has proinflammatory cytokines and stimulate cells in disease process. Therefore, in the present study we have evaluated the association of salivary vitamin D, LL-37, interleukins 6 and 17A in various levels of severity of dental caries. METHOD: Ethical approval was obtained (NU/CEC/2020/0339), 377 individuals reporting to department of conservative dentistry and endodontics, AB Shetty memorial institute of dental sciences were included based on inclusion criteria. The individuals were further divided into caries free(N = 105) and caries active(N = 272) based on their caries prevalence. The salivary were collected and evaluated for vitamin D, LL-37,IL-17A and IL-6.Results were statistically analysed with SPSS vs 22 (IBM Corp, USA). Normally distributed data were expressed as mean ± SD. Skewed data were expressed as median and interquartile range. To compare (mean) outcome measures between the two groups unpaired independent t-test was applied and for values in median IQR, Mann Whitney U test was used. All statistical analysis for P value were two-sided and significance was set to P ≤ 0.05. RESULTS: The study showed that, the salivary vitamin D statistically decreased with increasing severity of caries which showed that vitamin D plays an important role in prevention of caries. Antimicrobial peptide LL-37 was higher in caries free group but was not statistically significant, salivary IL-6 level was higher in caries active group but intergroup comparison did not show significant difference. Salivary IL-17A did not show statistically significant between caries active and caries free group. CONCLUSION: The salivary levels of vitamin D may play a vital role in prevalence of dental caries and its severity which can be a underlying cause in presence of other etiological factors.

Botulinum toxin type A ameliorates rat dorsal root ganglia neuron pyroptosis in postherpetic neuralgia by upregulating cathelicidin antimicrobial peptide to inhibit neutrophil elastase.

Botulinum toxin type A (BoNT/A) has exhibited efficacy in postherpetic neuralgia (PHN) treatment, and this study aims to uncover its underlying mechanisms. Resiniferatoxin (RTX)-induced PHN rats were given BoNT/A. Rat postoperative pain behaviors were assessed by Von Frey test. Cleaved-synaptosomal protein 25 kDa (cl-SNAP-25) or cathelicidin antimicrobial peptide (CAMP) expression in rat dorsal root ganglia (DRG) was detected by immunofluorescence or immunohistochemistry. Healthy rat-derived DRG neurons were transfected, incubated with lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) to stimulate pyroptosis and treated with BoNT/A. The CCK-8, Western blot, ELISA, and qRT-PCR were used to assess the viability, levels of pyroptosis-related proteins proinflammatory cytokine levels, as well as CAMP and ELANE mRNA levels. BoNT/A (30 U/kg) promoted cl-SNAP-25 expression in rat DRG and reversed RTX-induced decrease of rat paw withdrawal thresholds and CAMP expression and increase of pyroptosis-associated protein and inflammatory factor expression in rat DRG. CAMP interacted with ELANE in rat DRG neurons. BoNT/A attenuated LPS/ATP-stimulated inhibition of viability and CAMP expression and upregulation of inflammatory mediators, pyroptosis-related proteins, and ELANE expression in rat DRG neurons, which was counteracted by CAMP silencing. However, ELANE knockdown offset the effect of CAMP silencing in LPS/ATP/BoNT/A-treated rat DRG neurons. On the whole, BoNT/A alleviates rat DRG neuron pyroptosis during PHN by upregulating CAMP to inhibit ELANE.

Turning cationic antimicrobial peptide KR-12 into self-assembled nanobiotics with potent bacterial killing and LPS neutralizing activities.

