Antimicrobial Peptides and Their Anti-Leishmanial Efficacies on Leishmania tropica Promastigotes In vitro.

OBJECTIVE: Antimicrobial resistance is a real threat to humanity. Pentavalent antimonials are reported non-effective in leishmaniasis treatment today, in countries like India. New treatment options have been assessed worldwide lately. Antimicrobial peptides (AMP) are the leading antibiotic candidates due to their large spectrum, fast efficacy, and low resistance risks. Cathelicidins are the AMP with well-documented antimicrobial activities against bacteria, fungi, and protozoa, over their positively charged membranes. Here, we aim to design cathelicidine-like helical peptides (CLHP), and compare their anti-Leishmanial efficacies in vitro, with meglumine antimoniate (MA) on Leishmania tropica. METHODS: A total of five study [TN-1-5] and two control (MA and non-drug) groups were formed. Cryopreserved L. tropica isolate was thawed and cultivated in Novy-MacNeal-Nicolle medium and then in RPMI. Five different CLHPs (TN1-5) were diluted in dimethyl sulphoxide. A total of 150 uL of CLHPs and MA were added into the first wells of the test plaques, followed by serial dilutions that revealed doses within 4 and 512 ug/mL. Then, 100 uL of cultures including 1x108/mL of L. tropica promastigotes were added into each well. Viability of promastigotes was checked with XTT, while the parasite count was assessed at 24th and 48th hours. RESULTS: TN3 was effective at 32 ug/mL. All tested CLHPs exhibited varying degrees of anti-Leishmanial activities, except TN5, even at its highest dose. CONCLUSION: TN3 showed a particular efficacy against L. tropica in vitro. Further studies including in vivo testing of the candidate's both efficacy and toxicity are essential.

Evaluation of Novel HLM Peptide Activity and Toxicity against Planktonic and Biofilm Bacteria: Comparison to Standard Antibiotics.

BACKGROUND: Antibiotic resistance is one of the main concerns of public health, and the whole world is trying to overcome such a challenge by finding novel therapeutic modalities and approaches. This study has applied the sequence hybridization approach to the original sequence of two cathelicidin natural parent peptides (BMAP-28 and LL-37) to design a novel HLM peptide with broad antimicrobial activity. METHODS: The physicochemical characteristics of the newly designed peptide were determined. As well, the new peptide's antimicrobial activity (Minimum Inhibitory Concentration (MIC), Minimum Bacterial Eradication Concentration (MBEC), and antibiofilm activity) was tested on two control (Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922) and two resistant (Methicillin-resistant Staphylococcus aureus (MRSA) ATCC BAA41, New Delhi metallo-beta- lactamase-1 Escherichia coli ATCC BAA-2452) bacterial strains. Furthermore, synergistic studies have been applied to HLM-hybridized peptides with five conventional antibiotics by checkerboard assays. Also, the toxicity of HLM-hybridized peptide was studied on Vero cell lines to obtain the IC50 value. Besides the percentage of hemolysis action, the peptide was tested in freshly heparinized blood. RESULTS: The MIC values for the HLM peptide were obtained as 20, 10, 20, and 20 µM, respectively. Also, the results showed no hemolysis action, with low to slightly moderate toxicity action against mammalian cells, with an IC50 value of 10.06. The Biomatik corporate labs, where HLM was manufactured, determined the stability results of the product by Mass Spectrophotometry (MS) and High-performance Liquid Chromatography (HPLC) methods. The HLM-hybridized peptide exhibited a range of synergistic to additive antimicrobial activities upon combination with five commercially available different antibiotics. It has demonstrated the biofilm-killing effects in the same concentration required to eradicate the control strains. CONCLUSION: The results indicated that HLM-hybridized peptide displayed a broad-spectrum activity toward different bacterial strains in planktonic and biofilm forms. It showed synergistic or additive antimicrobial activity upon combining with commercially available different antibiotics.

[Progress of researches on the antiparasitic activity of antimicrobial peptide LL-37].

