Age-Related Loss of Innate Immune Antimicrobial Function of Dermal Fat Is Mediated by Transforming Growth Factor Beta.

Dermal fibroblasts (dFBs) resist infection by locally differentiating into adipocytes and producing cathelicidin antimicrobial peptide in response to Staphylococcus aureus (S. aureus). Here, we show that neonatal skin was enriched with adipogenic dFBs and immature dermal fat that highly expressed cathelicidin. The pool of adipogenic and antimicrobial dFBs declined after birth, leading to an age-dependent loss of dermal fat and a decrease in adipogenesis and cathelidicin production in response to infection. Transforming growth factor beta (TGF-ß), which acted on uncommitted embryonic and adult dFBs and inhibited their adipogenic and antimicrobial function, was identified as a key upstream regulator of this process. Furthermore, inhibition of the TGF-ß receptor restored the adipogenic and antimicrobial function of dFBs in culture and increased resistance of adult mice to S. aureus infection. These results provide insight into changes that occur in the skin innate immune system between the perinatal and adult periods of life.

Cathelicidin peptide analogues inhibit EV71 infection through blocking viral entry and uncoating.

Given the serious neurological complications and deaths associated with enterovirus 71 (EV71) infection, there is an urgent need to develop effective antivirals against this viral infection. In this study, we demonstrated that two Cathelicidin-derived peptides, LL-18 and FF-18 were more potent against EV71 infection than the parent peptide LL-37, which is the mature and processed form of Cathelicidin. These peptides could directly bind to the EV71 virus particles, but not to coxsackievirus, indicative of their high specificity. The binding of peptides with the virus surface occupied the viral canyon region in a way that could block virus-receptor interactions and inhibit viral uncoating. In addition, these peptide analogues could also relieve the deleterious effect of EV71 infection in vivo. Therefore, Cathelicidin-derived peptides might be excellent candidates for further development of antivirals to treat EV71 infection.

naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation.

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.

Antimicrobial peptide-producing dermal preadipocytes defend against Candida albicans skin infection via the FGFR-MEK-ERK pathway.

Dermal fibroblasts (dFBs) defend against deep bacterial skin infections by differentiating into preadipocytes (pAds) that produce the antimicrobial peptide cathelicidin; this differentiation is known as the dermal reactive adipogenesis response. However, the role of dFBs in fungal infection remains unknown. Here, we found that cathelicidin-producing pAds were present in high numbers in skin lesions from patients with cutaneous Candida granulomas. Second, we showed that dermal Candida albicans (C. albicans) infection in mice robustly triggered the dermal reactive adipogenesis response and induced cathelicidin expression, and inhibition of adipogenesis with pharmacological inhibitors of peroxisome proliferator-activated receptor γ (PPARγ) impaired skin resistance to C. albicans. In vitro, C. albicans products induced cathelicidin expression in pAds, and differentiating pAds markedly suppressed the growth of C. albicans by producing cathelicidin. Finally, we showed that C. albicans induced an antimicrobial response in pAds through the FGFR-MEK-ERK pathway. Together, our data reveal a previously unknown role of dFBs in the defense against skin infection caused by C. albicans.

LL37/FPR2 regulates neutrophil mPTP promoting the development of neutrophil extracellular traps in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists in vitro. In vivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, in vitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

Antimicrobial Peptide LL37 and MAVS Signaling Drive Interferon-ß Production by Epidermal Keratinocytes during Skin Injury.

Type 1 interferons (IFNs) promote inflammation in the skin but the mechanisms responsible for inducing these cytokines are not well understood. We found that IFN-ß was abundantly produced by epidermal keratinocytes (KCs) in psoriasis and during wound repair. KC IFN-ß production depended on stimulation of mitochondrial antiviral-signaling protein (MAVS) by the antimicrobial peptide LL37 and double stranded-RNA released from necrotic cells. MAVS activated downstream TBK1 (TANK-Binding Kinase 1)-AKT (AKT serine/threonine kinase 1)-IRF3 (interferon regulatory factor 3) signaling cascade leading to IFN-ß production and then promoted maturation of dendritic cells. In mice, the production of epidermal IFN-ß by LL37 required MAVS, and human wounded and/or psoriatic skin showed activation of MAVS-associated IRF3 and induction of MAVS and IFN-ß gene signatures. These findings show that KCs are an important source of IFN-ß and MAVS is critical to this function, and demonstrates how the epidermis triggers unwanted skin inflammation under disease conditions.