Gram-negative sepsis has become a substantial and escalating global healthcare challenge due to the growing antibiotic resistance crisis and the sluggish development of new antibiotics. LL-37, a unique Cathelicidin species found in humans, exhibits a wide range of bioactive properties, including direct bactericidal effects, inflammation regulation, and LPS neutralization. KR-12, the smallest yet potent peptide fragment of LL-37, has been modified to create more effective antimicrobials. In this study, we designed two myristoylated derivatives of KR-12, referred to as Myr-KR-12N and Myr-KR-12C. These derivatives displayed remarkable ability to spontaneously assemble into nanoparticles when mixed with deionized water. Myristoylated KR-12 derivatives exhibited broad-spectrum and intensified bactericidal activity by disrupting bacterial cell membranes. In particular, Myr-KR-12N showed superior capability to rescue mice from lethal E. coli-induced sepsis in comparison with the conventional antibiotic meropenem. We also confirmed that the myristoylated KR-12 nanobiotic possesses significant LPS binding capacity and effectively reduces inflammation in vitro. In an in vivo context, Myr-KR-12N outperformed polymyxin B in rescuing mice from LPS-induced sepsis. Crucially, toxicological assessments revealed that neither Myr-KR-12N nor Myr-KR-12C nanobiotics induced meaningful hemolysis or caused damage to the liver and kidneys. Collectively, our study has yielded an innovative nanobiotic with dual capabilities of bactericidal action and LPS-neutralization, offering substantial promise for advancing the clinical translation of antimicrobial peptides and the development of novel antibiotics. This addresses the critical need for effective solutions to combat Gram-negative sepsis, a pressing global medical challenge.

Cath-KP, a novel peptide derived from frog skin, prevents oxidative stress damage in a Parkinson's disease model.

Parkinson's disease (PD) is a neurodegenerative condition that results in dyskinesia, with oxidative stress playing a pivotal role in its progression. Antioxidant peptides may thus present therapeutic potential for PD. In this study, a novel cathelicidin peptide (Cath-KP; GCSGRFCNLFNNRRPGRLTLIHRPGGDKRTSTGLIYV) was identified from the skin of the Asiatic painted frog ( Kaloula pulchra). Structural analysis using circular dichroism and homology modeling revealed a unique αßß conformation for Cath-KP. In vitro experiments, including free radical scavenging and ferric-reducing antioxidant analyses, confirmed its antioxidant properties. Using the 1-methyl-4-phenylpyridinium ion (MPP +)-induced dopamine cell line and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice, Cath-KP was found to penetrate cells and reach deep brain tissues, resulting in improved MPP +-induced cell viability and reduced oxidative stress-induced damage by promoting antioxidant enzyme expression and alleviating mitochondrial and intracellular reactive oxygen species accumulation through Sirtuin-1 (Sirt1)/Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway activation. Both focal adhesion kinase (FAK) and p38 were also identified as regulatory elements. In the MPTP-induced PD mice, Cath-KP administration increased the number of tyrosine hydroxylase (TH)-positive neurons, restored TH content, and ameliorated dyskinesia. To the best of our knowledge, this study is the first to report on a cathelicidin peptide demonstrating potent antioxidant and neuroprotective properties in a PD model by targeting oxidative stress. These findings expand the known functions of cathelicidins, and hold promise for the development of therapeutic agents for PD.

Regulation of Cathelicidin Antimicrobial Peptide (CAMP) Gene Expression by TNFα and cfDNA in Adipocytes.

Understanding the complex interactions between metabolism and the immune system ("metaflammation") is crucial for the identification of key immunomodulatory factors as potential therapeutic targets in obesity and in cardiovascular diseases. Cathelicidin antimicrobial peptide (CAMP) is an important factor of innate immunity and is expressed in adipocytes. CAMP, therefore, might play a role as an adipokine in metaflammation and adipose inflammation. TNFα, cell-free nucleic acids (cfDNA), and toll-like receptor (TLR) 9 are components of the innate immune system and are functionally active in adipose tissue. The aim of the present study was to investigate the impact of TNFα and cfDNA on CAMP expression in adipocytes. Since cfDNA acts as a physiological TLR9 agonist, we additionally investigated TLR9-mediated CAMP regulation in adipocytes and adipose tissue. CAMP gene expression in murine 3T3-L1 and human SGBS adipocytes and in murine and human adipose tissues was quantified by real-time PCR. Adipocyte inflammation was induced in vitro by TNFα and cfDNA stimulation. Serum CAMP concentrations in TLR9 knockout (KO) and in wildtype mice were quantified by ELISA. In primary adipocytes of wildtype and TLR9 KO mice, CAMP gene expression was quantified by real-time PCR. CAMP gene expression was considerably increased in 3T3-L1 and SGBS adipocytes during differentiation. TNFα significantly induced CAMP gene expression in mature adipocytes, which was effectively antagonized by inhibition of PI3K signaling. Cell-free nucleic acids (cfDNA) significantly impaired CAMP gene expression, whereas synthetic agonistic and antagonistic TLR9 ligands had no effect. CAMP and TLR9 gene expression were correlated positively in murine and human subcutaneous but not in intra-abdominal/visceral adipose tissues. Male TLR9 knockout mice exhibited lower systemic CAMP concentrations than wildtype mice. CAMP gene expression levels in primary adipocytes did not significantly differ between wildtype and TLR9 KO mice. These findings suggest a regulatory role of inflammatory mediators, such as TNFα and cfDNA, in adipocytic CAMP expression as a novel putative molecular mechanism in adipose tissue innate immunity.

