Antimicrobial Peptide Gene Expression Research Articles

Role of serpin-25 in prophenoloxidase activation and expression of antimicrobial peptide genes in the silkworm Bombyx mori

Serine protease inhibitors (Serpins) are involved in diverse physiological and developmental processes, constituting one of the largest superfamilies of protease inhibitors. They are widely distributed across various organisms such as plants, animals, prokaryotes, and viruses. In insects, serpins exert their inhibitory effects on serine proteinase cascades, thereby regulating important pathways such as the Toll pathway and prophenoloxidase (proPO) in hemolymph. In this study, we cloned and analyzed the serpin-25 gene in Bombyx mori (Bmserpin-25). Bmserpin-25 mRNA was detected in all examined tissues, with high expression levels observed in the fat body, midgut, and epidermis. Upon injection of B. mori with four different microorganisms, including Gram-positive microbes (Micrococcus luteus), Gram-negative microbes (E. coli), fungi (Beauveria bassiana), and the B. mori nuclear polyhedrosis virus (BmNPV), the significant changes in Bmserpin-25 expression were observed specifically in response to E. coli and BmNPV treatments. Furthermore, the recombinant Bmserpin-25 was successfully expressed in Escherichia coli and purified for subsequent functional analysis. In vitro experiments showed that the recombinant Bmserpin-25 protein inhibited proPO activation but did not affect phenoloxidase activity. Additionally, injection of recombinant Bmserpin-25 protein into B. mori larvae resulted in a significant downregulation of antimicrobial peptide gene expression in the fat body. These findings emphasize the crucial role of serpin-25 in prophenoloxidase activation and the regulation of antimicrobial peptide gene expression in B. mori.

Gene expression of antimicrobial peptides in rat intestine under conditions of chronic stress

BACKGROUND: Severe stress causes an array of dysfunctions in the immune, neuroendocrine, cardiovascular, digestive and other systems, resulting in an emergence of various types of pathology. Common manifestations of a chronic stress are the disorders in the gastrointestinal tract, such as irritable bowel syndrome, functional dyspepsia, biliary dyskinesia, dysbiosis, inflammatory processes that determine the development of gastritis and one of the most widespread post-stress pathologies of the gastrointestinal tract — stomach ulcers. The disclosure of the molecular mechanisms of a pathogenesis of diseases associated with gastrointestinal dysfunction related to chronic stress as well as a search for new ways to correct these disorders are important tasks of fundamental and clinical medicine. The present work is focused on evaluating a participation of molecular factors of the innate immunity in intestine, such as antimicrobial peptides secreted by intestinal epithelial cells upon infection, in a response to the chronic stress. AIM: The aim of the study was to estimate the gene expression of a number of antimicrobial peptides: intestinal α- and β-defensins of laboratory animals (rats) under chronic stress conditions. MATERIALS AND METHODS: Modeling of a chronic stress was performed by daily forced swimming of laboratory animals in cold water. An expression of α- and β-defensin genes was evaluated using a real-time polymerase chain reaction. RESULTS: We found an increase in the level of expression of the rat α-defensin-5 and β-defensin-3 genes in response to chronic stress, while the expression of β-defensin-2 gene was not changed compared to the control. CONCLUSIONS: Considering that changes in the concentration and spectrum of peptides with antibacterial activity, caused by prolonged stress, can contribute to modification of the composition of the intestinal microbiota, the data obtained can expand our understanding of the molecular basis of the pathogenesis of diseases associated with disorders in the composition of microbiota under stress.

Comparative Transcriptomics of Fat Bodies between Symbiotic and Quasi-Aposymbiotic Adult Females of Blattella germanica with Emphasis on the Metabolic Integration with Its Endosymbiont Blattabacterium and Its Immune System.

We explored the metabolic integration of Blattella germanica and its obligate endosymbiont Blattabacterium cuenoti by the transcriptomic analysis of the fat body of quasi-aposymbiotic cockroaches, where the endosymbionts were almost entirely removed with rifampicin. Fat bodies from quasi-aposymbiotic insects displayed large differences in gene expression compared to controls. In quasi-aposymbionts, the metabolism of phenylalanine and tyrosine involved in cuticle sclerotization and pigmentation increased drastically to compensate for the deficiency in the biosynthesis of these amino acids by the endosymbionts. On the other hand, the uricolytic pathway and the biosynthesis of uric acid were severely decreased, probably because the reduced population of endosymbionts was unable to metabolize urea to ammonia. Metabolite transporters that could be involved in the endosymbiosis process were identified. Immune system and antimicrobial peptide (AMP) gene expression was also reduced in quasi-aposymbionts, genes encoding peptidoglycan-recognition proteins, which may provide clues for the maintenance of the symbiotic relationship, as well as three AMP genes whose involvement in the symbiotic relationship will require additional analysis. Finally, a search for AMP-like factors that could be involved in controlling the endosymbiont identified two orphan genes encoding proteins smaller than 200 amino acids underexpressed in quasi-aposymbionts, suggesting a role in the host-endosymbiont relationship.

AMPK activation modulates IL-36-induced inflammatory responses by regulating IκBζ expression in the skin.