The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiation.

Multiple sclerosis (MS) is a highly prevalent demyelinating autoimmune condition; the mechanisms regulating its severity and progression are unclear. The IL-17-producing Th17 subset of T cells has been widely implicated in MS and in the mouse model, experimental autoimmune encephalomyelitis (EAE). However, the differentiation and regulation of Th17 cells during EAE remain incompletely understood. Although evidence is mounting that the antimicrobial peptide cathelicidin profoundly affects early T cell differentiation, no studies have looked at its role in longer-term T cell responses. Now, we report that cathelicidin drives severe EAE disease. It is released from neutrophils, microglia, and endothelial cells throughout disease; its interaction with T cells potentiates Th17 differentiation in lymph nodes and Th17 to exTh17 plasticity and IFN-γ production in the spinal cord. As a consequence, mice lacking cathelicidin are protected from severe EAE. In addition, we show that cathelicidin is produced by the same cell types in the active brain lesions in human MS disease. We propose that cathelicidin exposure results in highly activated, cytokine-producing T cells, which drive autoimmunity; this is a mechanism through which neutrophils amplify inflammation in the central nervous system.

Antileishmanial Activity of Cathelicidin and its Modulation by Leishmania donovani in a cAMP Response Element Modulator-Dependent Manner in Infection.

Concerns regarding toxicity and resistance of current drugs in visceral leishmaniasis have been reported. Antimicrobial peptides are considered to be promising candidates and among them human cathelicidin hCAP18/LL-37 showed significant parasite killing on drug-sensitive and resistant Leishmania promastigotes, in addition to its apoptosis-inducing role. Administration of hCAP18/LL-37 to infected macrophages also decreased parasite survival and increased the host favorable cytokine interleukin 12. However, 1,25-dihydroxyvitamin D3 (vitamin D3)-induced endogenous hCAP18/LL-37 production was hampered in infected THP-1 cells. Infection also suppressed the vitamin D3 receptor (VDR), transcription factor of hCAP18/LL-37. cAMP response element modulator (CREM), the repressor of VDR, was induced in infection, resulting in suppression of both VDR and cathelicidin expression. PGE2/cAMP/PKA axis was found to regulate CREM induction during infection and silencing CREM in infected cells and BALB/c mice led to decreased parasite survival. This study documents the antileishmanial potential of cathelicidin and further identifies CREM as a repressor of cathelicidin in Leishmania infection.

Cathelicidin boosts the antifungal activity of neutrophils and improves prognosis during Aspergillus fumigatus keratitis.

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.

Bacterial pneumonia-induced shedding of epithelial heparan sulfate inhibits the bactericidal activity of cathelicidin in a murine model.

Bacterial pneumonia is a common clinical syndrome leading to significant morbidity and mortality worldwide. In the current study, we investigate a novel, multidirectional relationship between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Using an in vivo pneumonia model, we demonstrate that highly sulfated heparan sulfate (HS) oligosaccharides are shed into the airspaces in response to MRSA pneumonia. In vitro, these HS oligosaccharides do not directly alter MRSA growth or gene transcription. However, in the presence of an antimicrobial peptide (cathelicidin), increasing concentrations of HS inhibit the bactericidal activity of cathelicidin against MRSA as well as other nosocomial pneumonia pathogens (Klebsiella pneumoniae and Pseudomonas aeruginosa) in a dose-dependent manner. Surface plasmon resonance shows avid binding between HS and cathelicidin with a dissociation constant of 0.13 µM. These findings highlight a complex relationship in which shedding of airspace HS may hamper host defenses against nosocomial infection via neutralization of antimicrobial peptides. These findings may inform future investigation into novel therapeutic targets designed to restore local innate immune function in patients suffering from primary bacterial pneumonia.NEW & NOTEWORTHY Primary Staphylococcus aureus pneumonia causes pulmonary epithelial heparan sulfate (HS) shedding into the airspace. These highly sulfated HS fragments do not alter bacterial growth or transcription, but directly bind with host antimicrobial peptides and inhibit the bactericidal activity of these cationic polypeptides. These findings highlight a complex local interaction between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of bacterial pneumonia.

LL37/self-DNA complexes mediate monocyte reprogramming.

