Expression of Human ß-defensin 2 (hBD-2) in Pichia Pastoris and Investigation of Its Binding Efficiency with ACE-2.

COVID-19 is a disease that have affected the entire world, and it continues to spread with new variants. A patient's innate immune system plays a critical role in the mild and severe transition of COVID-19. Antimicrobial peptides (AMPs), which are important components of the innate immune system, are potential molecules to fight pathogenic bacteria, fungi, and viruses. Human ß-defensin 2 (hBD-2), a 41-amino-acid antimicrobial peptide, is one of the defensins inducibly expressed in the skin, lungs, and trachea in humans. In this study, it was aimed to investigate the interaction of hBD-2 produced recombinantly in Pichia pastoris with the human angiotensin-converting enzyme 2 (ACE-2) under in vitro conditions. First, hBD-2 was cloned in P. pastoris X-33 via the pPICZαA vector, a yeast expression platform, and its expression was confirmed by SDS-PAGE, western blotting, and qRT-PCR. Then, the interaction between recombinant hBD-2 and ACE-2 proteins was revealed by a pull-down assay. In light of these preliminary experiments, we suggest that the recombinantly produced hBD-2 may be protective against SARS-CoV-2 and be used as a supplement in treatment. However, current findings need to be supported by cell culture studies, toxicity analyses, and in vivo experiments.

Fecal HBD-2 and Claudin-3 may be potential biomarkers to predict the deterioration of necrotizing enterocolitis: A prospective study.

Background and purpose: Necrotizing enterocolitis (NEC) is a critical gastrointestinal disease. We aim to explore the value of fecal human ß-defensin 2 (HBD-2), Claudin-3, high-mobility group box-1 protein (HMGB-1), and resistin-like molecule ß (Relmß) as well as some laboratory metrics to predict the deterioration of NEC. Methods: Infants diagnosed with NEC at Stage II were enrolled in our study. Those who progressed to Stage III were included in the Stage III group and the rest were included in the Stage II group. Clinical data and laboratory metrics of the infants were collected. Fecal samples of HBD2, HMGB-1, Claudin-3, and Relmß collected during their enrollment were determined by using enzyme-linked immunosorbent assay (ELISA) kits. Student's t-test, the Mann-Whitney U test, the chi-square test, receiver operating characteristic (ROC), and logistic regression analysis were performed. Results: Sixty infants diagnosed with NEC at Stage II were enrolled in our study, with 27 in the Stage III group (n = 27) and 33 in the Stage II group (n = 33). Although many of these NEC cases were late preterm and term infants, the infants in the Stage III group had a lower gestational age (P < 0.05). The incidence of gestational diabetes mellitus, peritonitis, intestinal adhesion, and sepsis was higher and more infants in the Stage III group underwent surgeries (P < 0.05). The levels of HBD-2 and Claudin-3 were higher and neutrophil count was lower in the Stage III group than in the Stage II Group, and the area under the curve (AUC) was 0.754, 0,755, and 0.666, respectively (P < 0.05). HBD-2 ≥ 1649.02 ng/g and Claudin-3 ≥ 2488.71 pg/g were included in the multivariate stepwise logistic regression analysis (P < 0.05), and the AUC of the model was 0.805 (95% CI: 0.688-0.922). Conclusion: Fecal HBD-2 and Claudin-3 may be potential biomarkers to predict the deterioration of NEC from Stage II to Stage III.

Human Beta-Defensin 2 and 3 Inhibit HIV-1 Replication in Macrophages.

