Multi-omics resources for the Australian southern stuttering frog (Mixophyes australis) reveal assorted antimicrobial peptides.

The number of genome-level resources for non-model species continues to rapidly expand. However, frog species remain underrepresented, with up to 90% of frog genera having no genomic or transcriptomic data. Here, we assemble the first genomic and transcriptomic resources for the recently described southern stuttering frog (Mixophyes australis). The southern stuttering frog is ground-dwelling, inhabiting naturally vegetated riverbanks in south-eastern Australia. Using PacBio HiFi long-read sequencing and Hi-C scaffolding, we generated a high-quality genome assembly, with a scaffold N50 of 369.3 Mb and 95.1% of the genome contained in twelve scaffolds. Using this assembly, we identified the mitochondrial genome, and assembled six tissue-specific transcriptomes. We also bioinformatically characterised novel sequences of two families of antimicrobial peptides (AMPs) in the southern stuttering frog, the cathelicidins and ß-defensins. While traditional peptidomic approaches to peptide discovery have typically identified one or two AMPs in a frog species from skin secretions, our bioinformatic approach discovered 12 cathelicidins and two ß-defensins that were expressed in a range of tissues. We investigated the novelty of the peptides and found diverse predicted activities. Our bioinformatic approach highlights the benefits of multi-omics resources in peptide discovery and contributes valuable genomic resources in an under-represented taxon.

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.

Effective Healing of Staphylococcus aureus-Infected Wounds in Pig Cathelicidin Protegrin-1-Overexpressing Transgenic Mice.

Antimicrobial peptides (AMPs) are promising alternatives to existing treatments for multidrug-resistant bacteria-infected wounds. Therefore, the effect of protegrin-1 (PG1), a potent porcine AMP with broad-spectrum activity, on wound healing was evaluated. PG1-overexpressing transgenic mice were used as an in vivo model to evaluate its healing efficiency against Staphylococcus aureus-infected (106 colony forming units) wounds. We analyzed the wounds under four specific conditions in the presence or absence of antibiotic treatment. We observed the resolution of bacterial infection and formation of neo-epithelium in S. aureus-infected wounds of the mice, even without antibiotic treatment, whereas all wild-type mice with bacterial infection died within 8 to 10 days due to uncontrolled bacterial proliferation. Interestingly, the wound area on day 7 was smaller (p < 0.01) in PG1 transgenic mice than that in the other groups, including antibiotic-treated mice, suggesting that PG1 exerts biological effects other than bactericidal effect. Additionally, we observed that the treatment of primary epidermal keratinocytes with recombinant PG1 enhanced cell migration in in vitro scratch and cell migration assays. This study contributes to the understanding of broad-spectrum endogenous cathelicidins with potent antimicrobial activities, such as PG1, on wound healing. Furthermore, our findings suggest that PG1 is a potent therapeutic candidate for wound healing.

The LL-37 domain: A clue to cathelicidin immunomodulatory response?

Host defense peptides (HDPs) are naturally occurring polypeptide sequences that, in addition to being active against bacteria, fungi, viruses, and other parasites, may stimulate immunomodulatory responses. Cathelicidins, a family of HDPs, are produced by diverse animal species, such as mammals, fish, birds, amphibians, and reptiles, to protect them against pathogen infections. These peptides have variable C-terminal domains responsible for their antimicrobial and immunomodulatory activities and a highly conserved N-terminal pre-pro region homologous to cathelin. Although cathelicidins are the major components of innate immunity, the molecular basis by which they induce an immune response is still unclear. In this review, we will address the role of the LL-37 domain and its SK-24, IV-20, FK-13 and LL-37 fragments in the immunity response. Other cathelicidins also share structural and functional characteristics with the LL-37 domain, suggesting that these fragments may be responsible for interaction between these peptides and receptors in humans. Fragments of the LL-37 domain can give us clues about how homologous cathelicidins, in general, induce an immune response.