The interleukin (IL)-36 pathway is a critical player in the pathogenesis of pustular psoriasis. However, therapies targeting this pathway are limited or unaffordable (e.g. the anti-IL-36 receptor antibody). AMP-activated protein kinase (AMPK), a regulator of cellular energy and metabolism, is known to participate in inflammatory diseases. However, its role in IL-36-induced skin inflammation remains unclear. Therefore, we sought to investigate the role of AMPK signals in regulating IL-36-induced responses in the skin. IL-36-stimulated primary normal human epidermal keratinocytes (NHEKs) and IL-36-injected (intradermally) BALB/c mice served as the cell and animal models, respectively. Additionally, 5-aminoimidazole-4-carboxamide riboside (AICAR) and A769662 served as AMPK activators. AICAR and A769662 significantly suppressed the IL-36-induced IL-8 (CXCL8) and CCL20 production from NHEKs. IL-36-induced IκBζ protein expression was prominently reduced and IKK/IκBα phosphorylation was attenuated by AICAR and A769662. Conversely, AMPKα knockdown increased IκBζ protein expression and IKK/IκBα phosphorylation in IL-36-treated NHEKs. Furthermore, AICAR and A769662 enhanced IL-36-induced-IκBζ protein degradation via the proteasome-dependent but not the lysosome-dependent pathway. Pretreatment of NHEKs with IL-36 slightly suppressed the AICAR- and A769662-triggered phosphorylation of AMPK and acetyl-CoA carboxylase. In the mouse model, topical application of AICAR significantly reduced ear swelling, redness, epidermal thickening, neutrophil infiltration and inflammatory and antimicrobial peptide gene expression. AMPK activation suppresses IL-36-induced IL-8 and CCL20 release by regulating IκBζ expression in keratinocytes and reduces IL-36-induced skin inflammation in mice, suggesting that AMPK activation is a potential strategy for treating patients with IL-36-mediated inflammatory skin disorders.

Normalizing granuloma vasculature and matrix improves drug delivery and reduces bacterial burden in tuberculosis-infected rabbits

Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients.

Effect of transporting Oreochromis niloticus in water with and without sodium chloride on skin morphology and some immunity-related genes expression.

The aim of this study was to examine the effects of salt addition on the skin gene expression of Mucin, Antimicrobial peptides, cortisol, and glucose in Oreochromis niloticus after 5-hour transportation in water. Three groups were compared: Control, post-transport without salt (PT-S), and post-transport with 5g salt-1(PT + S), with a stocking density of 28.6 gL-1, 20 fish for each experimental group. The results showed that the PT-S group had more significant changes in gene expression than the PT + S group, suggesting that salt alleviated the stress and immune responses of O. niloticus. The PT-S group had higher expression of mucin- 2(MUC + 2) (7.58 folds) and mucin-5AC (MUC5-AC) (6.29 folds) than the PT + S group (3.30 folds and 4.16 folds, respectively). The PT-S group also had lower expression of β-defensin-1 (Dβ1) (0.42 folds), β-defensin-2 (Dβ2) (0.29 folds), and Cath1 (0.16 folds) than the PT + S group (0.82 folds, 0.69 folds, and 0.75 folds, respectively). The skin morphology of the PT-S group revealed some white patches with no goblet cell openings, while the PT + S group had better preservation of skin features with some goblet cell openings and slight white patches. This study indicates that O. niloticus can benefit from sodium chloride during transportation, as it helps to reduce stress and inflammation, balance mineral levels, enhance health and immunity, and regulate mucous secretion.

An ELISA-based method for Galleria mellonella apolipophorin-III quantification.

Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns. Galleria mellonella is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The G. mellonella immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the G. mellonella immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with Escherichia coli, Candida albicans, Sporothrix schenckii, Sporothrix globosa, and Sporothrix brasiliensis. The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique.

Silencing gram-negative bacteria binding protein 1 decreases the immunity of Tribolium castaneum against bacteria

Gram-negative bacteria binding proteins (GNBPs) have the ability to recognize molecular patterns associated with microbial pathogens (PAMPs), leading to the activation of immune responses downstream. In the genome of Tribolium castaneum, three GNBP genes have been identified; however, their immunological roles remain unexplored. In our study, a GNBP1, designated as TcGNBP1, were identified from the cDNA library of T. castaneum. The coding sequence of TcGNBP1 consisted of 1137 bps and resulted in the synthesis of a protein comprising 378 amino acids. This protein encompasses a signal peptide, a low-complexity region, and a glycoside hydrolase 16 domain. TcGNBP1 was strongly expressed in early adult stages, and mainly distributed in hemolymph and gut. Upon being challenged with Escherichia coli or Staphylococcus aureus, the transcript levels of TcGNBP1 were significantly changed at different time points. Through molecular docking and ELISA analysis, it was observed that TcGNBP1 has the ability to interact with lipopolysaccharides, peptidoglycan, and β-1, 3-glucan. Based on these findings, it was further discovered that recombinant TcGNBP1 can directly bind to five different bacteria in a Ca2+-dependent manner. After knockdown of TcGNBP1 with RNA interference, expression of antimicrobial peptide genes and prophenoloxidase (proPO) activity were suppressed, the susceptibility of T. castaneum to E. coli or S. aureus infection was enhanced, leading to low survival rate. These results suggest a regulatory mechanism of TcGNBP1 in innate immunity of T. castaneum and provide a potential molecular target for dsRNA-based insect pest management.