Cathelicidin Antimicrobial Peptide Acts as a Tumor Suppressor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is associated with high rates of metastasis and recurrence, and is one of the most common causes of cancer-associated death worldwide. This study examined the protein changes within circulating exosomes in patients with HCC against those in healthy people using isobaric tags for a relative or absolute quantitation (iTRAQ)-based quantitative proteomics analysis. The protein levels of von Willebrand factor (VWF), cathelicidin antimicrobial peptide (CAMP), and proteasome subunit beta type-2 (PSMB2) were altered in HCC. The increased levels of VWF and PSMB2 but decreased CAMP levels in the serum of patients with HCC were validated by enzyme-linked immunosorbent assays. The level of CAMP (the only cathelicidin found in humans) also decreased in the circulating exosomes and buffy coat of the HCC patients. The serum with reduced levels of CAMP protein in the HCC patients increased the cell proliferation of Huh-7 cells; this effect was reduced following the addition of CAMP protein. The depletion of CAMP proteins in the serum of healthy people enhances the cell proliferation of Huh-7 cells. In addition, supplementation with synthetic CAMP reduces cell proliferation in a dose-dependent manner and significantly delays G1-S transition in Huh-7 cells. This implies that CAMP may act as a tumor suppressor in HCC.

Endopeptidase O promotes Streptococcus suis immune evasion by cleaving the host- defence peptide cathelicidins.

Streptococcus suis is a zoonotic Gram-positive bacterium that causes invasive infections such as sepsis and meningitis, threatening public health worldwide. For successful establishment of infection, the bacterium should subvert the innate effectors of immune defence, including the cathelicidin family of host-defence peptides that combat pathogenic bacteria by directly disrupting cell membranes and coordinating immune responses. Here, our study shows that an extracellular endopeptidase O (PepO) of S. suis contributes to assisting the bacterium to resist cathelicidin-mediated killing, as the deletion of the pepO gene makes S. suis more sensitive to the human cathelicidin LL-37, as well as its mouse equivalent, mCRAMP. This protease targets and cleaves both LL-37 and mCRAMP, degrading them into shorter peptides with only a few amino acids, thereby abrogating their ability to kill S. suis. By cleaving LL-37 and mCRAMP, PepO impairs their chemotactic properties for neutrophil migration and undermines their anti-apoptosis activity, which is required for prolonging neutrophil lifespan. Also, PepO inhibits the ability of LL-37 and mCRAMP to promote lysosome development in macrophages. Moreover, the loss of PepO attenuates organ injury and decreases bacterial burdens in a murine model of S. suis bacteraemia. Taken together, these data provide novel insights into the role of the intrinsic proteolytic characteristics of PepO in S. suis-host interaction. Our findings demonstrate that S. suis utilizes the PepO protease to cleave cathelicidins, which is an immunosuppressive strategy adopted by this bacterium to facilitate pathogenesis.

Antimicrobial peptide LL-37 disrupts plasma membrane and calcium homeostasis in Candida albicans via the Rim101 pathway.

IMPORTANCE: Candida albicans is a major human fungal pathogen, and antimicrobial peptides are key components of innate immunity. Studying the interplay between C. albicans and human antimicrobial peptides would enhance a better understanding of pathogen-host interactions. Moreover, potential applications of antimicrobial peptides in antifungal therapy have aroused great interest. This work explores new mechanisms of LL-37 against C. albicans and reveals the complex connection among calcium homeostasis, oxidative stress, signaling, and possibly organelle interaction. Notably, these findings support the possible use of antimicrobial peptides to prevent and treat fungal infections.

Evaluation of the differences in proteomics of high-quality bovine colostrum and low-quality bovine colostrum.