Expression, purification and investigation of antibacterial activity of a novel hybrid peptide LL37/hBD-129 by applied comprehensive computational and experimental approaches.

Antibiotic-resistant pathogens have become a great universal health concern. Antimicrobial peptides (AMPs) are small amphipathic and cationic polypeptides with high therapeutic potential against various microorganisms containing drug-resistant strains. Two major groups of these peptides, which have antibacterial activity against Gram-positive and Gram-negative bacteria, antiviral activity, and even antifungal activity, are defensins and cathelicidins. Hybridization of various AMPs is an appropriate approach to achieving new fusion AMPs with high antibacterial activity but low cellular toxicity. In the current research, the amino-acid sequence of human cathelicidin LL-37 (2-31) and Human beta-defensin (hBD)-129 were combined, and the fusion protein was evaluated by bioinformatics tool. The designed AMP gene sequence was commercially synthesized and cloned in the pET-28a expression vector. The LL-37/hBD-129 fusion protein was expressed in E.coli BL21-gold (DE3). The expression of the recombinant protein was evaluated using the SDS-PAGE method. The LL37/hBD-129 was successfully expressed as a recombinant hybrid AMP in E.coli BL21-gold (DE3) strain. Purification of the expressed AMP was performed by Ni-NTA column affinity chromatography, and the purified AMP was validated using the Western blot technic. Finally, the antimicrobial activity of the fusion AMP against Staphylococcus aureus and Escherichia coli bacteria was assessed. Based on the in silico analysis and experimental evaluations, the fusion AMP showed a significant antimicrobial effect on E. coli and Staphylococcus aureus bacteria.

High-Throughput CAMP Assay (HiTCA): A Novel Tool for Evaluating the Vitamin D-Dependent Antimicrobial Response.

Vitamin D is known to modulate human immune responses, and vitamin D deficiency is associated with increased susceptibility to infection. However, what constitutes sufficient levels or whether vitamin D is useful as an adjuvant therapeutic is debated, much in part because of inadequate elucidation of mechanisms underlying vitamin D's immune modulatory function. Cathelicidin antimicrobial peptide (CAMP) has potent broad-spectrum activity, and the CAMP gene is regulated in human innate immune cells by active 1,25(OH)2D3, a product of hydroxylation of inactive 25(OH)D3 by CYP27B1-hydroxylase. We developed a CRISPR/Cas9-edited human monocyte-macrophage cell line containing the mCherry fluorescent reporter gene at the 3' end of the endogenous CAMP gene. The High Throughput CAMP Assay (HiTCA) developed here is a novel tool for evaluating CAMP expression in a stable cell line that is scalable for a high-throughput workflow. Application of HiTCA to serum samples from a small number of human donors (n = 10) showed individual differences in CAMP induction that were not fully accounted for by the serum vitamin D metabolite status of the host. As such, HiTCA may be a useful tool that can advance our understanding of the human vitamin D-dependent antimicrobial response, which is being increasingly appreciated for its complexity.

Evolutionary diversification of defensins and cathelicidins in birds and primates.

Divergent evolution for more than 310 million years has resulted in an avian immune system that is complex and more compact than that of primates, sharing much of its structure and functions. Not surprisingly, well conserved ancient host defense molecules, such as defensins and cathelicidins, have diversified over time. In this review, we describe how evolution influenced the host defense peptides repertoire, its distribution, and the relationship between structure and biological functions. Marked features of primate and avian HDPs are linked to species-specific characteristics, biological requirements, and environmental challenge.

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.

Cathelicidins and defensins antimicrobial host defense peptides in the treatment of TB and HIV: Pharmacogenomic and nanomedicine approaches towards improved therapeutic outcomes.