Human beta-defensins (hBDs) are broad-spectrum antimicrobial peptides, secreted by epithelial cells of the skin and mucosae, and astrocytes, which we and others have shown to inhibit HIV-1 in primary CD4+ T cells. Although loss of CD4+ T cells contributes to mucosal immune dysfunction, macrophages are a major source of persistence and spread of HIV and also contribute to the development of various HIV-associated complications. We hypothesized that, besides T cells, hBDs could protect macrophages from HIV. Our data in primary human monocyte-derived macrophages (MDM) in vitro show that hBD2 and hBD3 inhibit HIV replication in a dose-dependent manner. We determined that hBD2 neither alters surface expression of HIV receptors nor induces expression of anti-HIV cytokines or beta-chemokines in MDM. Studies using a G-protein signaling antagonist in a single-cycle reporter virus system showed that hBD2 suppresses HIV at an early post-entry stage via G-protein coupled receptor (GPCR)-mediated signaling. We find that MDM express the shared chemokine-hBD receptors CCR2 and CCR6, albeit at variable levels among donors. However, cell surface expression analyses show that neither of these receptors is necessary for hBD2-mediated HIV inhibition, suggesting that hBD2 can signal via additional receptor(s). Our data also illustrate that hBD2 treatment was associated with increased expression of APOBEC3A and 3G antiretroviral restriction factors in MDM. These findings suggest that hBD2 inhibits HIV in MDM via more than one CCR thus adding to the potential of using ß-defensins in preventive and therapeutic approaches.

Serum Human ß-Defensin 2 Is Increased in Angioedema Accompanying Chronic Spontaneous Urticaria.

INTRODUCTION: Chronic spontaneous urticaria (CSU) is a common cutaneous disease caused by mast-cell degranulation. Human ß-defensin 2 (HBD2) is a well-known antimicrobial peptide that is also a pruritogen inducing vascular permeability via non-IgE-mediated mast-cell degranulation. OBJECTIVE: We investigated the associations between serum HBD2 levels and the clinical characteristics of CSU patients. METHODS: Serum samples from 124 CSU patients and 56 healthy controls were screened for the levels of HBD2 and translationally controlled tumor protein (TCTP)_ by using ELISA. The urticaria activity score over 7 days (UAS7) was used to measure disease activity in CSU patients. Accompanying angioedema was self-reported. RESULTS: Serum HBD2 levels were higher in the CSU group than in healthy subjects (median [interquartile range], 84.1 [43.5, 142.5] vs. 59.5 [26.7, 121.5], p = 0.034). In CSU patients, serum HBD2 level was negatively correlated with the peripheral basophil percentages (Spearman's rho = -0.229, p = 0.01) and vitamin D levels (-0.262, p = 0.02), but positively correlated with TCTP levels (0.252, p = 0.006). In CSU patients, HBD2 level was higher in those with than without angioedema (101.7 [50.9, 184.2] vs. 66.7 [37.9, 132.0], p = 0.019). It did not differ by aspirin hypersensitivity or atopy status, or autologous serum skin test positivity. CONCLUSION: A known mast-cell degranulator, HBD2 was elevated in the sera from CSU patients compared to healthy controls and may be involved in the pathogenesis of accompanying angioedema.

Relationship Between Human ß-Defensin 2 and the Vaginal Environment.

As one of the main antimicrobial peptides, human ß-defensin 2 (HBD2) plays multiple roles in the lower genital tract. Based on the Nugent score as a diagnostic criterion for bacterial vaginosis, we sought to clarify the correlations among the Nugent score and interleukin-6 (IL-6) and HBD2 levels in vaginal secretions in association with various types of infection. Ninety-eight women were recruited for this study. Levels of HBD2 and IL-6 in vaginal wash were measured by enzyme-linked immunosorbent assays. According to the Nugent method, the number of Lactobacillus morphotypes per field of view was well correlated with the HBD2 level. The amount of HBD2 was also well correlated with the presence of Candida spp. (P < 0.01). In vitro experiments revealed that the expression of HBD2 from the human vaginal epithelial cell line, VK2/E6E7, was induced by the addition of heat-killed C. albicans (HKCA). The addition of HKCA induced expression of Dectin-1 mRNA. A luciferase assay for nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) responsive elements showed that HKCA activated NF-κB signaling. These results suggested that C. albicans induced the activation of Dectin-1 and (NF-κB) signaling, resulting in HBD2 expression. In conclusion, the expression of HBD2 positively correlated with the presence of Lactobacillus and Candida spp.

