Human Β-defensin Research Articles

The Antimicrobial Peptides Human β-Defensins Induce the Secretion of Angiogenin in Human Dermal Fibroblasts.

The skin produces a plethora of antimicrobial peptides that not only show antimicrobial activities against pathogens but also exhibit various immunomodulatory functions. Human β-defensins (hBDs) are the most well-characterized skin-derived antimicrobial peptides and contribute to diverse biological processes, including cytokine production and the migration, proliferation, and differentiation of host cells. Additionally, hBD-3 was recently reported to promote wound healing and angiogenesis, by inducing the expression of various angiogenic factors and the migration and proliferation of fibroblasts. Angiogenin is one of the most potent angiogenic factors; however, the effects of hBDs on angiogenin production in fibroblasts remain unclear. Here, we investigated the effects of hBDs on the secretion of angiogenin by human dermal fibroblasts. Both in vitro and ex vivo studies demonstrated that hBD-1, hBD-2, hBD-3, and hBD-4 dose-dependently increased angiogenin production by fibroblasts. hBD-mediated angiogenin secretion involved the epidermal growth factor receptor (EGFR), Src family kinase, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-kappa B (NF-κB) pathways, as evidenced by the inhibitory effects of specific inhibitors for these pathways. Indeed, we confirmed that hBDs induced the activation of the EGFR, Src, JNK, p38, and NF-κB pathways. This study identified a novel role of hBDs in angiogenesis, through the production of angiogenin, in addition to their antimicrobial activities and other immunomodulatory properties.

Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides.

The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. To improve the pMAL expression vector, we introduced the His6 tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His6 -Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His6 tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His6 and His6 -enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human β-defensin. We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory.

Anti-cancer Properties of Potential Probiotics and Their Cell-free Supernatants for the Prevention of Colorectal Cancer: an In Vitro Study.

This study aimed to characterize the anti-cancer properties of potential probiotics (Lacticaseibacillus paracasei SD1, Lacticaseibacillus rhamnosus SD4, Lacticaseibacillus rhamnosus SD11, and Lacticaseibacillus rhamnosus GG) and their cell-free supernatants (CFS) for the prevention of colorectal cancer (CRC), which including anti-bacterial and anti-inflammation activities against pathogens associated with CRC (Fusobacterium nucleatum, Porphyromonas gingivalis, ETEC, and Salmonella enterica). The expression of human β-defensin (2-4) and IL-10 after being stimulated with probiotics was also examined. In addition, anti-cancer activity of CFS and probiotic growth under intestinal conditions were determined. An in vitro study was conducted in the Caco-2 and HIEC-6 cells. Results showed that probiotic cells and their CFS displayed different antibacterial activity, and L. rhamnosus SD11 showed the strongest inhibition of the growth of pathogens. Additionally, both probiotic cell walls and their CFS suppressed pro-inflammatory cytokines after being stimulated with pathogens in Caco-2 and HIEC-6 cells. L. paracasei SD1 and L. rhamnosus SD11 showed significantly higher suppression levels than others and also both strains can stimulate highly expression of hBD (2-4) and IL-10. The CFS of L. paracasei SD1 and L. rhamnosus SD11 inhibited significantly high growth of Caco-2 cells but not much in HIEC-6 cells. Furthermore, all probiotics adhered to Caco-2 and HIEC-6 cells, and L. rhamnosus SD4 showed the highest adhesion to both cells. They could survive more than 70% in intestinal conditions. In conclusion, results indicate that potential probiotics tested exhibited various anti-cancer properties, which may be good candidates used as biotherapy for the prevention or to delay the progression of CRC.

Novel human skin surface antimicrobial peptide quantification method using a skin patch test chamber: A pilot study.