Tuberculosis (TB) and human immunodeficiency virus (HIV) represent a significant burden of disease on a global scale. Despite improvements in the global epidemic status, largely facilitated by increased access to pharmacotherapeutic interventions, slow progress in the development of new clinical interventions coupled with growing antimicrobial resistance to existing therapies represents a global health crisis. There is an urgent need to expand the armamentarium of TB and HIV therapeutic strategies. Host mediated immune responses represent an untapped reservoir of novel approaches for TB and HIV. Antimicrobial peptides (AMPs) are an essential aspect of the immune system. Cathelicidins and defensins AMPs have been studied for their potential applications in TB and HIV therapeutic interventions. Genetic polymorphism across different population groups may affect endogenous expression or activity of AMPs, potentially influencing therapeutic outcomes. However, certain genetic polymorphisms in autophagy pathways may alter the downstream effects of nano-delivery of cathelicidin. On the other hand, certain genetic polymorphisms in beta-defensins may provide a protective role in reducing HIV-1 mother-to-child-transmission. Pharmaceutical development of cathelicidins and defensins is disadvantaged with complex challenges. Nanoparticle formulations improve pharmacokinetics and biocompatibility while facilitating targeted drug delivery, potentially minimising the risk of immunogenicity or non-specific haemolytic activity. This review aims to explore the potential viability of using cathelicidins and defensins as novel pharmacotherapy in the management of TB and HIV, highlight potential pharmacogenomic implications in host mediated immunity and AMP therapeutic applications, as well as propose novel drug delivery strategies represented by nanomedicine for AMPs.

Sequence polymorphisms of PR39 cathelicidins and extensive copy variations in commercial pig breeds.

Copy number polymorphisms (CNPs) of antimicrobial peptides (AMPs) in livestock can influence the innate immune response of individuals. We conducted a high-resolution analysis of the genomic variations of porcine cathelicidin PR39 using cloned PR39 amplicons corresponding to the 5' untranslated region (UTR) to 3' UTR from four individuals of three different pig breeds. We identified 15 different sequences corresponding to 9 different coding domain sequences (CDSs), encoding 7 different protein sequences consisting of 3 functional and 4 non-functional forms. Subsequently, we developed a PR39 CNP typing method using real-time polymerase chain reaction (PCR) and analyzed the PR39 copy numbers from 44 pigs of six breeds. Significant variations in PR39 copies ranging from 2 to 10 copies, with a mean copy number of 5, were observed among all commercial breeds, except the wild boar. Among the different breeds, the PR39 copy number was highest (10) in Korean native pigs. Gene expression analysis showed that PR39 expression was correlated with the copy number. Moreover, the comparative analysis of the cathelicidin cluster-containing region among eight mammalian species showed the complete evolutionary conservation of the region, except for differences in the degree of cathelicidin expansion in each species. Therefore, characterization of CNPs in AMP genes could aid in improving the genetic potential of innate immune responses in livestock animals.

Indolicidin revisited: biological activity, potential applications and perspectives of an antimicrobial peptide not yet fully explored.

The emergence of multidrug-resistant bacteria, viruses and tumors is a serious threat to public health. Among natural peptides, indolicidin, a 13-residue peptide belonging to the cathelicidin family, deserves special attention. Indolicidin has a broad spectrum of biological activity and is active against a wide range of targets, such as bacteria (Gram+ and Gram-), fungi and viruses. Here, we review the most important features of the biological activity, potential applications and perspectives of indolicidin and its analogs. Although not yet approved for commercialization, this peptide has great potential to be applied in different areas, including the medical, biomedical, food industry and other unexplored areas. To achieve this goal, a multidisciplinary team of researchers must work together to fine tune peptides that overall lead to novel analogs and formulations to combat existing and possibly future diseases.

Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity.

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

Expression of mBD4, mBD3 and CRAMP during type II collagen-induced arthritis/CIA and their association with inflammation and bone-remodeling markers.