BACKGROUND: Although there are studies on colostrum and milk proteomics of different species in the literature, there is no published report about different quality bovine colostrums' proteomics. OBJECTIVES: The aim of this study was to compare the proteome content of high- and low-quality bovine colostrums for the first time. METHODS: Colostrum samples were collected from 32 Holstein cows from the same farm that had just calved. Brix% levels of colostrums were measured, and then, those with a Brix% value of ≥27% were classified as high-quality and those with a Brix% value of <22% as low-quality. Three samples from high-quality and low-quality colostrums were selected and proteomic analyses were performed by pooling separately. RESULTS: Totally 95 proteins were identified in the colostrums, and 19 of them showed significant changes between high- and low-quality colostrums. Expressions in colostrum of glycosylation-dependent cell adhesion molecule-1, cofilin-1, alpha-S2-casein, alpha-lactalbumin, alpha-1B-glycoprotein, actin_cytoplasmic-1, nucleobindin-1, cathelicidin-4, inter-alpha-trypsin inhibitor heavy chain H4, chitinase-3-like protein 1 and monocyte differentiation antigen CD14 were lower, whereas tetranectin, secreted frizzled-related protein-1 (SFRP1), perilipin-2, coatomer subunit epsilon (COPE), butyrophilin subfamily 1 member A1, polyubiquitin-B, lactadherin and albumin levels were higher in high-quality colostrum than low-quality colostrum. Moreover, SFRP1, COPE and cathelicidin-4 proteins were identified for the first time in bovine colostrum. In high-quality colostrum, the most prominently down-regulated proteins were cathelicidin-4 (26.01-fold) and cofilin-1 (17.42-fold), and the most prominently up-regulated proteins were COPE (3.37-fold) and tetranectin (3.07-fold). CONCLUSIONS: It was detected that the proteome contents of high- and low-quality bovine colostrums were different from each other. As new functions are added to the protein databases regarding these proteins detected in colostrums, the interactions of proteins with each other and with other molecules will be detailed and the effects of high-quality colostrums on passive transfer immunity and calf health will be understood in full detail.

Tasmanian devil cathelicidins exhibit anticancer activity against Devil Facial Tumour Disease (DFTD) cells.

The Tasmanian devil (Sarcophilus harrisii) is endangered due to the spread of Devil Facial Tumour Disease (DFTD), a contagious cancer with no current treatment options. Here we test whether seven recently characterized Tasmanian devil cathelicidins are involved in cancer regulation. We measured DFTD cell viability in vitro following incubation with each of the seven peptides and describe the effect of each on gene expression in treated cells. Four cathelicidins (Saha-CATH3, 4, 5 and 6) were toxic to DFTD cells and caused general signs of cellular stress. The most toxic peptide (Saha-CATH5) also suppressed the ERBB and YAP1/TAZ signaling pathways, both of which have been identified as important drivers of cancer proliferation. Three cathelicidins induced inflammatory pathways in DFTD cells that may potentially recruit immune cells in vivo. This study suggests that devil cathelicidins have some anti-cancer and inflammatory functions and should be explored further to determine whether they have potential as treatment leads.

Evaluation of biocompatibility and angiogenic potential of extracellular matrix hydrogel biofunctionalized with the LL-37 peptide.

BACKGROUND: Biomaterials must allow revascularization for a successful tissue regeneration process. Biomaterials formulated from the extracellular matrix (ECM) have gained popularity in tissue engineering because of their superior biocompatibility, and due to their rheological properties, ECM-hydrogels can be easily applied in damaged areas, allowing cell colonization and integration into the host tissue. Porcine urinary bladder ECM (pUBM) retains functional signaling and structural proteins, being an excellent option in regenerative medicine. Even some small molecules, such as the antimicrobial cathelicidin-derived LL-37 peptide have proven angiogenic properties. OBJECTIVE: The objective of this study was to evaluate the biocompatibility and angiogenic potential of an ECM-hydrogel derived from the porcine urinary bladder (pUBMh) biofunctionalized with the LL-37 peptide (pUBMh/LL37). METHODS: Macrophages, fibroblasts, and adipose tissue-derived mesenchymal stem cells (AD-MSC) were exposed pUBMh/LL37, and the effect on cell proliferation was evaluated by MTT assay, cytotoxicity by quantification of lactate dehydrogenase release and the Live/Dead Cell Imaging assays. Moreover, macrophage production of IL-6, IL-10, IL-12p70, MCP-1, INF-γ, and TNF-α cytokines was quantified using a bead-based cytometric array. pUBMh/LL37 was implanted directly by dorsal subcutaneous injection in Wistar rats for 24 h to evaluate biocompatibility, and pUBMh/LL37-loaded angioreactors were implanted for 21 days for evaluation of angiogenesis. RESULTS: We found that pUBMh/LL37 did not affect cell proliferation and is cytocompatible to all tested cell lines but induces the production of TNF-α and MCP-1 in macrophages. In vivo, this ECM-hydrogel induces fibroblast-like cell recruitment within the material, without tissue damage or inflammation at 48 h. Interestingly, tissue remodeling with vasculature inside angioreactors was seen at 21 days. CONCLUSIONS: Our results showed that pUBMh/LL37 is cytologically compatible, and induces angiogenesis in vivo, showing potential for tissue regeneration therapies.