Antimicrobial peptides (AMPs) on the skin surface are related to the innate immunity of the skin in preventing external infection. Skin rinsing and tape stripping (TS) are acceptable methods for analyzing AMPs on the skin surface but have limitations, such as causing skin damage. In this study, we proposed a noninvasive method to measure AMPs on the skin surface with minimal skin damage. Using the patch test assay, we aimed to analyze the skin surface human β-defensin (hBDs) levels without damaging the skin barrier. The concentrations of hBDs on the skin surface were evaluated through the skin patch testing of 13 healthy subjects, and hBD-1 concentrations were compared with those obtained using the TS method in this proof-of-concept study. In addition, changes in skin physiology and concentration of hBDs under 1% sodium lauryl sulfate stimulation were monitored in 14 healthy subjects (8 young and 6 elderly subjects) for 150 h. The correlation between the two methods had a Pearson's coefficient of 0.640, and skin patch analysis led to a relatively less impaired barrier with no significant increase in transepidermal water loss after analysis. Age-specific comparisons suggested that higher skin surface hBD-2 concentrations were present in the young group as compared with the elderly group. Skin surface expression of hBD-2 after skin barrier disruption was also higher in the young group. Our findings show that skin patch analysis is a convenient method to analyze hBDs on the skin surface. hBDs are factors of innate immunity that can be used as an index to predict a decreased chemical immune response of skin due to aging.

Effects of potential probiotics on the expression of cytokines and human β-defensins in human gingival epithelial cells and in vivo efficacy in a dog model

ObjectivesThe aims of this study were to examine the effects of probiotic strains on the expression of cytokines and human β-defensins-2–4 in human gingival epithelial cells and to investigate the in vivo efficacy of probiotics in a dog model. DesignSelected probiotics, Lacticaseibacillus paracasei SD1, L. rhamnosus SD4, L. rhamnosus SD11 and Limosilactobacillus fermentum SD7, were examined for the expression of human β-defensins-2–4, and cytokine responses after stimulated by various periodontopathogens. Subsequently, an in vivo study was set as a randomized, double-blind, placebo-controlled intervention in a dog model. A total of 20 dogs with mild gingivitis were randomly assigned to either the probiotic or control group. The effects of probiotics on periodontopathogenic- and cytokine levels were analyzed after 4 weeks of intervention. ResultsIt showed that all probiotics could induce human β-defensins-2–4, interleukin-1β, interleukin-6, interleukin-8, and tumor necrosis factor-α expressions in human gingival epithelial cells; however, a significant difference had been found among the strains. When individual probiotic strain was combined to periodontopathogens, a significant reduction of IL-8 expression was found. Results of the in vivo study demonstrated that the bacterial levels, especially Porphyromonas gingivalis, and the interleukin-8 levels were significantly decreased after receiving the probiotic products compared to the controls. ConclusionThe results of the in vitro and in vivo studies in dogs are encouraging to support the effectiveness of a mixture of probiotic treatments for improvement of periodontal health by reducing periodontopathogens and interleukin-8 levels. Therefore, such probiotics preparation could possibly have therapeutic potential in human periodontitis.

Cyclic-di-GMP stimulates keratinocyte innate immune responses and attenuates methicillin-resistant Staphylococcus aureus colonization in a murine skin wound infection model

BackgroundStaphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model.ResultsStimulation of HaCaT cells with 125 μM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human β-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 μM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice.ConclusionsThese results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.

Enhanced In Vitro Expression of Filaggrin and Antimicrobial Peptides Following Application of Glycosaminoglycans and a Sphingomyelin-Rich Lipid Extract.