Parasitic diseases caused by protozoan and helminth infections are still widespread across the world, notably in tropical and subtropical areas, which threaten the children and adult health. Long-term use of anti-parasitic drugs may result in reduced drug susceptibility and even drug resistance. Antimicrobial peptides have been demonstrated to inhibit parasite growth and development, which has potential antiparasitic values. LL-37, the only human antimicrobial peptide in the cathelicidin family, has been widely investigated. This paper reviews the progress of researches on the antiparasitic activity of LL-37, and discusses the prospects of LL-37 in the research of parasites.

Rational Design of a Potent Antimicrobial Peptide Based on the Active Region of a Gecko Cathelicidin.

The emergence of multidrug-resistant (MDR) bacteria presents a significant challenge to public health, increasing the risk of infections that are resistant to current antibiotic treatment. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics in the prevention of MDR bacterial infections. In the present study, we identified a novel cathelicidin AMP from Gekko japonicus, which exhibited broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging from 2.34 to 4.69 µg/mL. To improve its potential therapeutic application, a series of peptides was synthesized based on the active region of the gecko-derived cathelicidin. The lead peptide (RH-16) showed an antimicrobial activity comparable to that of the parent peptide. Structural characterization revealed that RH-16 adopted an amphipathic α-helical conformation. Furthermore, RH-16 demonstrated neither hemolytic nor cytotoxic activity but effectively killed a wide range of clinically isolated, drug-resistant bacteria. The antimicrobial activity of RH-16 was attributed to the nonspecific targeting of bacterial membranes, leading to rapid bacterial membrane permeabilization and rupture. RH-16 also retained its antibacterial activity in plasma and exhibited mild toxicity in vivo. Notably, RH-16 offered robust protection against skin infection in a murine model. Therefore, this newly identified cathelicidin AMP may be a strong candidate for future pharmacological development targeting multidrug resistance. The use of a rational design approach for isolating the minimal antimicrobial unit may accelerate the transition of natural AMPs to clinically applicable antibacterial agents.

Complexation of fungal extracellular nucleic acids by host LL-37 peptide shapes neutrophil response to Candida albicans biofilm.

Candida albicans remains the predominant cause of fungal infections, where adhered microbial cells form biofilms - densely packed communities. The central feature of C. albicans biofilms is the production of an extracellular matrix (ECM) consisting of polymers and extracellular nucleic acids (eDNA, eRNA), which significantly impedes the infiltration of host cells. Neutrophils, as crucial players in the innate host defense, employ several mechanisms to eradicate the fungal infection, including NETosis, endocytosis, or the release of granules containing, among others, antimicrobial peptides (AMPs). The main representative of these is the positively charged peptide LL-37 formed from an inactive precursor (hCAP18). In addition to its antimicrobial functions, this peptide possesses a propensity to interact with negatively charged molecules, including nucleic acids. Our in vitro studies have demonstrated that LL-37 contacting with C. albicans nucleic acids, isolated from biofilm, are complexed by the peptide and its shorter derivatives, as confirmed by electrophoretic mobility shift assays. We indicated that the generation of the complexes induces discernible alterations in the neutrophil response to fungal nucleic acids compared to the effects of unconjugated molecules. Our analyses involving fluorescence microscopy, flow cytometry, and Western blotting revealed that stimulation of neutrophils with DNA:LL-37 or RNA:LL-37 complexes hamper the activation of pro-apoptotic caspases 3 and 7 and fosters increased activation of anti-apoptotic pathways mediated by the Mcl-1 protein. Furthermore, the formation of complexes elicits a dual effect on neutrophil immune response. Firstly, they facilitate increased nucleic acid uptake, as evidenced by microscopic observations, and enhance the pro-inflammatory response, promoting IL-8 production. Secondly, the complexes detection suppresses the production of reactive oxygen species and attenuates NETosis activation. In conclusion, these findings may imply that the neutrophil immune response shifts toward mobilizing the immune system as a whole, rather than inactivating the pathogen locally. Our findings shed new light on the intricate interplay between the constituents of the C. albicans biofilm and the host's immune response and indicate possible reasons for the elimination of NETosis from the arsenal of the neutrophil response during contact with the fungal biofilm.

