Breastfeeding and circulating immunological markers during the first 3 years of life: the DIABIMMUNE study.

AIMS/HYPOTHESIS: Our aim was to study the association between duration of breastfeeding and circulating immunological markers during the first 3 years of life in children with HLA-conferred susceptibility to type 1 diabetes. METHODS: We performed a longitudinal analysis of 38 circulating immunological markers (cytokines, chemokines and growth factors) in serum samples from Finnish (56 individuals, 147 samples), Estonian (56 individuals 148 samples) and Russian Karelian children (62 individuals, 149 samples) at 3, 6, 12, 18, 24 and 36 months of age. We also analysed gut inflammation markers (calprotectin and human ß defensin-2) at 3 (n = 96) and 6 months (n = 153) of age. Comparisons of immunological marker medians were performed between children who were breastfed for 6 months or longer vs children who were breastfed for less than 6 months. RESULTS: Breastfeeding for 6 months or longer vs less than 6 months was associated with lower median of serum immunological markers at 6 months (granulocyte-macrophage colony-stimulating factor [GMCSF], macrophage inflammatory protein [MIP-3α]), 12 months (IFN-α2, vascular endothelial growth factor, GMCSF, IFN-γ, IL-21), 18 months (FGF-2, IFN-α2) and 24 months of age (CCL11 [eotaxin], monocyte chemoattractant protein-1, TGFα, soluble CD40 ligand, IL-13, IL-21, IL-5, MIP-1α) (all p < 0.01) but not at 36 months of age. Breastfeeding was not associated with gut inflammation markers at 3 and 6 months of age. CONCLUSIONS/INTERPRETATION: Children who were breastfed for 6 months or longer had lower medians for 14 immunological markers at one or more age points during the first 2 years of life compared with children who were breastfed for less than 6 months. The clinical meaning of the findings is not clear. However, the present study contributes to the understanding of immunological differences in children that have been breastfed longer, and thus provides a mechanistic suggestion for the previously observed associations between breastfeeding and risk of type 1 diabetes.

Multifaceted immune functions of human defensins and underlying mechanisms.

Defensins have been long recognized as natural antimicrobial peptides, but they also possess diverse and versatile immune functions. Defensins can both induce inflammation and suppress inflammatory responses by acting on specific cells through distinct mechanisms. Defensins can also modulate the immune response by forming a complex with cellular molecules including proteins, nucleic acids, and carbohydrates. The mechanisms of defensin-mediated immune modulation appear to be cell-type and context specific. Because the levels of human defensins are often altered in response to infection or disease states, suggesting their clinical relevance, this review summarizes the complex immune functions of human defensins and their underlying mechanisms of action, which have implications for the development of new therapeutics.

Soluble expression and purification of human ß-defensin DEFB136 in Escherichia coli and identification of its bioactivity.

Human ß-defensins are an important family of innate host defense peptides with pleiotropic activities. Human ß-defensin 36 (DEFB136) is a novel member of the ß-defensin family which have not been characterized so far. In the present research, the DEFB136 peptide was expressed successfully and purified using the IMPACT-TWIN 1 expression system. The purified DEFB136 peptide was identified by MALDI-TOF mass spectrometry and circular dichroism spectroscopy. While the recombinant DEFB136 peptide exhibited a broad spectrum of antimicrobial activity against E. coli, Staphylococcus aureus and Candida albicans strains, but had low cytotoxicity to human erythrocytes. In addition, the result of the octet assay showed that the DEFB136 had a high lipopolysaccharide (LPS)-binding affinity, suggesting the DEFB136 may be involved in immunoregulation through its LPS neutralization. These results may help lay the groundwork to understand better the complex interaction between innate host defense and the diversity of the defensin family.

Isoleucine improved growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli × Leiocassis longirostris.