Filaggrin is an epidermal protein involved in skin barrier formation and hydration, whose expression is altered in canine atopic dermatitis (CAD). CAD patients also present an abnormal immune response with an altered expression of antimicrobial peptides (AMPs), such as β-defensins and cathelicidins. Sphingolipids and glycosaminoglycans (GAGs) have been reported to improve the skin barrier in several animal species, including dogs. Our objective was to evaluate the in vitro effects of a sphingomyelin-rich lipid extract (LE), a hyaluronic acid-rich GAG matrix, and their combination, on the expression of filaggrin and human β-defensin 2 (hBD-2). Filaggrin expression was quantified in a reconstructed human epidermis (RHE), and hBD-2 in normal human epidermal keratinocyte (NHEK) cultures. LE and GAGs were tested at 0.02 mg/mL, with or without adding a cytokine mix. A significant increase in mean hBD-2, compared to the control (99 pg/mL) was achieved with LE (138 pg/mL) and LE+GAGs (165 pg/mL). Filaggrin increased with GAGs (202% ± 83) and LE (193% ± 44) vs. the stimulated control, but this difference was statistically significant (p < 0.05) only with LE+GAGs (210% ± 39). In conclusion, the tested GAGs and LE enhance filaggrin and AMP expression in vitro, which might benefit CAD patients if applied in vivo.

S230: HUMAN ΒETA-DEFENSIN 2 TREATMENT MODULATES THE INTESTINAL MICROBIOME AND THE ALLOGENEIC T CELL RESPONSE TO LIMIT ACUTE GRAFT-VERSUS-HOST DISEASE

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy option for patients with hematological malignancies. Acute graft-versus-host disease (GVHD) is primarily driven by allogeneic donor T cells and remains a serious cause of morbidity and mortality post allo-HCT. In particular, acute GVHD of the gastrointestinal tract (GI GVHD) is associated with a high mortality rate and treatment options are limited. Human β-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide, which is induced by inflammatory stimuli and possesses both antimicrobial and immunomodulatory functions. Aims: In this study, we aimed to investigate the expression of beta defensins in human and murine GI GVHD and the therapeutic effect of recombinant hBD-2 on acute GVHD development in mouse models. Methods: To investigate the functional role of hBD-2 post allo-HCT, we employed established murine GVHD and graft-versus-leukemia (GVL) models and analyzed the allogeneic T cell response using transcriptome and kinome profiling. Shotgun metagenomic sequencing was used to examine the effect of hBD-2 on the intestinal microbiome. Furthermore, we studied beta-defensin expression in two independent acute GVHD patient cohorts and in mice with acute GVHD. Results: We found that expression of murine beta-defensin 4 (mBD-4), the murine orthologue for hBD-2, was reduced in the colon and ileum of mice developing acute GVHD. Oral treatment of mice with recombinant hBD-2 post allo-HCT reduced weight loss and acute GVHD severity and mortality. Furthermore, hBD-2 treatment affected the intestinal microbial composition, including a shift towards higher abundance of Bacteroides species in hBD-2 treated compared to vehicle treated mice. The changes in the microbiome resulted in reduced neutrophil infiltration of the ileum during acute GVHD induction in hBD-2 treated mice. Additionally, hBD-2 dampened pro-inflammatory Th1 cytokine production (TNF, IFN-gamma) by allogeneic T cells in vivo, while preserving the beneficial GVL effect in two different leukemia models. Mechanistically, oral hBD-2 treatment decreased alloreactive T cell infiltration and the expression of genes involved in T cell receptor (TCR) signaling in the ileum of mice with acute GVHD. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation and metabolism by reducing proximal TCR signaling. In patients with acute GI GVHD, intestinal hBD-2 expression was inadequately induced in response to inflammation at the mRNA and protein level when compared to healthy subjects and patients with ulcerative colitis. Summary/Conclusion: In conclusion, our study demonstrates that hBD-2 reduces acute GVHD severity, likely through its effects in shaping the intestinal microbiota, reducing neutrophil infiltration in the ileum and dampening allogeneic T cell responses. Both human and murine acute GVHD are characterized by a lack of intestinal beta-defensin induction and recombinant hBD-2 represents a potential novel therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Recombinant human β-defensin 2 delivery improves smoking-induced lung neutrophilia and bacterial exacerbation.