The aim of this study was to analyze the expression of mBD4, mBD3 and CRAMP in joint of mice with type II collagen-induced arthritis/CIA and to explore its possible association with IL-10, IL-4, IFN-γ, IL-17, MMP3, RANK/RANKL/OPG and histological parameters. METHODS: CIA was induced in 44 DBA/1 J mice. The joints from mice were classified into the onset, peak and remission phase of CIA. Histological sections were stained with hematoxylin-eosin and safranin O. The expression of CRAMP, mBD-3, mBD-4, and MMP-3 was evaluated using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The expression of IL-10, IL-4, IFN-γ, IL-17, RANK/RANKL/OPG was analyzed by RT-PCR. RESULTS: We observed that inflammation and immunostained cells for CRAMP increased in the peak and remission phases compared to the control group. In addition, increments in relative expressions of CRAMP were detected for the remission phase and in IL-4 and IL-17 in the peak phase compared to the control and onset phase. In addition, an increase in IL-10 in a peak phase compared to the control, as well as the relative expression of IFN-γ in remission phase was higher than in the onset phase. This was accompanied by an increase in cartilage damage in the peak phase compared to the control. Cells immunostained to MMP3 increased in the peak phase compared to the onset and control group, and relative expression of MMP3 was detected in the peak phase compared to the onset, remission, and control group. We observed that the relative expression of RANK and RANKL in the peak phase was higher than in control and onset phase. Finally, the relative expression of OPG in the peak phase compared to the onset, remission, and control group was detected. Regarding CRAMP behavior in the different phases studied, it was positively correlated with IL-4 and RANK, and showed a negative correlation with IFN-γ, IL-17, IL-10, RANKL, OPG and RANKL/OPG ratio in the control group. Also was positively correlated with IFN-γ, IL-17, IL-4, IL-10, as well as with RANK, RANKL, and OPG in the onset and peak phases of the CIA. In the peak phase, CRAMP showed a positive association with MMP3, and we observed a direct correlation between CRAMP and IFN-γ and RANKL/OPG ratio in remission phase. mBD3 correlates positively with IFN-γ, IL-17, IL-10, RANKL, OPG and RANKL/OPG ratio, and showed a negative correlation with CRAMP, MMP3, and RANK in the control group. Also, it was directly associated with IFN-γ, IL-17, IL-4, IL-10 and RANKL in the onset phase while it was inversely associated with CRAMP, MMP-3, RANK, RANKL, and OPG in the peak phase. Finally, mBD3 was inversely correlated with MMP3 in the remission phase and was directly associated with CRAMP, IFN-γ and RANKL/OPG ratio in this phase. mBD4 was directly associated with CRAMP, IFN-γ, IL-17, IL-4, IL-10, RANKL / OPG in the onset phase, and with CRAMP, IFN-γ, IL-17, IL-4, IL-10, MMP3, RANK, RANKL and OPG in the peak phase. Finally, mBD4 was positively associated with mBD3, IFN-γ, IL-17, IL-10, RANK, RANKL OPG and RANKL/OPG in the CIA remission phase. CONCLUSIONS: Our results demonstrate that CRAMP plays an important role in CIA progress and suggest that its abundance is associated with local pro- and anti-inflammatory status. This makes us propose CRAMP as a possible contributor of bone reconstruction in the last stage of CIA.

Long-term exposure to particulate matter from air pollution alters airway ß-defensin-3 and -4 and cathelicidin host defense peptides production in a murine model.