This study was performed to determine effects of dietary isoleucine (Ile) on growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli × Leiocassis longirostris. Six hundred and thirty fish (33.11 ± 0.09 g) were randomly divided into seven experimental groups with three replicates each, and respectively fed seven diets with 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0 g Ile kg-1 diets for 8 weeks. The results showed improvement of growth performance, feed intake, feed utilization, relative gut length (RGL), and intestinal fold height and width by dietary Ile (P < 0.05). Meanwhile, dietary Ile (12.5 g kg-1 diet) improved the activities of lysozyme (LZM), acid phosphatase, alkaline phosphatase and the contents of complement 3 (C3), C4, and immunoglobulin M (IgM) (P < 0.05). The c-type-lectin, c-LZM, g-LZM, and hepcidin mRNA expressions in the intestine were up-regulated in fish fed diets with 10.0-20.0 g Ile kg-1 diet (P < 0.05). Dietary Ile (10.0-12.5 g Ile kg-1 diet) increased intestinal ß-defensin mRNA expression partially in association with Sirt1/ERK/90RSK signaling pathway. Dietary Ile (12.5-15.0 g Ile kg-1 diet) decreased oxidative damage and improved antioxidant ability by increasing activities and expressions of superoxide dismutase, glutathione peroxidase, and glutathione reductase, glutathione-S-transferase (P < 0.05). The occludin, ZO-1, ZO-2, claudin3, and claudin 7 mRNA expressions in the intestine were up-regulated in fish fed diets with 10.0 and 12.5 g Ile kg-1 diet (P < 0.05), whereas the myosin light chain kinase gene expression was decreased in fish fed diets with 7.5-17.5 g Ile kg-1 diet. Dietary Ile (10-12.5 g Ile kg-1 diet) decreased apoptotic responses by reducing the expression of caspase3 and caspase 9 via the AKT/TOR signaling pathway. Based on the quadratic regression analysis of PWG, the dietary Ile requirement of hybrid catfish was estimated to be 12.43 g Ile kg-1 diet, corresponding to 32.05 g Ile kg-1 dietary protein. Collectively, dietary Ile improved growth performance and immunological and physical barrier function of intestine in hybrid catfish.

Molecular and functional identification of a ß-defensin homolog in large yellow croaker (Larimichthys crocea).

ß-defensin (BD) is a cysteine-rich cationic antibacterial peptide that is active against a wide range of bacteria. Here, a ß-defensin homolog (LcBD2) was identified in large yellow croaker (Larimichthys crocea). The open reading frame of LcBD2 contains 195 nucleotides, encoding a protein of 64 amino acids that possesses a typical arrangement of six conserved cysteine residues (C31 , C37 , C41 , C53 , C59 and C60 ). LcBD2 transcripts were constitutively expressed in all examined tissues and significantly increased in head kidney, spleen and gills by Vibrio alginolyticus. The synthetic LcBD2 peptide imparted antimicrobial effects on both Gram-negative bacteria (V. campbellii, V. parahaemolyticus, V. alginolyticus, V. harveyi and Pseudomonas plecoglossicida) and Gram-positive bacteria (Bacillus subtilis). We also observed that after treatment with synthetic LcBD2 peptide, numerous blisters appeared on the membrane of P. plecoglossicida, which in turn may result in cell membrane breakage and bacterial death. Moreover, the synthetic LcBD2 peptide significantly upregulated the expression levels of TNF-α2, IL-1ß and CXCL8_L1 in monocytes/macrophages, while downregulated expression level of IL-10. The LcBD2 peptide also remarkedly enhanced the phagocytosis of monocytes/macrophages. These results indicate that LcBD2 not only protects large yellow croaker against multiple bacterial pathogens but also plays a role in activation of monocytes/macrophages.

Dietary supplementation of extracts of red sea weed (Kappaphycus alvarezii) improves growth, intestinal morphology, expression of intestinal genes and immune responses in broiler chickens.

BACKGROUND: Effects of supplementation of dried alkaline (referred to as MVP1) and aqueous (referred to as PBD1) extracts of Kappaphycus alvarezii, were evaluated in broiler (Vencobb 400) chickens (1-35 days post-hatch). In experiment I, each of the seven diets (basal diet with three levels (0.5, 1.5 or 5.0 g kg-1 diet) of MVP1 or PBD1 and a negative control was fed to 12 pen replicates containing five birds in each. In experiment II, each of three diets [a negative control, and PBD1 at two levels (1.0 or 1.5 g kg-1 diet)] was fed to 16 pen replicates of five chicks in each. RESULTS: Concentrations of total phenolics, phycobillins and free radical scavenging activity were higher (P < 0.01) whereas carrageenan was lower in PBD1 than in MVP1. In the experiment I, PBD1 at 1.5 g kg-1 diet improved (P < 0.05) body weight (BW) (7.11% higher). In the experiment II, both the treatments improved (P < 0.01) BW (9.18% and 8.47%, respectively) compared to the control. The group fed with PBD1@ 1.0 g kg-1 had higher (P < 0.05) haemagglutination inhibition titre, expression of intestinal claudin 2, TLR2A, NOD1, avian beta defensin 4, interleukin 2 and interleukin 6 genes than control. Treatments did not influence feed efficiency or levels of most of the antioxidant enzymes. Villus width and crypt depth were significantly higher in the group fed with 1.5 g kg-1 of PBD1. CONCLUSION: Supplementing dried aqueous extract of K. alvarezii at 1 g kg-1 diet may be an effective strategy to increase growth and immunity in broiler chickens. © 2020 Society of Chemical Industry.