Treatment of the cigarette smoke-associated lung diseases, such as chronic obstructive pulmonary disease (COPD), has largely focused on broad-spectrum anti-inflammatory therapies. However, these therapies, such as high-dose inhaled corticosteroids, enhance patient susceptibility to lung infection and exacerbation. Our objective was to assess whether the cationic host defense peptide, human β-defensin 2 (hBD-2), can simultaneously reduce pulmonary inflammation in cigarette smoke-exposed mice while maintaining immune competence during bacterial exacerbation. Mice were exposed to cigarette smoke acutely (4 days) or chronically (5 days/wk for 7 wk) and administered hBD-2 intranasally or by gavage. In a separate model of acute exacerbation, chronically exposed mice treated with hBD-2 were infected with nontypeable Haemophilus influenzae before euthanasia. In the acute exposure model, cigarette smoke-associated pulmonary neutrophilia was significantly blunted by both local and systemic hBD-2 administration. Similarly, chronically exposed mice administered hBD-2 therapeutically exhibited reduced pulmonary neutrophil infiltration and downregulated proinflammatory signaling in the lungs compared with vehicle-treated mice. Finally, in a model of acute bacterial exacerbation, hBD-2 administration effectively limited neutrophil infiltration in the lungs while markedly reducing pulmonary bacterial load. This study shows that hBD-2 treatment can significantly attenuate lung neutrophilia induced by cigarette smoke exposure while preserving immune competence and promoting an appropriate host-defense response to bacterial stimuli.

Human β-defensin 2 (hBD-2) improves neutrophilia and bacterial clearance during cigarette smoke exposure

Abstract Rationale Treatment of cigarette smoke-associated lung diseases has largely focused on broad-spectrum anti-inflammatory therapies, such as inhaled corticosteroids. However, these therapies have recently been shown to enhance patient susceptibility to pneumonia. Objective Assess whether the anti-microbial peptide, human β-defensin 2 (hBD-2), can simultaneously reduce pulmonary inflammation in cigarette smoke-exposed mice while maintaining immune competence during bacterial exacerbation. Methods Mice were exposed to cigarette smoke acutely (4 days) or chronically (5 days/week for 7 weeks) and administered hBD-2 intranasally or by gavage. In a separate model of acute exacerbation, chronically exposed mice treated with hBD-2 were infected with non-typeable Haemophilus influenzae prior to sacrifice. Results In the acute exposure model, cigarette smoke-associated pulmonary neutrophilia was significantly blunted by both local and systemic hBD-2 administration. Similarly, chronically exposed mice administered hBD-2 therapeutically exhibited reduced pulmonary neutrophil infiltration and downregulated pro-inflammatory signaling in the lungs compared to vehicle-treated mice, as assessed via microarray. Finally, in a model of bacterial exacerbation, hBD-2 administration effectively limited neutrophil infiltration in the lungs while markedly reducing pulmonary bacterial load. Conclusions This study shows that hBD-2 treatment can significantly attenuate lung neutrophilia induced by cigarette smoke exposure while preserving immune competence and promoting an appropriate host-defense response to bacterial stimuli. Supported by CIHR

In-silico analysis of non-synonymous single nucleotide polymorphisms in human β-defensin type 1 gene reveals their impact on protein-ligand binding sites

Single nucleotide polymorphism (SNPs) is an important genetic biomarker to assess protein function and its possible contribution to genetic diseases, such as the β- defensin 1 gene (DEFB1)-associated non-synonymous SNPs (nsSNPs). Defensins are antimicrobial and immunomodulatory peptides, acting as part of innate immunity, and killing bacteria by interacting phosphatidylinositol 4,5-bisphosphate (PIP2). Therefore, we apply cutting-edge computational algorithms to identify detrimental SNPs in the DEFB1 gene that potentially impact PIP2 binding sites. Furthermore, 4 most important nsSNPs in the DEFB1 gene were discovered (C67S, T58S, G62W, and Y35C) and only two of them were found to be linked to the PIP2 binding site-forming residues (Thr58 and Tyr35). Additional molecular docking and molecular dynamics simulations confirmed the decreased binding affinity of DEFB1 to bacterial PIP2 due to these mutations. Overall, this computational study analyzing nsSNPs in DEFB1 provides more understanding of how these missense mutations could impair or change protein functions by altering the PIP2 binding site.