Cathelicidin LL-37 promotes wound healing in diabetic mice by regulating TFEB-dependent autophagy.

Diabetic patients often experience impaired wound healing. Human cathelicidin LL-37 possesses various biological functions, such as anti-microbial, anti-inflammatory, and pro-wound healing activities. Autophagy has important effects on skin wound healing. However, little is known about whether LL-37 accelerates diabetic wound healing by regulating autophagy. In the study, we aimed to investigate the role of autophagy in LL-37-induced wound healing and uncover the underlying mechanisms involved. A full-thickness wound closure model was established in diabetic mice to evaluate the effects of LL-37 and an autophagy inhibitor (3-MA) on wound healing. The roles of LL-37 and 3-MA in regulating keratinocyte migration were assessed using transwell migration and wound healing assays. The activation of transcription factor EB (TFEB) was measured using western blotting and immunofluorescence (IF) assays of its nuclear translocation. The results showed that LL-37 treatment improved wound healing in diabetic mice, whereas these effects were reversed by 3-MA. In vitro, 3-MA decreased the effects of LL-37 on promoting HaCat keratinocyte migration in the presence of high glucose (HG). Mechanistically, LL-37 promoted TFEB activation and resulted in subsequent activation of autophagy, as evidenced by increased nuclear translocation of TFEB and increased expression of ATG5, ATG7, and beclin 1 (BECN1), whereas these changes were blocked by TFEB knockdown. As expected, TFEB knockdown damaged the effects of LL-37 on promoting keratinocyte migration. Collectively, these results suggest that LL-37 accelerates wound healing in diabetic mice by activating TFEB-dependent autophagy, providing new insights into the mechanism by which LL-37 promotes diabetic wound healing.

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.

LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides.

An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) Escherichia coli strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against Staphylococcus aureus, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae bacteria clinical isolates at a lower minimum inhibition concentration level (10-33 µM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50-100 µM). KEY POINTS: • The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker. • The peptide was expressed with the carrier proteins SmbP and CusF3H+. • The hybrid peptide shows antibacterial potency against clinical bacterial isolates.

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.

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.

A snake cathelicidin enhances transcription factor EB-mediated autophagy and alleviates ROS-induced pyroptosis after ischaemia-reperfusion injury of island skin flaps.

BACKGROUND AND PURPOSE: Ischaemia-reperfusion (I/R) injury is a major contributor to skin flap necrosis, which presents a challenge in achieving satisfactory therapeutic outcomes. Previous studies showed that cathelicidin-BF (BF-30) protects tissues from I/R injury. In this investigation, BF-30 was synthesized and its role and mechanism in promoting survival of I/R-injured skin flaps explored. EXPERIMENTAL APPROACH: Survival rate analysis and laser Doppler blood flow analysis were used to evaluate I/R-injured flap viability. Western blotting, immunofluorescence, TdT-mediated dUTP nick end labelling (TUNEL) and dihydroethidium were utilized to examine the levels of apoptosis, pyroptosis, oxidative stress, transcription factor EB (TFEB)-mediated autophagy and molecules related to the adenosine 5'-monophosphate-activated protein kinase (AMPK)-transient receptor potential mucolipin 1 (TRPML1)-calcineurin signalling pathway. KEY RESULTS: The outcomes revealed that BF-30 enhanced I/R-injured island skin flap viability. Autophagy, oxidative stress, pyroptosis and apoptosis were related to the BF-30 capability to enhance I/R-injured flap survival. Improved autophagy flux and tolerance to oxidative stress promoted the inhibition of apoptosis and pyroptosis in vascular endothelial cells. Activation of TFEB increased autophagy and inhibited endothelial cell oxidative stress in I/R-injured flaps. A reduction in TFEB level led to a loss of the protective effect of BF-30, by reducing autophagy flux and increasing the accumulation of reactive oxygen species (ROS) in endothelial cells. Additionally, BF-30 modulated TFEB activity via the AMPK-TRPML1-calcineurin signalling pathway. CONCLUSION AND IMPLICATIONS: BF-30 promotes I/R-injured skin flap survival by TFEB-mediated up-regulation of autophagy and inhibition of oxidative stress, which may have possible clinical applications.