Regulatory effect of 17ß-estradiol on the expression of ß-defensin-2 and proinflammatory cytokines in human oral epithelial cells.

BACKGROUND: Although estrogen deficiency has been proposed as a risk factor for oral mucosal inflammatory diseases in post-menopausal women, the mechanisms involved remain unclear. This study aimed to investigate the effect of 17ß-estradiol (E2) on the inflammatory response stimulated by interleukin-1 beta (IL-1ß) in human oral mucosal epithelial cells (hOMECs) and its possible mechanism. METHODS: Primary hOMECs were obtained from female infants and cultured in keratinocyte growth medium. The hOMECs at second passage were collected and stimulated by 10-7  mol/L ICI182,780 or 10-7  mol/L G1 for 1 hour, E2 (10-7  mol/L, 10-8  mol/L, 10-9  mol/L) for 36 hour, 100 ng/mL IL-1ß for 12 hours, respectively. Human beta-2 defensin (hBD-2), tumor necrosis factor-alpha (TNF)-α, IL-6, IL-8, estrogen receptor-alpha (ERα), estrogen receptor-beta (ERß), and G protein-coupled receptor 30 (GPR30) mRNA levels and protein levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western Blot (WB), respectively. RESULTS: Expression of hBD-2 and inflammatory cytokines increased after IL-1ß stimulation, which was down-regulated by E2 pre-treatment. With ICI182,780, the suppression of E2 on hBD-2 mRNA was attenuated. With G1, the mRNA expression and protein expression of hBD-2 were reduced. CONCLUSION: Pre-treatment of hOMECs with E2 at physiological concentrations inhibited the IL-1ß-induced expression of hBD-2 and inflammatory cytokines. The protective effects of E2 suggest its potential use treating oral inflammatory diseases in clinical practice.

Recombinant human interleukin 17A enhances the anti-Candida effect of human oral mucosal epithelial cells by inhibiting Candida albicans growth and inducing antimicrobial peptides secretion.

BACKGROUND: Candida albicans (C albicans) is the most common fungal pathogen causing opportunistic infections. IL17 (IL17A) is a vital mediator of antifungal immunity. The aim of the study was to investigate the effect of recombinant human interleukin 17A (rhIL17A) on human oral mucosal epithelial cells (hOMECs) defending against C albicans infection. METHODS: Human oral mucosal epithelial cells were divided into four groups: C albicans+ (MOI = 0.1), rhIL17A+ (100 µg/L), rhIL17A + C albicans+ (MOI = 0.1, rhIL17A:100 µg/L) and blank control. Then, C albicans growth was observed after 24 hours. Human beta-2 defensin (hBD-2), S100A8 and LL-37 in supernatants and their mRNAs in cells were measured by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. RESULTS: In C albicans+ group, C albicans hyphae formation and the death of infected hOMECs were observed. However, in the rhIL17A + C albicans+ group, IL17 inhibited both hypha formation, and C albicans from infecting hOMECs and its further growth. There was no statistical significance in adhesion rates of C albicans to hOMECs. Compared with the control group, the level of hBD-2 mRNA has increased, while hBD-2 and hBD-2 mRNA levels in the rhIL17A + C albicans+ group were the highest. Both hBD-2 and hBD-2 mRNA levels were higher in the rhIL17A+ group than in the C albicans+ group. S100A8 and LL-37 mRNAs have similar trend, and both upregulated after treatment with rhIL17A; however, protein levels were undetectable. CONCLUSION: Recombinant human interleukin 17A may inhibit C albicans from infecting hOMECs by affecting the growth and reproduction of C albicans as well as the formation of hyphae. Besides, rhIL17A might induce hBD-2, S100A8 and LL-37 secretion from hOMECs to strengthen their anti-infective ability.