Human β-Defensin-3 is Associated With Platelet-Derived Extracellular Vesicles and is a Potential Contributor to Endothelial Dysfunction.

While platelets are the essential mediators of hemostasis, they are being increasingly recognized for their potential of contributing to host defenses. Here, using immunofluorescent microscopy, western blot, and ELISA, we found that human β-defensin 3 (hBD-3), an important antimicrobial peptide produced by epithelial cells, can be detected in human platelets and megakaryocytes. Flow cytometry and immuno-electron microscopy revealed hBD-3 on the surface of thrombin activated platelets. Moreover, hBD-3 was also found in platelet derived extracellular vesicles (p-EVs), isolated from platelet poor plasma and from platelet supernatants following thrombin stimulation. Incubation of platelets with hBD-3 peptide resulted in modest platelet activation and pre-incubation of platelets with synthetic hBD-3 prior to exposure to thrombin appeared to increase hBD-3 content in platelet lysates as well as in p-EVs, suggesting that hBD-3 can be initially taken up by platelets, perhaps via their open canalicular system. Interestingly, in vitro exposure of primary human endothelial cells to either hBD-3 peptide or purified p-EVs, caused significant endothelial dysfunction as documented by diminished levels of phosphorylated endothelial nitric oxide synthase (eNOS), Krüppel like factor-2 (KLF-2), and elevated relative expression of von Willebrand Factor (vWF). Pre-incubation of platelets with hBD-3 appeared to augment endothelial dysfunction caused by p-EVs. Overall, the current study provides evidence that hBD-3 enriched EVs can be released by activated platelets and may play a role in positive feedback of platelet activation as well as in endothelial dysfunction. Theoretically, these effects could contribute to both cellular recruitment to the endothelium creating a pro-thrombotic vascular microenvironment which serve as a bridge between innate immunity and hemostasis.

Human β-Defensin 2 (HBD-2) Displays Oncolytic Activity but Does Not Affect Tumour Cell Migration.

Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human β-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P2 interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development.

Therapeutic Potential of Synthetic Human β-Defensin 1 Short Motif Pep-B on Lipopolysaccharide-Stimulated Human Dental Pulp Stem Cells.

Dental pulp inflammation is a widespread public problem usually caused by caries or trauma. Alleviating inflammation is critical to inflamed pulp repair. Human β-defensin 1 short motif Pep-B is a cationic peptide that has anti-inflammatory, antibacterial, and immunoregulation properties, but its repair effect on human dental pulp stem cells (hDPSCs) under inflammation remains unclear. In this study, we aimed to investigate anti-inflammatory function of Pep-B and explore its therapeutic potential in lipopolysaccharide-(LPS-) induced hDPSCs. CCK-8 assay and transwell assay evaluated effects of Pep-B on hDPSC proliferation and chemotaxis. Inflammatory response in hDPSCs was induced by LPS; after Pep-B application, lactate dehydrogenase release, intracellular ROS, inflammatory factor genes expression and possible signaling pathway were measured. Then, osteo-/odontoblast differentiation effect of Pep-B on LPS-induced hDPSCs was detected. The results showed that Pep-B promoted hDPSC proliferation and reduced LPS-induced proinflammatory marker expression, and western blot result indicated that Pep-B inhibited inflammatory activation mediated by NF-κB and MAPK pathways. Pep-B also enhanced the expression of the osteo-/odontogenic genes and proteins, alkaline phosphatase activity, and nodule mineralization in LPS-stimulated hDPSCs. These findings indicate that Pep-B has anti-inflammatory activity and promote osteo-/odontoblastic differentiation in LPS-induced inflammatory environment and may have a potential role of hDPSCs for repair and regeneration.