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.

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.

Antifungal properties of cathelicidin LL-37: current knowledge and future research directions.

The threat of fungal diseases is substantially underestimated worldwide, but they have serious consequences for humans, animals, and plants. Given the limited number of existing antifungal drugs together with the emergence of drug-resistant strains, many researchers have actively sought alternatives or adjuvants to antimycotics. The best way to tackle these issues is to unearth potential antifungal agents with new modes of action. Antimicrobial peptides are being hailed as a promising source of novel antimicrobials since they exhibit rapid and broad-spectrum microbicidal activities with a reduced likelihood of developing drug resistance. Recent years have witnessed an explosion in knowledge on microbicidal activity of LL-37, the sole human cathelicidin. Herein, we provide a summary of the current understanding about antifungal properties of LL-37, with particular emphasis on its molecular mechanisms. We further illustrate fruitful areas for future research. LL-37 is able to inhibit the growth of clinically and agronomically relevant fungi including Aspergillus, Candida, Colletotrichum, Fusarium, Malassezia, Pythium, and Trichophyton. Destruction of the cell wall integrity, membrane permeabilization, induction of oxidative stress, disruption of endoplasmic reticulum homeostasis, formation of autophagy-like structures, alterations in expression of numerous fungal genes, and inhibition of cell cycle progression are the key mechanisms underlying antifungal effects of LL-37. Burgeoning evidence also suggests that LL-37 may act as a potential anti-virulence peptide. It is hoped that this review will not only motivate researchers to conduct more detailed studies in this field, but also inspire further innovations in the design of LL-37-based drugs for the treatment of fungal infections.

Niacinamide enhances cathelicidin mediated SARS-CoV-2 membrane disruption.

The continual emergence of SARS-CoV-2 variants threatens to compromise the effectiveness of worldwide vaccination programs, and highlights the need for complementary strategies for a sustainable containment plan. An effective approach is to mobilize the body's own antimicrobial peptides (AMPs), to combat SARS-CoV-2 infection and propagation. We have found that human cathelicidin (LL37), an AMP found at epithelial barriers as well as in various bodily fluids, has the capacity to neutralise multiple strains of SARS-CoV-2. Biophysical and computational studies indicate that LL37's mechanism of action is through the disruption of the viral membrane. This antiviral activity of LL37 is enhanced by the hydrotropic action of niacinamide, which may increase the bioavailability of the AMP. Interestingly, we observed an inverse correlation between LL37 levels and disease severity of COVID-19 positive patients, suggesting enhancement of AMP response as a potential therapeutic avenue to mitigate disease severity. The combination of niacinamide and LL37 is a potent antiviral formulation that targets viral membranes of various variants and can be an effective strategy to overcome vaccine escape.

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.

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.

Tree Frog-Derived Cathelicidin Protects Mice against Bacterial Infection through Its Antimicrobial and Anti-Inflammatory Activities and Regulatory Effect on Phagocytes.

Due to excessive use or abuse in the food industry, agriculture, and medicine, many pathogens are developing resistance against conventional antibiotics. Antimicrobial peptides (AMPs) hold promise as effective therapeutic options for the treatment of bacterial infections. Herein, a novel cathelicidin antimicrobial peptide (Zs-CATH) was identified from the tree frog Zhangixalus smaragdinus. Zs-CATH mainly adopted an amphipathic ß-sheet structure in a membrane-mimetic environment. It showed broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria in vitro and significantly protected mice from lethal infections induced by Gram-negative bacteria Escherichia coli ATCC 25922 or Gram-positive bacteria Staphylococcus aureus ATCC 25923 in vivo. In addition, Zs-CATH exerted a strong anti-inflammatory effect by neutralizing lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and promoting macrophage M2 polarization, thus inhibiting the secretion of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) and enhancing the production of M2 macrophage markers IL-10, IL-4, and CD206. The MAPK and NF-κB inflammatory signaling pathways and transcriptional activator 6 (STAT6) were involved in this effect. In mice, Zs-CATH rapidly recruited neutrophils and monocytes/macrophages to the abdominal cavity but not T and B lymphocytes. Zs-CATH did not exhibit a direct chemoattractant effect on phagocytes but significantly promoted phagocyte migration in the presence of macrophages. Zs-CATH stimulated macrophages to secrete chemokines CXCL1, CXCL2, and CCL2, which mediated the recruitment of phagocytes. Furthermore, Zs-CATH promoted the production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs), which are oxygen-dependent and oxygen-independent mechanisms of the microbicidal activity of neutrophils, respectively. Zs-CATH exhibited no toxic side effects on mammalian cells and mice. These findings show that in addition to direct antibacterial activity, Zs-CATH also possesses the ability to modulate immune and inflammatory processes during bacterial infection, showing potential for development as anti-infective and/or anti-inflammatory agents.