Ubiquitously expressed Human Beta Defensin 1 (hBD1) forms bacteria-entrapping nets in a redox dependent mode of action.

Ever since the discovery of endogenous host defense antimicrobial peptides it has been discussed how these evolutionary conserved molecules avoid to induce resistance and to remain effective. Human ß-defensin 1 (hBD1) is an ubiquitously expressed endogenous antimicrobial peptide that exhibits qualitatively distinct activities between its oxidized and reduced forms. Here, we explore these antimicrobial mechanisms. Surprisingly, using electron microscopy we detected a so far unknown net-like structure surrounding bacteria, which were treated with the reduced but not the oxidized form of hBD1. A transmigration assay demonstrated that hBD1-derived nets capture bacteria and inhibit bacterial transmigration independent of bacterial killing. The presence of nets could completely prevent migration of hBD1 resistant pathogens and are stable in the presence of human duodenal secretion with a high amount of proteases. In contrast to HD6, cysteins are necessary for net formation. This redox-dependent function serves as an additional mechanism of action for hBD1 and differs from net formation by other defensins such as Paneth cell-derived human α-defensin 6 (HD6). While hBD1red and hBD1ox have distinct antimicrobial profiles and functions, only the reduced form provides additional host protection by entrapping bacteria in extracellular net structures preventing bacterial invasion. Better understanding of the modes of action of endogenous host peptides will help to find new antimicrobial strategies.

Potassium is a key signal in host-microbiome dysbiosis in periodontitis.

Dysbiosis, or the imbalance in the structural and/or functional properties of the microbiome, is at the origin of important infectious inflammatory diseases such as inflammatory bowel disease (IBD) and periodontal disease. Periodontitis is a polymicrobial inflammatory disease that affects a large proportion of the world's population and has been associated with a wide variety of systemic health conditions, such as diabetes, cardiovascular and respiratory diseases. Dysbiosis has been identified as a key element in the development of the disease. However, the precise mechanisms and environmental signals that lead to the initiation of dysbiosis in the human microbiome are largely unknown. In a series of previous in vivo studies using metatranscriptomic analysis of periodontitis and its progression we identified several functional signatures that were highly associated with the disease. Among them, potassium ion transport appeared to be key in the process of pathogenesis. To confirm its importance we performed a series of in vitro experiments, in which we demonstrated that potassium levels a increased the virulence of the oral community as a whole and at the same time altering the immune response of gingival epithelium, increasing the production of TNF-α and reducing the expression of IL-6 and the antimicrobial peptide human ß-defensin 3 (hBD-3). These results indicate that levels of potassium in the periodontal pocket could be an important element in of dysbiosis in the oral microbiome. They are a starting point for the identification of key environmental signals that modify the behavior of the oral microbiome from a symbiotic community to a dysbiotic one.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Histone deacetylase inhibition enhances antimicrobial peptide but not inflammatory cytokine expression upon bacterial challenge.

Antimicrobial peptides (AMP) are defense effectors of the innate immunity playing a crucial role in the intestinal homeostasis with commensals and protection against pathogens. Herein we aimed to investigate AMP gene regulation by deciphering specific characteristics allowing their enhanced expression among innate immune genes, particularly those encoding proinflammatory mediators. Our emphasis was on epigenetic regulation of the gene encoding the AMP ß-defensin 2 (HBD2), taken as a model of possibly specific induction, upon challenge with a commensal bacterium, compared with the proinflammatory cytokine IL-8. Using an in vitro model of colonic epithelial cells challenged with Escherichia coli K12, we showed that inhibition of histone deacetylases (HDAC) by trichostatin A dramatically enhanced induction of HBD2 expression, without affecting expression of IL-8. This mechanism was supported by an increased phosphorylation of histone H3 on serine S10, preferentially at the HBD2 promoter. This process occurred through activation of the IκB kinase complex, which also led to activation of NF-κB. Moreover, we demonstrated that NF-κB was modified by acetylation upon HDAC inhibition, partly by the histone acetyltransferase p300, and that both NF-κB and p300 supported enhanced induction of HBD2 expression. Furthermore, we identified additional genes belonging to antimicrobial defense and epithelial restitution pathways that showed a similar pattern of epigenetic control. Finally, we confirmed our finding in human colonic primary cells using an ex vivo organoid model. This work opens the way to use epigenetic pharmacology to achieve induction of epithelial antimicrobial defenses, while limiting the deleterious risk of an inflammatory response.