S-Layer Protein of Lactobacillus helveticus SBT2171 Promotes Human ß-Defensin 2 Expression via TLR2-JNK Signaling.

Antimicrobial peptides that contribute to innate immunity are among the most important protective measures against infection in many organisms. Several substances are known to regulate the expression of antimicrobial peptides. In this study, we investigated the factors in lactic acid bacteria (LAB) that induce antimicrobial peptide expression in the host. We found that Lactobacillus helveticus SBT2171 (LH2171) induced the expression of human ß-defensin (hBD)2 in Caco-2 human colonic epithelial cells. Specifically, surface layer protein (SLP) of LH2171 stimulated hBD2 expression by activating c-Jun N-terminal kinase (JNK) signaling via Toll-like receptor (TLR)2 in Caco-2 cells. SLPs extracted from other lactobacilli similarly increased hBD2 expression, suggesting that this stimulatory effect is common feature of Lactobacillus SLPs. Interestingly, Lactobacillus strains that strongly induced hBD2 expression also potently activated JNK signaling. Thus, upregulation of hBD2 induced by TLR2-JNK signaling contributes to protection of the host against infection.

Recombinant Oncolytic Vaccinia Viruses Expressing Human ß-Defensin 2 Enhance Anti-tumor Immunity.

Cancer is still a leading of cause of death worldwide. Among the bio-therapy strategies for cancer, vaccinia virus (VV) has been widely used as an expression vector because of its potent oncolytic activities in addition to its large capacity for insertion of foreign genes and excellent safety records. In the present study, a novel recombinant VV, VV-HBD2-lacZ, expressing human ß-defensin 2 (HBD2), an anti-microbial peptide of the innate immune system, was constructed. First, the chemotaxis characteristics of HBD2 expressed on VV-HBD2-lacZ-infected cells toward dendritic cells (DCs) in vitro and in vivo were demonstrated. The anti-tumor effects of VV-HBD2-lacZ in vitro and in vivo in a mouse melanoma cancer model were then investigated. It was found that VV-HBD2-lacZ was able to inhibit tumor growth and metastasis significantly. It was further demonstrated that VV-HBD2-lacZ induced potent cytotoxic activity by increasing the tumor-infiltrating CD4+ and CD8+ T cells. These results indicate that HBD2-expressing VV recruited plasmacytoid DCs (pDCs) to the tumor location, leading to cytotoxic T cell response against the tumor, and thus inhibited tumor growth in vitro and in vivo. In conclusion, oncolytic HBD2-expressing VV provides an effective treatment for tumors by triggering innate and adaptive immunity.

Effect of Substance P in Staphylococcus aureus and Staphylococcus epidermidis Virulence: Implication for Skin Homeostasis.

Staphylococcus aureus and Staphylococcus epidermidis are two major skin associated bacteria, and Substance P (SP) is a major skin neuropeptide. Since bacteria are known to sense and response to many human hormones, we investigated the effects of SP on Staphylococci virulence in reconstructed human epidermis model and HaCaT keratinocytes. We show that SP is stimulating the virulence of S. aureus and S. epidermidis in a reconstructed human epidermis model. qRT-PCR array analysis of 64 genes expressed by keratinocytes in the response to bacterial infection revealed a potential link between the action of SP on Staphylococci and skin physiopathology. qRT-PCR and direct assay of cathelicidin and human ß-defensin 2 secretion also provided that demonstration that the action of SP on bacteria is independent of antimicrobial peptide expression by keratinocytes. Considering an effect of SP on S. aureus and S. epidermidis, we observed that SP increases the adhesion potential of both bacteria on keratinocytes. However, SP modulates the virulence of S. aureus and S. epidermidis through different mechanisms. The response of S. aureus is associated with an increase in Staphylococcal Enterotoxin C2 (SEC2) production and a reduction of exolipase processing whereas in S. epidermidis the effect of SP appears mediated by a rise in biofilm formation activity. The Thermo unstable ribosomal Elongation factor Ef-Tu was identified as the SP-interacting protein in S. aureus and S. epidermidis. SP appears as an inter-kingdom communication factor involved in the regulation of bacterial virulence and essential for skin microflora homeostasis.