Vitamin D modulates expression of antimicrobial peptides and proinflammatory cytokines to restrict Zika virus infection in macrophages.

Although the impact of Zika virus (ZIKV) infection on human health has been well documented, we still have no vaccine or effective treatment. This fact highlights the importance of searching for alternative therapy for treating ZIKV. To search for ZIKV antivirals, we examined the effect of vitamin D in monocyte-derived macrophages (MDMs) differentiated in the presence of vitamin D (D3-MDM) and explored the molecular mechanisms by analyzing transcriptional profiles. Our data show the restriction of ZIKV infection in D3-MDMs as compared to MDMs. Transcriptional profiles show that vitamin D alters about 19% of Zika response genes (8.2% diminished and 10.8% potentiated). Among the genes with diminished expression levels, we found proinflammatory cytokines and chemokines such as IL6, TNF, IL1A, IL1B, and IL12B, CCL1, CCL4, CCL7, CXCL3, CXCL6, and CXCL8. On the other hand, genes with potentiated expression were related to degranulation such as Lysozyme, cathelicidin (CAMP), and Serglycin. Since the CAMP gene encodes the antimicrobial peptide LL-37, we treated MDMs with LL-37 and infected them with ZIKV. The results showed a decrease in the proportion of infected cells. Our data provide new insights into the role of vitamin D in restricting ZIKV infection in macrophages that are mediated by induction of cathelicidin/LL-37 expression and downregulation of proinflammatory genes. Results highlight the biological relevance of vitamin D-inducible peptides as an antiviral treatment for Zika fever.

The neutralising and stimulatory effects of antimicrobial peptide LL-37 in human gingival fibroblasts.

OBJECTIVES: To investigate the effects of LL-37, a broad spectrum antimicrobial peptide expressed in periodontal tissues, on human gingival fibroblast responsiveness to microbial challenge and to explore the direct effects of LL-37 on human gingival fibroblasts. DESIGN: The effect of LL-37 on bacterial lipopolysaccharide-induced expression of Interleukin (IL-6) and chemokine C-X-C motif ligand (CXCL) 8 was determined by enzyme linked immunosorbent assay (ELISA). LL-37's influence on bacterial lipopolysaccharide-induced IκBα degradation was investigated by western blot. DNA microarray analysis initially determined the direct effects of LL-37 on gene expression, these findings were subsequently confirmed by quantitative polymerase chain reaction and ELISA analysis of selected genes. RESULTS: Bacterial lipopolysaccharide-induced IL-6 and CXCL8 production by human gingival fibroblasts was significantly reduced in the presence of LL-37 at concentrations in the range of 1-10 µg/ml. LL-37 led to a reduction in lipopolysaccharide-induced IκBα degradation by Escherichia coli lipopolysaccharide and Porphyromonas gingivalis lipopolysaccharide (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, Interleukin-24 (IL-24), CXCL8, Chemokine (C-C motif) Ligand 2, and Suppressor of Cytokine Signalling 3 mRNA were significantly upregulated by LL-37. LL-37 also significantly stimulated expression of CXCL8, hepatocyte growth factor and CXCL1 at the protein level. CONCLUSION: LL-37 plays an important regulatory role in the immunomodulatory activity of gingival fibroblasts by inhibiting lipopolysaccharide -induced expression of inflammatory cytokines and directly stimulating the expression of an array of bioactive molecules involved in inflammation and repair.