Role of Toll-like receptor 2 in mediating the production of cytokines and human beta-defensins in oral mucosal epithelial cell response to Leptospiral infection.

BACKGROUND: Pathogenic Leptospira spp. is the causative agent of leptospirosis. Oral mucosal cavity is one of portal entry for this bacterium. Oral mucosal epithelium provides a physical barrier and secretes cytokines, chemokines and antimicrobial peptides (AMPs) in response to microbial infection. Human ß-defensins (hBDs); hBD1, hBD2, and hBD3 are predominantly AMPs expressed in the oral cavity. Toll-like receptors (TLRs) have been reported in hBD regulation. TLR2 recognizes leptospiral lipopolysaccharide, and plays a key role in the early control of leptospirosis. OBJECTIVE: The aim of this study is to investigate the role of TLR2 in mediating the production of cytokines and hBDs in oral mucosal epithelial cell response to leptospiral infection. METHODS: Cultivated oral mucosal epithelial cells were prepared, characterized, and compared with oral mucosal tissues. The TLR1-10 and hBD mRNA expressions were examined. Pro-inflammatory cytokine and hBD1-3 expressions in response to leptospires were determined by quantitative (q) RT-PCR. RESULTS: The cultivated oral epithelium expressed TLR2 and hBD1-3. The induction of IL-ßIL-8, TNF-α, and hBD2 were increased in response to Leptospira via TLR2 recognition. CONCLUSION: The characteristics of primary epithelial cells and tissue were similar in terms of TLR expression. All primary epithelial cells expressed TLR2 and hBD1-3. We used primary epithelial cells to study response to L. interrogans. Our results yielded the first evidence that human TLR2 regulates hBD2 expression in oral mucosa epithelial responded to L. interrogans. Expression of hBD2 may act to neutralize the virulence or prevent the invasion of L. interrogans at the portal of entry.

Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial ß-Defensin 1.

The antimicrobial peptide human ß-defensin 1 (hBD1) is continuously produced by epithelial cells in many tissues. Compared to other defensins, hBD1 has only minor antibiotic activity in its native state. After reduction of its disulfide bridges, however, it becomes a potent antimicrobial agent against bacteria, while the oxidized native form (hBD1ox) shows specific activity against Gram-negative bacteria. We show that the killing mechanism of hBD1ox depends on aerobic growth conditions and bacterial enzymes. We analyzed the different activities of hBD1 using mutants of Escherichia coli lacking one or more specific proteins of their outer membrane, cytosol, or redox systems. We discovered that DsbA and DsbB are essential for the antimicrobial activity of hBD1ox but not for that of reduced hBD1 (hBD1red). Furthermore, our results strongly suggest that hBD1ox uses outer membrane protein FepA to penetrate the bacterial periplasm space. In contrast, other bacterial proteins in the outer membrane and cytosol did not modify the antimicrobial activity. Using immunogold labeling, we identified the localization of hBD1ox in the periplasmic space and partly in the outer membrane of E. coli However, in resistant mutants lacking DsbA and DsbB, hBD1ox was detected mainly in the bacterial cytosol. In summary, we discovered that hBD1ox could use FepA to enter the periplasmic space, where its activity depends on presence of DsbA and DsbB. HBD1ox concentrates in the periplasm in Gram-negative bacteria, which finally leads to bleb formation and death of the bacteria. Thus, the bacterial redox system plays an essential role in mechanisms of resistance against host-derived peptides such as hBD1.

Human ß-defensin 2 plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity.