Epidemiological studies have associated long-term exposure to environmental air pollution particulate matter (PM) with the development of diverse health problems. They include infectious respiratory diseases related to the deregulation of some innate immune response mechanisms, such as the host defense peptides' expression. Herein, we evaluated in BALB/c mice the effect of long-standing exposure (60 days) to urban-PM from the south of Mexico City, with aerodynamic diameters below 2.5 µm (PM2.5) and 10 µm (PM10) on the lung's gene expression and production of three host defense peptides (HDPs); murine beta-defensin-3, -4 (mBD-3, mBD-4) and cathelin-related antimicrobial peptide (CRAMP). We also evaluated mRNA levels of Il1b and Il10, two cytokines related to the expression of host defense peptides. Exposure to PM2.5 and PM10 differentially induced lung inflammation, being PM2.5, which caused higher inflammation levels, probably associated with a differential deposition on the airways, that facilitate the interaction with alveolar macrophages. Inflammation levels were associated with an early upregulation of the three HDPs assessed and an increment in Il1b mRNA levels. Interestingly, after 28 days of exposure, Il10 mRNA upregulation was observed and was associated with the downregulation of HDPs and Il1b mRNA levels. The upregulation of Il10 mRNA and suppression of HDPs might facilitate microbial colonization and the development of diseases associated with long-term exposure to PM.

Bioinformatic Analysis of Genome-Predicted Bat Cathelicidins.

Bats are unique in their potential to serve as reservoir hosts for intracellular pathogens. Recently, the impact of COVID-19 has relegated bats from biomedical darkness to the frontline of public health as bats are the natural reservoir of many viruses, including SARS-Cov-2. Many bat genomes have been sequenced recently, and sequences coding for antimicrobial peptides are available in the public databases. Here we provide a structural analysis of genome-predicted bat cathelicidins as components of their innate immunity. A total of 32 unique protein sequences were retrieved from the NCBI database. Interestingly, some bat species contained more than one cathelicidin. We examined the conserved cysteines within the cathelin-like domain and the peptide portion of each sequence and revealed phylogenetic relationships and structural dissimilarities. The antibacterial, antifungal, and antiviral activity of peptides was examined using bioinformatic tools. The peptides were modeled and subjected to docking analysis with the region binding domain (RBD) region of the SARS-CoV-2 Spike protein. The appearance of multiple forms of cathelicidins verifies the complex microbial challenges encountered by these species. Learning more about antiviral defenses of bats and how they drive virus evolution will help scientists to investigate the function of antimicrobial peptides in these species.

Cathelicidin antimicrobial peptide (CAMP) gene promoter methylation induces chondrocyte apoptosis.

OBJECTIVE: The occurrence of osteoarthritis is related to genetic and environmental factors. Among them, the change of chondrocyte gene expression pattern regulated by epigenetic modification is an important participant. This study analyzed the effect of CAMP gene methylation on the level of oxidative stress and inflammation of chondrocytes. METHODS: We analyzed the changes of the transcriptome in the articular cartilage tissue of osteoarthritis (OA) patients from the GSE117999 dataset. The GSE48422 dataset was used to analyze the changes in the methylation level of osteoarthritis cells. Cell Counting Kit-8 (CCK-8) and flow cytometry analysis of short hairpin RNA (shRNA) silencing CAMP gene and 5-µM 5-Aza-2'-Deoxycytidine (AZA) treatment on the proliferation and apoptosis of Human chondrocytes osteoarthritis (HC-OA) cells. The Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was used to detect the level of reactive oxygen species (ROS), and the expression level of inflammatory factors was analyzed by Western Blot. RESULTS: The expression of CAMP in cartilage tissue of OA patients was upregulated, and the level of methylation was downregulated. CAMP was highly expressed in osteoarthritis articular cartilage cells. Silencing CAMP inhibited the proliferation of HC-OA cells and promoted their apoptosis. CAMP gene methylation inhibited ROS levels and tumor necrosis factor-α (TNF-α) expression levels in HC-OA cells, and promoted transforming growth factor beta (TGF-ß) expression. CAMP gene methylation inhibited the proliferation of HC-OA cells and promoted their apoptosis. CONCLUSION: CAMP gene promoter methylation inhibits ROS levels and inflammation and induces chondrocyte apoptosis.

Genetic diversity of Japanese quail cathelicidins.