Molecular Mechanisms of Staphylococcus and Pseudomonas Interactions in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that is characterized by recurrent and chronic infections of the lung predominantly by the opportunistic pathogens, Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. While S. aureus is the main colonizing bacteria of the CF lungs during infancy and early childhood, its incidence declines thereafter and infections by P. aeruginosa become more prominent with increasing age. The competitive and cooperative interactions exhibited by these two pathogens influence their survival, antibiotic susceptibility, persistence and, consequently the disease progression. For instance, P. aeruginosa secretes small respiratory inhibitors like hydrogen cyanide, pyocyanin and quinoline N-oxides that block the electron transport pathway and suppress the growth of S. aureus. However, S. aureus survives this respiratory attack by adapting to respiration-defective small colony variant (SCV) phenotype. SCVs cause persistent and recurrent infections and are also resistant to antibiotics, especially aminoglycosides, antifolate antibiotics, and to host antimicrobial peptides such as LL-37, human β-defensin (HBD) 2 and HBD3; and lactoferricin B. The interaction between P. aeruginosa and S. aureus is multifaceted. In mucoid P. aeruginosa strains, siderophores and rhamnolipids are downregulated thus enhancing the survival of S. aureus. Conversely, protein A from S. aureus inhibits P. aeruginosa biofilm formation while protecting both P. aeruginosa and S. aureus from phagocytosis by neutrophils. This review attempts to summarize the current understanding of the molecular mechanisms that drive the competitive and cooperative interactions between S. aureus and P. aeruginosa in the CF lungs that could influence the disease outcome.

Human β-defensin 2 enhances IL-1β production and pyroptosis through P2X7-mediated NLRP3 expression in macrophages

Periodontal disease is the leading cause of tooth loss, which is also a high-risk factor for other diseases including oral cancer and cardiovascular disease. Periodontitis is one of the most common type of periodontal diseases. Interleukin-1β (IL-1β) plays a key role in the pathogenesis of periodontitis. However, the mechanism how IL-1β is produced during periodontitis is still unclear. In the present study, we found that human β-defensin 2 (hBD2) enhances IL-1β production through an LPS-primed human acute monocytic leukemia (THP-1) macrophage model. Inhibition of P2X purinoceptor 7 (P2X7) reduced hBD2-enhanced IL-1β production. Incubation of LPS-primed THP-1 macrophages with potassium chloride also suppressed hBD2-enhanced IL-1β production. Silence of inflammasome adaptor Nod-like receptor family pyrin domain containing 3 (NLRP3) led to reduced hBD2-enhanced IL-1β production. Likewise, inhibition of caspase-1 also resulted in the decrease of IL-1β. Moreover, an ethidium bromide uptake test indicated that hBD2-activated caspase-1 mediated pyroptotic pore formation. Subsequent lactate dehydrogenase detection and flow cytometric analysis indicated that hBD2 also induced pyroptosis. In brief, these findings illustrated not only the mechanism of hBD2 in enhancing the inflammatory response, but also provided novel therapeutic targets for periodontitis.

Inflammatory response elicited by Ureaplasma parvum colonization in human cervical epithelial, stromal, and immune cells.

Ureaplasma parvum is a commensal bacterium in the female reproductive tract but has been associated with pregnancy complications such as preterm prelabor rupture of membranes and preterm birth (PTB). However, the pathologic effects of U. parvum in the cervix, which prevents ascending infections during pregnancy, are still poorly understood. To determine the impact of U. parvum on the cervix, ectocervical (ecto) and endocervical (endo) epithelial and stromal cells were incubated with U. parvum. Macrophages were also tested as a proxy for cervical macrophages to determine the antigenicity of U. parvum. The effects of U. parvum, including influence on cell cycle and cell death, antimicrobial peptide (AMP) production, epithelial-to-mesenchymal transition (EMT), and inflammatory cytokine levels, were assessed. U. parvum colonized cervical epithelial and stromal cells 4 h post-infection. Like uninfected control, U. parvum neither inhibited cell cycle progression and nor caused cell death in cervical epithelial and stromal cells. U. parvum increased the production of the AMPs cathelicidin and human β-defensin 3 and exhibited weak signs of EMT evidenced by decreased cytokeratin 18 and increased vimentin expression in cervical epithelial cells. U. parvum induced a proinflammatory environment (cytokines) and increased MMP-9 in cervical epithelial cells but promoted pro- and anti-inflammatory response in cervical stromal cells and macrophages. U. parvum may colonize the cervical epithelial layer, but induction of AMPs and anti-inflammatory response may protect the cervix and may prevent ascending infections that can cause PTB. These findings suggest that U. parvum is a weak inducer of inflammation in the cervix.