BACKGROUND: Antimicrobial peptides (AMPs) are primarily known for their innate immune defense against invading microorganisms, including viruses. In addition, recent research has suggested their modulatory activity in immune induction. Given that most subunit vaccines require an adjuvant to achieve effective immune induction through the activation of innate immunity, AMPs are plausible candidate molecules for stimulating not only innate immune but also adaptive immune responses. RESULTS: In this study, we investigated the ability of human ß-defensin (HBD) 2 to promote antiviral immunity in vitro and in vivo using a receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) spike protein (S RBD) as a model antigen (Ag). When HBD 2-conjugated S RBD was used to treat THP-1 human monocytic cells, the expression levels of antiviral (IFN-ß, IFN-γ, MxA, PKR, and RNaseL) and primary immune-inducing (NOD2, TNF-α, IL-1ß, and IL-6) molecules were enhanced compared to those expressed after treatment with S RBD only. The expression of chemokines capable of recruiting leukocytes, including monocytes/macrophages, natural killer cells, granulocytes, T cells, and dendritic cells, was also increased following HBD 2-conjugated S RBD treatment. More important, immunization of mice with HBD 2-conjugated S RBD enhanced the immunogenicity of the S RBD and elicited a higher S RBD-specific neutralizing antibody response than S RBD alone. CONCLUSIONS: We conclude that HBD 2 activates the primary antiviral innate immune response and may also mediate the induction of an effective adaptive immune response against a conjugated Ag.

Gingival crevicular fluid levels of human beta-defensin 1 in individuals with and without chronic periodontitis.

BACKGROUND AND OBJECTIVE: Human beta-defensins (hBDs) contribute to innate immunity antimicrobial activity. They are also effective in the adaptive immune response and may play a crucial role in the susceptibility to diseases of the oral cavity. This study aimed to evaluate the levels of hBD-1 in the gingival crevicular fluid of individuals with and without chronic periodontitis. MATERIAL AND METHODS: Twenty periodontally healthy individuals (H) and 20 individuals with chronic periodontitis were recruited. Gingival crevicular fluid samples were collected from: healthy sites (Hh) from periodontally healthy individuals; and healthy sites (Ph), sites with gingivitis (Pg), and sites with periodontitis (Pp) from individuals with periodontitis. The levels of hBD-1 (pg/mL) were measured using enzyme-linked immunosorbent assay. Comparisons of hBD-1 between individuals (H and chronic periodontitis) and among sites (Hh, Ph, Pg, Pp) were performed through hierarchical linear modeling. RESULTS: Gingival crevicular fluid levels of hBD-1 were: Hh = 229.52 ± 138.96 (median 199.26), Ph = 53.88 ± 58.17 (median 35.75), Pg = 57.11 ± 40.18 (median 39.90) and Pp = 55.31 ± 37.28 (median 54.19). No influence of site diagnosis (level 1; health/gingivitis/periodontitis) was observed; however, individual diagnosis (level 2; health/periodontitis) influenced the levels of hBD-1 (P < .001). CONCLUSION: Periodontally healthy individuals showed higher gingival crevicular fluid levels of hBD-1 when compared to individuals with chronic periodontitis. This suggests a potential protective role of hBD-1 in the susceptibility to chronic periodontitis.

Induction and antiviral activity of human ß-defensin 3 in intestinal cells with picornavirus infection.

Antimicrobial peptides produced by epithelial and immune cells protect tissues from various infections. Whether enterovirus infection leads to stimulation of antimicrobial peptide expression is unknown. We examined antimicrobial peptide mRNA and protein production in HT-29 colon adenocarcinoma cells infected with picornaviruses. The antiviral activity of increased antimicrobial peptide production was evaluated by using a recombinant peptide and corresponding gene overexpression. Enterovirus infection enhanced both the mRNA expression and secretion of human ß-defensin (hBD) 3 in intestinal epithelial cells but did not increase expression of human neutrophil peptide 1-3 (HNP 1-3), HNP4, human defensin 5 (HD5), HD6, hBD1, hBD2, and hBD5. The recombinant but not the intracellularly overexpressed hBD3 inhibited enterovirus (EV) 71, coxsackievirus A16 (CVA16), CVB5 and poliovirus 1 (PV1) infecting HT-29 cells. Our results suggest that enterovirus infection induces hBD3 production in human intestinal epithelial cells and that hBD3 can exert extracellular anti-enterovirus activity.

Differential Regulation of Mas-Related G Protein-Coupled Receptor X2-Mediated Mast Cell Degranulation by Antimicrobial Host Defense Peptides and Porphyromonas gingivalis Lipopolysaccharide.