Cathelicidin (LL-37) causes expression of inflammatory factors in coronary artery endothelial cells of Kawasaki disease by activating TLR4-NF-κB-NLRP3 signaling.

BACKGROUND: Kawasaki disease (KD) is a type of vasculitis with an unidentified etiology. Cathelicidin (LL-37) may be involved in the development of the KD process; therefore, further research to investigate the molecular mechanism of LL-37 involvement in KD is warranted. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, NLRP3, and LL-37 in the sera of healthy subjects, children with KD, and children with pneumonia. Subsequently, human recombinant LL-37 or/and toll-like receptors 4 (TLR4)-specific inhibitor TAK-242 stimulated human coronary artery endothelial cells (HCAECs), CCK-8 was used to detect cell proliferation, flow cytometry to detect apoptosis, transmission electron microscopy to observe cytoskeletal changes, Transwell to measure cell migration ability, ELISA to detect inflammatory factor levels, Western blot analysis to analyze protein levels of toll-like receptors 4 (TLR4) and NF-κB p-65, and quantitative real-time polymerase chain reaction (qRT-PCR) to determine LL-37, NLRP3 mRNA levels. RESULTS: In this study, we found that the level of LL-37 was highly expressed in the serum of children with KD, and after LL-37 stimulation, apoptosis was significantly increased in HCAECs, and the expression levels of TLR4, NLRP3 and inflammatory factors in cells were significantly enhanced. Intervention with the TLR4-specific inhibitor TAK-242 significantly alleviated the LL-37 effects on cellular inflammation, TLR4, NLRP3 promotion effect. CONCLUSIONS: Our data suggest that LL-37 induces an inflammatory response in KD coronary endothelial cells via TLR4-NF-κB-NLRP3, providing a potential target for the treatment of KD.

Epithelial-Immune Cell Crosstalk Determines the Activation of Immune Cells In Vitro by the Human Cathelicidin LL-37 at Low Physiological Concentrations.

The only human cathelicidin, LL-37, is a host defense antimicrobial peptide with antimicrobial activities against protozoans, fungi, Gram(+) and Gram(-) bacteria, and enveloped viruses. It has been shown in experiments in vitro that LL-37 is able to induce the production of various inflammatory and anti-inflammatory cytokines and chemokines by different human cell types. However, it remains an open question whether such cytokine induction is physiologically relevant, as LL-37 exhibited its immunomodulatory properties at concentrations that are much higher (>20 µg/mL) than those observed in non-inflamed tissues (1-5 µg/mL). In the current study, we assessed the permeability of LL-37 across the Caco-2 polarized monolayer and showed that this peptide could pass through the Caco-2 monolayer with low efficiency, which predetermined its low absorption in the gut. We showed that LL-37 at low physiological concentrations (<5 µg/mL) was not able to directly activate monocytes. However, in the presence of polarized epithelial monolayers, LL-37 is able to activate monocytes through the MAPK/ERK signaling pathway and induce the production of cytokines, as assessed by a multiplex assay at the protein level. We have demonstrated that LL-37 is able to fulfill its immunomodulatory action in vivo in non-inflamed tissues at low physiological concentrations. In the present work, we revealed a key role of epithelial-immune cell crosstalk in the implementation of immunomodulatory functions of the human cathelicidin LL-37, which might shed light on its physiological action in vivo.