LL37 alone and in complex with self-DNA triggers inflammatory responses in myeloid cells and plays a crucial role in the development of systemic autoimmune diseases, like psoriasis and systemic lupus erythematosus. We demonstrated that LL37/self-DNA complexes induce long-term metabolic and epigenetic changes in monocytes, enhancing their responsiveness to subsequent stimuli. Monocytes trained with LL37/self-DNA complexes and those derived from psoriatic patients exhibited heightened glycolytic and oxidative phosphorylation rates, elevated release of proinflammatory cytokines, and affected naïve CD4+ T cells. Additionally, KDM6A/B, a demethylase of lysine 27 on histone 3, was upregulated in psoriatic monocytes and monocytes treated with LL37/self-DNA complexes. Inhibition of KDM6A/B reversed the trained immune phenotype by reducing proinflammatory cytokine production, metabolic activity, and the induction of IL-17-producing T cells by LL37/self-DNA-treated monocytes. Our findings highlight the role of LL37/self-DNA-induced innate immune memory in psoriasis pathogenesis, uncovering its impact on monocyte and T cell dynamics.

Vitamin D triggers hCAP18/LL-37 production: Implications for LL-37-induced human osteoblast cytotoxicity.

The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3-4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 µM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.

Cathelicidin CATH-B1 Inhibits Pseudorabies Virus Infection via Direct Interaction and TLR4/JNK/IRF3-Mediated Interferon Activation.

Pseudorabies virus (PRV), the causative pathogen of Aujeszky's disease, is one of the most important pathogens threatening the global pig industry. Although vaccination has been used to prevent PRV infection, the virus cannot be eliminated in pigs. Thus, novel antiviral agents as complementary to vaccination are urgently needed. Cathelicidins (CATHs) are host defense peptides that play an important role in the host immune response against microbial infections. In the study, we found that the chemical synthesized chicken cathelicidin B1 (CATH-B1) could inhibit PRV regardless of whether CATH-B1 was added pre-, co-, or post-PRV infection in vitro and in vivo. Furthermore, coincubation of CATH-B1 with PRV directly inactivated virus infection by disrupting the virion structure of PRV and mainly inhibited virus binding and entry. Importantly, pretreatment of CATH-B1 markedly strengthened the host antiviral immunity, as indicated by the increased expression of basal interferon-ß (IFN-ß) and several IFN-stimulated genes (ISGs). Subsequently, we investigated the signaling pathway responsible for CATH-B1-induced IFN-ß production. Our results showed that CATH-B1 induced phosphorylation of interferon regulatory transcription factor 3 (IRF3) and further led to production of IFN-ß and reduction of PRV infection. Mechanistic studies revealed that the activation of Toll-like receptor 4 (TLR4), endosome acidification, and the following c-Jun N-terminal kinase (JNK) was responsible for CATH-B1-induced IRF3/IFN-ß pathway activation. Collectively, CATH-B1 could markedly inhibit PRV infection via inhibiting virus binding and entry, direct inactivation, and regulating host antiviral response, which provided an important theoretical basis for the development of antimicrobial peptide drugs against PRV infection. IMPORTANCE Although the antiviral activity of cathelicidins could be explained by direct interfering with the viral infection and regulating host antiviral response, the specific mechanism of cathelicidins regulating host antiviral response and interfering with pseudorabies virus (PRV) infection remains elusive. In this study, we investigated the multiple roles of cathelicidin CATH-B1 against PRV infection. Our study showed that CATH-B1 could suppress the binding and entry stages of PRV infection and direct disrupt PRV virions. Remarkably, CATH-B1 significantly increased basal interferon-ß (IFN-ß) and IFN-stimulated gene (ISG) expression levels. Furthermore, TLR4/c-Jun N-terminal kinase (JNK) signaling was activated and involved in IRF3/IFN-ß activation in response to CATH-B1. In conclusion, we elucidate the mechanisms by which the cathelicidin peptide direct inactivates PRV infection and regulates host antiviral IFN-ß signaling.

Cathelicidins Target HSP60 To Restrict CVB3 Transmission via Disrupting the Exosome and Reducing Cardiomyocyte Apoptosis.