Human beta-defensin 1, a new animal toxin-like blocker of potassium channel.

The discovery of human ß-defensin 2 (hBD2), as a Kv1.3 channel inhibitor with the unique molecular mechanism and novel immune modulatory function, suggests that human ß-defensins are a novel class of channel ligands. Here, the function and mechanism of the human ß-defensin 1 (hBD1) binding to potassium channels was investigated. Based on the structural similarity between hBD1 and Kv1.3 channel-sensitive hBD2, hBD1 was found to selectively inhibit human and mouse Kv1.3 channels with IC50 values of 11.8 ± 3.1 µM and 13.2 ± 4.0 µM, respectively. Different from hBD2 modifying Kv1.3 channel activation and increasing activation time constant, hBD1 did not affect the activation feature of both human and mouse Kv1.3 channels. In comparison with hBD2 simultaneously interacting with the extracellular S1-S2 linker and pore region of Kv1.3 channel, the chimeric channel and mutagenesis experiments showed that hBD1 only bound to the extracellular pore region of Kv1.3 channel instead of extracellular S1-S2 linker or S3-S4 linker. Together, these findings enhance knowledge of hBD1 as a new immune-related Kv1.3 channel blocker and highlight the major functional differences between hBD1 and hBD2 to explore in future research.

Human ß-defensin 2 may inhibit internalisation of bacillus Calmette-Guérin (BCG) in bladder cancer cells.

OBJECTIVE: To investigate whether secretion of human ß-defensin 2 (HBD-2) is induced by bacillus Calmette-Guérin (BCG) and to determine whether HBD-2 affects BCG internalisation in bladder cancer cells. MATERIALS AND METHODS: Reverse transcription-polymerase chain reaction analysis was used to determine whether HBD-2 mRNA increases after incubation with BCG. HBD-2 proteins in 5637 and T24 human bladder cancer cell lines were assayed by enzyme-linked immunosorbent assay. The internalisation rate was evaluated by double immunofluorescence assay and confocal microscopy to test the optimal dose of HBD-2 for BCG internalisation. We also investigated the difference in internalisation rates and cell viability between recombinant HBD-2 protein, anti-HBD-2 antibody, and HBD-2 plus anti-HBD-2 antibody pretreatments. RESULTS: BCG induced HBD-2 mRNA expression and HBD-2 production dose and time-dependently in bladder cancer cells and affected BCG internalisation. Pretreatment with recombinant HBD-2 protein lowered internalisation of BCG dose-dependently. Moreover, anti-HBD-2 antibody prevented the effect of HBD-2 on BCG internalisation in bladder cancer cells. The internalisation rate of BCG pretreated with anti-HBD-2 antibody was higher than that in the control in 5637 (P < 0.01) and T24 cells (P < 0.05). The BCG internalisation rate in cells pretreated with anti-HBD-2 antibody plus recombinant HBD-2 protein was higher than that in the control in 5637 (P < 0.01) and T24 cells (P < 0.05). Mycobacterium bovis BCG decreased bladder cancer cell viability, and anti-HBD-2 antibody prevented the inhibitory role of HBD-2 on the anti-proliferative effects of M. bovis BCG in bladder cancer cells CONCLUSION: Bladder cancer cells produce HBD-2 when they are infected by BCG to defend themselves against BCG internalisation, which plays an important role during the initiation and propagation of the immunotherapeutic response in bladder cancer cells.