LL-37 Triggers Antimicrobial Activity in Human Platelets.

Platelets play a crucial role in hemostasis and the immune response, mainly by recognizing signals associated with vascular damage. However, it has recently been discovered that the antimicrobial peptide LL-37 activates platelets in functions related to thrombus formation and inflammation. Therefore, this work aims to evaluate the effect of LL-37 on the activation of antimicrobial functions of human platelets. Our results show that platelets treated with LL-37 increase the surface expression of receptors (Toll-like receptors (TLRs) 2 and -4, CD32, CD206, Dectin-1, CD35, LOX-1, CD41, CD62P, and αIIbß3 integrins) for the recognition of microorganisms, and molecules related to antigen presentation to T lymphocytes (CD80, CD86, and HLA-ABC) secrete the antimicrobial molecules: bactericidal/permeability-increasing protein (BPI), azurocidin, human neutrophil peptide (HNP) -1, and myeloperoxidase. They also translate azurocidin, and have enhanced binding to Escherichia coli, Staphylococcus aureus, and Candida albicans. Furthermore, the supernatant of LL-37-treated platelets can inhibit E. coli growth, or platelets can employ their LL-37 to inhibit microbial growth. In conclusion, these findings demonstrate that LL-37 participates in the antimicrobial function of human platelets.

Cathelicidin LL-37 promotes EMT, migration and metastasis of hepatocellular carcinoma cells in vitro and mouse model.

The effect of cathelicidin hCAP18/LL-37 in hepatocellular carcinoma (HCC) metastasis remains unclear. Here, we confirmed that LL-37 expression enhanced endothelial-mesenchymal transition (EMT), migration and invasion in HCC cells. And the HER2/EGFR-MAPK/ERK signal participated in the process above. More frequent lung metastases were observed in an LL-37-overexpressing hematogenous metastasis model. Interestingly, 1,25(OH)2D3 together with si-LL-37 significantly enhanced 1,25(OH)2D3-induced inhibition of migration and invasion in PLC/PRF-5 cells, and also enhanced reversion of the EMT process. Therefore, LL-37 is involved in HCC metastases, and may act as an important factor to attenuate the inhibitory activity of 1,25(OH)2D3 on HCC metastasis. Targeting hCAP18/LL-37 may offer a potential strategy to improve the anticancer activity of 1,25(OH)2D3 in HCC therapy.

Oral vitamin D modulates the epidermal expression of the vitamin D receptor and cathelicidin in children with atopic dermatitis.

Although vitamin D (VD) is known to have multiple effects on the skin and immunity, its effects on atopic dermatitis (AD) severity remain unclear. We investigated whether oral cholecalciferol (VD3) supplementation changes stratum corneum expression of the vitamin D receptor (vdr), and the epidermal alarmins Cathelicidin Antimicrobial Peptide (camp/LL-37) and Thymic Stromal Lymphopoietin (tslp) in children with AD. We conducted an open-label supplementation study with weekly oral VD3 for six weeks in children with AD. Serum 25-hydroxyvitamin D (25OHD), lesional Staphylococcus aureus colonization, and AD severity evaluated by SCORAD index were evaluated before and after supplementation. Tape stripping (TS) was performed on non-lesional and lesional skin to measure mRNA expression of vdr, camp, and tslp through RT-qPCR and LL-37 peptide by ELISA. Twenty-two children with moderate-severe AD received weekly oral VD3 for six weeks. Total serum 25OHD increased from 45.1 ± 23 to 93.5 ± 24.3 nmoL/L (p < 0.0001), while SCORAD decreased from 41.4 ± 13.5 to 31.5 ± 15.8 (p < 0.0001). After treatment, epidermal gene expression of camp increased significantly in non-lesional (p = 0.014) and lesional (p = 0.0007) tape stripping samples, while vdr only increased in lesional skin samples (p < 0.0001). LL-37 peptide increased significantly only in lesional skin samples (p = 0.008). Gene expression of tslp did not change after oral VD3 treatment. In children with AD, oral VD3 supplementation was associated with improved VD status and AD severity, as well as increased VDR and Cathelicidin expression in lesional skin, which provide mechanistic clues on its effects.

In silico assessment of missense point mutations on human cathelicidin LL-37.