Cathelicidin antimicrobial peptides (mouse, CRAMP; human, LL-37) have broad-spectrum antiviral activities against enveloped viruses, but their mechanisms of action against nonenveloped viruses remain to be elucidated. Coxsackievirus B3 (CVB3), a member of nonenveloped virus belonging to the Enterovirus genus of Picornaviridae, is an important pathogen of viral myocarditis and dilated cardiomyopathy. Here, we observed that cardiac CRAMP expression was significantly upregulated in mice after CVB3 infection. The administration of CRAMP or LL-37 markedly suppressed CVB3 infection in mice, and CRAMP deficiency increased the susceptibility of mice to CVB3. CRAMP and LL-37 inhibited CVB3 replication in primary cardiomyocytes. However, they did not inactivate CVB3 particles and did not regulate the response of cardiomyocytes against CVB3 infection. Intriguingly, they inhibited CVB3 transmission through the exosome, but not virus receptor. In detail, CRAMP and LL-37 directly induced the lysis of exosomes by interfering with exosomal heat shock protein 60 (HSP60) and then blocked the diffusion of exosomes to recipient cells and inhibited the establishment of productive infection by exosomes. In addition, the interaction of CRAMP and LL-37 with HSP60 simultaneously inhibited HSP60-induced apoptosis in cardiomyocytes and reduced HSP60-enhanced CVB3 replication. Our findings reveal a novel mechanism of cathelicidins against viral infection and provide a new therapeutic strategy for CVB3-induced viral myocarditis. IMPORTANCE The relative mechanisms that cathelicidin antimicrobial peptides use to influence nonenveloped virus infection are unclear. We show here that cathelicidin antimicrobial peptides (CRAMP and LL-37) directly target exosomal HSP60 to destroy exosomes, which in turn block the diffusion of exosomes to recipient cardiomyocytes and reduced HSP60-induced apoptosis, thus restricting coxsackievirus B3 infection. Our results provide new insights into the mechanisms cathelicidin antimicrobial peptides use against viral infection.

Insight into the inhibitory activity and mechanism of bovine cathelicidin BMAP 27 against Salmonella Typhimurium.

This study synthesized an antimicrobial peptide based on the bovine cathelicidin BMAP 27 sequence. It was found to have a broad spectrum of antibacterial activity, with exceptionally high activity against Salmonella. However, the antibacterial mechanism of BMAP 27 against Salmonella remains unclear. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of BMAP 27 against Salmonella enterica serovar Typhimurium were determined to be 2 µM and 4 µM, respectively. After treatment with 2 MIC of BMAP 27, the absorbance of DNA in centrifugal supernatant increased from 0.244 to 1.464, and that of protein rose from 0.174 to 0.774, respectively. BMAP 27 has compromised the cell membrane as observed through field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM), and confirmed by the propidium iodide (PI) test. The alkaline phosphatase (AKP) enzyme activity in the supernatant of the 2 MIC treatment group was 2.15 times higher than the control group, indicating extracellular membrane damage. BMAP 27 treatment increased intracellular ROS levels as tested by dichlorofluorescein diacetate (DCFH) staining. DNA interaction analysis revealed that BMAP 27 has a binding affinity towards DNA, causing its characteristic bands to disappear and peak intensity at 260 nm to reduce. Molecular docking identified its potential binding mode with DNA. The crystal violet biofilm staining results demonstrated that BMAP 27 inhibited S. Typhimurium biofilm formation by 43.1 % and cleared mature biofilms by 53.62 %. Confocal Laser scanning electron microscopy (CLSM) observed that BMAP 27 could kill bacteria within the biofilm and dislodge bacteria from the surface of glasses. Swimming tests identified that the motor capacity of S. Typhimurium was diminished by BMAP 27. By counting the total bacteria, BMAP 27 was revealed to exert bacteriostatic effects in chilled pork and orange juice, which might provide a basis for its application in the inhibition of Salmonella.

Pancreatic ß-Cells Limit Autoimmune Diabetes via an Immunoregulatory Antimicrobial Peptide Expressed under the Influence of the Gut Microbiota.

Antimicrobial peptides (AMPs) expressed by epithelial and immune cells are largely described for the defense against invading microorganisms. Recently, their immunomodulatory functions have been highlighted in various contexts. However how AMPs expressed by non-immune cells might influence autoimmune responses in peripheral tissues, such as the pancreas, is unknown. Here, we found that insulin-secreting ß-cells produced the cathelicidin related antimicrobial peptide (CRAMP) and that this production was defective in non-obese diabetic (NOD) mice. CRAMP administrated to prediabetic NOD mice induced regulatory immune cells in the pancreatic islets, dampening the incidence of autoimmune diabetes. Additional investigation revealed that the production of CRAMP by ß-cells was controlled by short-chain fatty acids produced by the gut microbiota. Accordingly, gut microbiota manipulations in NOD mice modulated CRAMP production and inflammation in the pancreatic islets, revealing that the gut microbiota directly shape the pancreatic immune environment and autoimmune diabetes development.