Development of multivalent vaccine targeting M segment of Crimean Congo Hemorrhagic Fever Virus (CCHFV) using immunoinformatic approaches

Crimean-Congo Hemorrhagic Fever (CCHF) is a tick-borne viral infection with no licensed vaccine or therapeutics available for its treatment. In the present study we have developed the first multi-epitope subunit vaccine effective against all the seven genotypes of CCHF virus (CCHFV). The vaccine contains five B-cell, two MHC-II (HTL), and three MHC-I (CTL) epitopes screened from two structural glycoproteins (Gc and Gn in M segment) of CCHFV with an N-terminus human β-defensin as an adjuvant, as well as an N-terminus EAAAK sequence. The epitopes were rigorously investigated for their antigenicity, allergenicity, IFN gamma induction, anti-inflammatory responses, stability, and toxicity. The three-dimensional structure of the vaccine was predicted and docked with TLR-3, TLR-8, and TLR-9 receptors to find the strength of the binding complexes via molecular dynamics simulation. After codon adaptation, the subunit vaccine construct was developed in a pDual-GC plasmid and has population coverage of 98.47% of the world's population (HLA-I & II combined). The immune simulation studies were carried out on the C-ImmSim in-silico interface showing a marked increase in the production of cellular and humoral response (B-cell and T-cell) as well as TGFβ, IL-2, IL-10, and IL-12 indicating that the proposed vaccine would be able to sufficiently provoke both humoral and cell-mediated immune responses. Thus, making it a new and promising vaccine candidate against CCHFV.

A novel smaller β-defensin-derived peptide is active against multidrug-resistant bacterial strains.

Antibiotic resistance is becoming a severe obstacle in the fight against acute and chronic infectious diseases that accompany most degenerative illnesses from neoplasia to osteo-arthritis and obesity. Currently, the race is on to identify pharmaceutical molecules or combinations of molecules able to prevent or reduce the insurgence and/or progression of infectivity. Attempts to substitute antibiotics with antimicrobial peptides have, thus far, met with little success against multidrug-resistant (MDR) bacterial strains. During the last decade, we designed and studied the activity and features of human β-defensin analogs, which are salt-resistant, and hence active also under high salt concentrations as, for instance, in cystic fibrosis. Herein, we describe the design, synthesis, and major features of a new 21 aa long molecule, peptide γ2. The latter derives from the γ-core of the β-defensin natural molecules, a small fragment of these molecules still bearing high antibacterial activity. We found that peptide γ2, which contains only one disulphide bond, recapitulates most of the biological properties of natural human β-defensins and can also counteract both Gram-positive and Gram-negative MDR bacterial strains and biofilm formation. Moreover, it has great stability in human serum thereby enhancing its antibacterial presence and activity without cytotoxicity in human cells. In conclusion, peptide γ2 is a promising new weapon also in the battle against intractable infectious diseases.

Human β-Defensin 2 and Its Postulated Role in Modulation of the Immune Response.

Studies described so far suggest that human β-defensin 2 is an important protein of innate immune response which provides protection for the human organism against invading pathogens of bacterial, viral, fungal, as well as parasitical origin. Its pivotal role in enhancing immunity was proved in infants. It may also be considered a marker of inflammation. Its therapeutic administration has been suggested for maintenance of the balance of systemic homeostasis based on the appropriate composition of the microbiota. It has been suggested that it may be an important therapeutic tool for modulating the response of the immune system in many inflammatory diseases, offering new treatment modalities. For this reason, its properties and role in the human body discussed in this review should be studied in more detail.