Cathelicidin antimicrobial peptides are a diverse family of cationic amphipathic peptides with multiple activities. In humans, cathelicidin LL-37 is one of the main host defense peptides with a remarkable medical and biotechnological potential. Deregulation of LL-37 expression has been associated with inflammatory diseases. However the effects of point mutations driven by single nucleotide polymorphisms (SNPs) on LL-37 are unknown. Here we applied an array of computational tools to investigate the effects of such mutations on LL-37 structure and activity. Due to the fact that, on cathelicidins, the prodomain is more conserved than the mature peptide, the SNP effect predictions were biased and, overall, resulted in neutral effects; and due to the slight changes in physicochemical properties, the antimicrobial predictions indicated the maintenance of such activity. Nonetheless, R07P, R07W, R29Q, R29W mutations reduced the peptide net charge, which in turn could result in less active LL-37 variants. Molecular dynamics data indicated that R07Q and N30Y mutations altered the LL-37 structure, leading to potential deleterious effects. In addition, the helix dipole is altered in G03A, R07P, R07W and L31P mutations, which could also alter the antimicrobial activity. Our results indicated that despite the mutations did not alter the residues from LL-37 active core, they could influence the antimicrobial activity and consequently, could be involved in inflammatory diseases.

Cathelicidin Antimicrobial Peptide LL37 Induces Toll-Like Receptor 8 and Amplifies IL-36γ and IL-17C in Human Keratinocytes.

LL37 is produced by skin injury and bacterial infection and plays an important role in the early stages of psoriasis. In particular, the intracellular receptors toll-like receptors (TLR)3, TLR7, TLR8, and TLR9 are thought to be involved in the pathogenesis of psoriasis in conjunction with LL37, but the interaction between TLR7/8 and LL37 in keratinocytes (KCs) remains unclear. This study aimed to clarify the relationship between LL37 and TLR7/8 in KCs and their involvement in the pathogenetic pathways seen in psoriasis using cultured KCs and skin samples of patients with psoriasis. TLR7/8 was induced by LL37 in KCs. TLR8 but not TLR7 functionally induced many psoriasis-related molecules, whereas IL-17C was not altered by the blockade of TLR7/8. Although costimulation of LL37 with self-RNA/DNA did not show any interaction, LL37 itself would promote psoriasis-related genes. IL-36 receptor antagonistic antibody suppressed IL-17C induced by LL37. In psoriatic epidermis, LL37, TLRs, IL-17C, and IL-36γ expressions were increased and coexpressed with each other. Thus, we concluded that LL37 activates TLR8 in KCs and induces IL-17C through the induction of IL-36γ. Regulation of TLR8 or LL37 in KCs could be a potential therapeutic strategy for psoriatic inflammation.

Cathelicidin LL-37 Activates Human Keratinocyte Autophagy through the P2X7, Mechanistic Target of Rapamycin, and MAPK Pathways.

Human cathelicidin LL-37 is a multifunctional antimicrobial peptide that exhibits antimicrobial and immunomodulatory activities. LL-37 regulates skin barrier function and was recently reported to activate autophagy in macrophages. Because autophagy deficiency is associated with skin diseases characterized by a dysfunctional epidermal barrier, we hypothesized that LL-37 might regulate the skin barrier through autophagy modulation. We showed that LL-37 activated autophagy in human keratinocytes and three-dimensional skin equivalent models as indicated by increases in LC3 puncta formation, decreases in p62, and autophagosome and autolysosome formation. LL-37‒induced autophagy was suppressed by P2X7 receptor, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 inhibitors, suggesting that the P2X7, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 pathways are involved. Moreover, LL-37 enhanced the phosphorylation of adenosine monophosphate‒activated protein kinase and unc-51-like kinase 1. In addition, LL-37‒mediated autophagy involves the mechanistic target of rapamycin and MAPK pathways. Interestingly, the LL-37‒induced distribution of tight junction proteins and improvement in the tight junction barrier were inhibited in autophagy-deficient keratinocytes and keratinocytes and skin models treated with autophagy inhibitors, indicating that the LL-37‒mediated tight junction barrier is associated with autophagy activation. Collectively, these findings suggest that LL-37 is a potential therapeutic target for skin diseases characterized by dysfunctional autophagy and skin barriers.