Gut-Pancreatic Axis AMPlified in Islets of Langerhans.

Microbially derived metabolites in the intestine regulate host immunity and impact disease pathophysiology in various organs. Sun et al. (2015) suggest a direct effect of microbial metabolites on pancreatic endocrine cells in regulating type 1 diabetes pathophysiology.

Mitochondrial DNA-LL-37 Complex Promotes Atherosclerosis by Escaping from Autophagic Recognition.

Atherosclerosis is a chronic inflammatory disease of arterial wall. Mitochondrial DNA (mtDNA) and human antimicrobial peptide LL-37 (Cramp in mice) are involved in atherosclerosis. Recently, mtDNA has been found to escape from autophagy and cause inflammation. Normally, mtDNA as an inflammatogenic factor cannot escape from autophagy and degradation by DNase II. In this study, we found elevated amounts of LL37-mtDNA complex in atherosclerotic plasma and plaques. The complex was resistant to DNase II degradation and escaped from autophagic recognition, leading to activation of Toll-like receptor 9 (TLR9)-mediated inflammatory responses. Mouse model studies indicated that Cramp-mtDNA complex aggravated atherosclerotic lesion formation in apolipoprotein E-deficient mice and antibody treatment against the complex alleviated the lesion. These findings suggest that the LL-37-mtDNA complex acts as a key mediator of atherosclerosis formation, and thus represents a promising therapeutic target.

Zika virus infection suppresses CYP24A1 and CAMP expression in human monocytes.

Monocytes are the primary targets of Zika virus (ZIKV) and are associated with ZIKV pathogenesis. Currently, there is no effective treatment for ZIKV infection. It is known that 1,25-dihydroxy vitamin D3 (VitD3) has strong antiviral activity in dengue virus-infected macrophages, but it is unknown whether VitD3 inhibits ZIKV infection in monocytes. We investigated the relationship between ZIKV infection and the expression of genes of the VitD3 pathway, as well as the inflammatory response of infected monocytes in vitro. ZIKV replication was evaluated using a plaque assay, and VitD3 pathway gene expression was analyzed by RT-qPCR. Pro-inflammatory cytokines/chemokines were quantified using ELISA. We found that VitD3 did not suppress ZIKV replication. The results showed a significant decrease in the expression of vitamin D3 receptor (VDR), cytochrome P450 family 24 subfamily A member 1 (CYP24A1), and cathelicidin antimicrobial peptide (CAMP) genes upon ZIKV infection. Treatment with VitD3 was unable to down-modulate production of pro-inflammatory cytokines, except TNF-α, and chemokines. This suggests that ZIKV infection inhibits the expression of VitD3 pathway genes, thereby preventing VitD3-dependent inhibition of viral replication and the inflammatory response. This is the first study to examine the effects of VitD3 in the context of ZIKV infection, and it has important implications for the role of VitD3 in the control of viral replication and inflammatory responses during monocyte infection.

Human Cathelicidin Peptide LL-37 Induces Cell Death in Autophagy-Dysfunctional Endothelial Cells.

Human cathelicidin LL-37 is an antimicrobial peptide that has a broad spectrum of antimicrobial activities but also acts on host cells to exert immunomodulatory functions. It has been suggested that the increase of LL-37 in atherosclerotic aortas and the dysregulated autophagy of endothelial cells are involved in the pathogenesis of atherosclerosis. In this study, to elucidate the role of LL-37 in atherosclerosis, we investigated the effect of LL-37 on autophagy in endothelial cells using HUVECs. First, LL-37 upregulated LC3-II (an autophagosomal membrane marker) and enhanced the formation of LC3-positive puncta in the cells, suggesting that LL-37 induces autophagy in endothelial cells. Second, LL-37 was associated with p62, which recognizes ubiquitinated proteins and transfers them to autophagosomes, suggesting that LL-37 is ubiquitinated and recognized by p62. Third, the degradation of LL-37 was delayed, and LL-37 induced cell death in atg7 knockdown cells, which was accompanied by the formation of protein aggregates in the cells. Taken together, these observations suggest that LL-37 induces autophagy in endothelial cells but enhances cell death in autophagy-dysfunctional conditions, in which the intracellular degradation of LL-37 is disturbed. Thus, LL-37 may exert an adverse action on autophagy-dysfunctional endothelial cells to induce cell death in the pathogenesis of atherosclerosis.