Japanese quail is a low-fat, meat-bird species exhibiting high disease resistance. Cathelicidins (CATHs) are host defense peptides conserved across numerous vertebrate species that play an important role in innate immunity. The activity of host defense peptides can be affected by amino acid substitutions. However, no polymorphisms in avian CATH genes have been reported to date. The aim of this study was to clarify the polymorphisms in CATHs in Japanese quail. DNA for genomic analyses was extracted from the peripheral blood of 99 randomly selected quail from 6 inbred lines. A total of 6, 4, 6, and 4 CjCATH1, -2, -3, and -B1 alleles were identified, respectively. Nine haplotypes, including 4 strain-specific haplotypes, were identified by combining alleles at the CjCATH1, -2, -3, and -B1 loci. In addition, 2 and 1 amino acid substitutions (I145F, Q148H, and P245H) predicted by PROVEAN and PolyPhen-2 to have deleterious effects were detected in CjCATH2 and -B1, respectively. Synthetic CjCATH2 and -B1 peptides exhibited greater antibacterial activity against Escherichia coli than chicken CATH2 and -B1, respectively. Furthermore, the CjCATHB1∗04 peptide exhibited less potent antimicrobial activity than other CjCATHB1 peptides examined. This is the first report of amino acid substitutions accompanied by changes in antibacterial activity in avian CATHs. These findings could be employed as indicators of improvements in innate immune response in poultry.

LL-37-mediated activation of host receptors is critical for defense against group A streptococcal infection.

Group A Streptococcus (GAS) causes diverse human diseases, including life-threatening soft-tissue infections. It is accepted that the human antimicrobial peptide LL-37 protects the host by killing GAS. Here, we show that GAS extracellular protease ScpC N-terminally cleaves LL-37 into two fragments of 8 and 29 amino acids, preserving its bactericidal activity. At sub-bactericidal concentrations, the cleavage inhibits LL-37-mediated neutrophil chemotaxis, shortens neutrophil lifespan, and eliminates P2X7 and EGF receptors' activation. Mutations at the LL-37 cleavage site protect the peptide from ScpC-mediated splitting, maintaining all its functions. The mouse LL-37 ortholog CRAMP is neither cleaved by ScpC nor does it activate P2X7 or EGF receptors. Treating wild-type or CRAMP-null mice with sub-bactericidal concentrations of the non-cleavable LL-37 analogs promotes GAS clearance that is abolished by the administration of either P2X7 or EGF receptor antagonists. We demonstrate that LL-37-mediated activation of host receptors is critical for defense against GAS soft-tissue infections.

Regulation of CAMP (cathelicidin antimicrobial peptide) expression in adipocytes by TLR 2 and 4.

Recent data argue for a pro-inflammatory role of CAMP (cathelicidin antimicrobial peptide) in adipocytes and adipose tissue (AT) and for regulatory circuits involving TLRs. In order to investigate regulatory effects of TLR2 and TLR4, 3T3-L1 adipocytes were stimulated with TLR2 agonistic lipopeptide MALP-2 and with TLR4 agonist LPS in presence or absence of signal transduction inhibitors. CAMP gene expression was analysed by quantitative real-time PCR in adipocytes and in murine AT compartments and cellular subfractions. CAMP expression was higher in gonadal than in subcutaneous AT and there was a gender-specific effect with higher levels in males. Adipocytes had higher CAMP expression than the stroma-vascular cell (SVC) fraction. MALP-2 up-regulated CAMP expression significantly, mediated by STAT3 and PI3K and potentially (non-significant trend) by NF-κB and MAPK, but not by raf-activated MEK-1/-2. Moreover, LPS proved to act as a potent inducer of CAMP via NF-κB, PI3K and STAT3, whereas specific inhibition of MAPK and MEK-1/-2 had no effect. In conclusion, activation of TLR2 and TLR4 by classical ligands up-regulates adipocyte CAMP expression involving classical signal transduction elements. These might represent future drug targets for pharmacological modulation of CAMP expression in adipocytes, especially in the context of metabolic and infectious diseases.