Porphyromonas gingivalis is a keystone pathogen that contributes to periodontal pathogenesis by disrupting host-microbe homeostasis and promoting dysbiosis. The virulence of P. gingivalis likely reflects an alteration in the lipid A composition of its lipopolysaccharide (LPS) from the penta-acylated (PgLPS1690) to the tetra-acylated (PgLPS1435/1449) form. Mast cells play an important role in periodontitis, but the mechanisms of their activation and regulation remain unknown. The expression of epithelium- and neutrophil-derived host defense peptides (HDPs) (LL-37 and human ß-defensin-3), which activate mast cells via Mas-related G protein-coupled receptor X2 (MRGPRX2), is increased in periodontitis. We found that MRGPRX2-expressing mast cells are present in normal gingiva and that their numbers are elevated in patients with chronic periodontitis. Furthermore, HDPs stimulated degranulation in a human mast cell line (LAD2) and in RBL-2H3 cells stably expressing MRGPRX2 (RBL-MRGPRX2). PgLPS1690 caused substantial inhibition of HDP-induced mast cell degranulation, but PgLPS1435/1449 had no effect. A fluorescently labeled HDP (FAM-LL-37) bound to RBL-MRGPRX2 cells, and PgLPS1690 inhibited this binding, but PgLPS1435/1449 had no effect. These findings suggest that low-level inflammation induced by HDP/MRGPRX2-mediated mast cell degranulation contributes to gingival homeostasis but that sustained inflammation due to elevated levels of both HDPs and MRGPRX2-expressing mast cells promotes periodontal disease. Furthermore, differential regulation of HDP-induced mast cell degranulation by PgLPS1690 and PgLPS1435/1449 may contribute to the modulation of disease progression.

The Evolution of Innate Immune Genes: Purifying and Balancing Selection on ß-Defensins in Waterfowl.

In disease dynamics, high immune gene diversity can confer a selective advantage to hosts in the face of a rapidly evolving and diverse pathogen fauna. This is supported empirically for genes involved in pathogen recognition and signalling. In contrast, effector genes involved in pathogen clearance may be more constrained. ß-Defensins are innate immune effector genes; their main mode of action is via disruption of microbial membranes. Here, five ß-defensin genes were characterized in mallards (Anas platyrhynchos) and other waterfowl; key reservoir species for many zoonotic diseases. All five genes showed remarkably low diversity at the individual-, population-, and species-level. Furthermore, there was widespread sharing of identical alleles across species divides. Thus, specific ß-defensin alleles were maintained not only spatially but also over long temporal scales, with many amino acid residues being fixed across all species investigated. Purifying selection to maintain individual, highly efficacious alleles was the primary evolutionary driver of these genes in waterfowl. However, we also found evidence for balancing selection acting on the most recently duplicated ß-defensin gene (AvBD3b). For this gene, we found that amino acid replacements were more likely to be radical changes, suggesting that duplication of ß-defensin genes allows exploration of wider functional space. Structural conservation to maintain function appears to be crucial for avian ß-defensin effector molecules, resulting in low tolerance for new allelic variants. This contrasts with other types of innate immune genes, such as receptor and signalling molecules, where balancing selection to maintain allelic diversity has been shown to be a strong evolutionary force.

CCR6/CCL20 chemokine axis in human immunodeficiency virus immunity and pathogenesis.

Recent studies in human immunodeficiency virus (HIV) have garnered interest for the role of CC chemokine receptor 6 (CCR6) and its known ligands, CC chemokine ligand 20 (CCL20) and human ß-defensins, in viral entry, dissemination and antiviral immunity. Several studies have suggested that CCR6 may also act as a weak co-receptor of HIV entry, in addition to the canonical CXC chemokine receptor 4 (CXCR4) and CCR5. However, the pathogenic significance has yet to be demonstrated as the observations for preferential infection of CD4+CCR6+ over CD4+CCR6- T cells appear to be independent of CCR6 expression. This indicates means for preferential infection other than CCR6 co-receptor use. Attention has also turned to the inadvertent role of the CCR6/CCL20 axis in attracting key immune cells, including TH17 cells and dendritic cells, to sites of infection and propagating the virus to other sites of the body. This review article will summarize the latest evidence that the CCR6/CCL20 chemokine axis is playing an important role in HIV pathogenesis and immunity. Further work with in vivo studies is needed to establish the biological and, hence, therapeutic significance of these findings.