Evidence that the loss of colonic anti-microbial peptides may promote dysbiotic Gram-negative inflammaging-associated bacteria in aging mice.

Introduction: Aging studies in humans and mice have played a key role in understanding the intestinal microbiome and an increased abundance of "inflammaging" Gram-negative (Gn) bacteria. The mechanisms underlying this inflammatory profile in the aging microbiome are unknown. We tested the hypothesis that an aging-related decrease in colonic crypt epithelial cell anti-microbial peptide (AMP) gene expression could promote colonic microbiome inflammatory Gn dysbiosis and inflammaging. Methods: As a model of aging, C57BL/6J mice fecal (colonic) microbiota (16S) and isolated colonic crypt epithelial cell gene expression (RNA-seq) were assessed at 2 months (mth) (human: 18years old; yo), 15mth (human: 50yo), and 25mth (human: 84yo). Informatics examined aging-related microbial compositions, differential colonic crypt epithelial cell gene expressions, and correlations between colonic bacteria and colonic crypt epithelial cell gene expressions. Results: Fecal microbiota exhibited significantly increased relative abundances of pro-inflammatory Gn bacteria with aging. Colonic crypt epithelial cell gene expression analysis showed significant age-related downregulation of key AMP genes that repress the growth of Gn bacteria. The aging-related decrease in AMP gene expressions is significantly correlated with an increased abundance in Gn bacteria (dysbiosis), loss of colonic barrier gene expression, and senescence- and inflammation-related gene expression. Conclusion: This study supports the proposed model that aging-related loss of colonic crypt epithelial cell AMP gene expression promotes increased relative abundances of Gn inflammaging-associated bacteria and gene expression markers of colonic inflammaging. These data may support new targets for aging-related therapies based on intestinal genes and microbiomes.

The interaction between lipid and vitamin D3 impacts lipid metabolism and innate immunity in Chinese mitten crabs Eriocheir sinensis

As a fat-soluble vitamin, vitamin D3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D3: low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D3 [1α,25(OH)2D3] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH)2D3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH)2D3, mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder.

Humoral immune response of Galleria mellonella after mono- and co-injection with Hypericum perforatum extract and Candida albicans.

Galleria mellonella is used as a model organism to study the innate immune response of insects. In this study, the humoral immune response was assessed by examining phenoloxidase activity, fungal burden, and the expression of phenoloxidase and antimicrobial peptide genes at different time point following separate and combined injections of Hypericum perforatum extract and a nonlethal dose of Candida albicans. The administration of a plant extract at low doses increased phenoloxidase activity, while higher doses had no effect. Similarly, co-injection of a low dose of the extract with the pathogen allowed half of the yeast cells to survive after 24 h. Co-injection of plant extract with the pathogen decreased the phenoloxidase activity at the end of 4 h compared to C. albicans mono-injection. The phenoloxidase gene expressions was reduced in all experimental conditions with respect to the control. When plant extracts and the pathogen were administered together, gallerimycin and hemolin gene expressions were considerably higher compared to mono-injections of plant extracts and the pathogen. The results of this study reveal that gene activation and regulatory mechanisms may change for each immune gene, and that recognition and signaling pathways may differ depending on the involved immunoregulator.

BmPGPR-L4 is a negative regulator of the humoral immune response in the silkworm Bombyx mori.

Toll, immune deficiency and prophenoloxidase cascade represent vital immune signaling pathways in insects. Peptidoglycan recognition proteins (PGRPs) are innate immune receptors that activate and regulate the immune signaling pathways. Previously, we reported that BmPGPR-L4 was induced in the silkworm Bombyx mori larvae by bacteria and peptidoglycan challenges. Here, we focused on the function of BmPGRP-L4 in regulating the expression of antimicrobial peptides (AMPs). The hemolymph from BmPGRP-L4-silenced larvae exhibited an enhanced inhibitory effect on the growth of Escherichia coli, either by growth curve or inhibitory zone experiments. Coincidentally, most of the AMP genes were upregulated after RNAi of BmPGRP-L4. Oral administration of heat-inactivated E. coli and Staphylococcus aureus after RNAi of BmPGRP-L4 resulted in the increased expression of BmPGRP-L4 in different tissues of the silkworm larvae, revealing an auto-regulatory mechanism. By contrast, the expression of most AMP genes was downregulated by oral bacterial administration after RNAi of BmPGRP-L4. The above results demonstrate that BmPGRP-L4 recognizes bacterial pathogen-associated molecular patterns and negatively regulates AMP expression to achieve immunological homeostasis. As a negative regulator, BmPGPR-L4 is proposed to be involved in the feedback regulation of the immune signaling pathways of the silkworm to prevent excessive activation of the immune response.

DEVELOPMENT OF A PERSONALIZED HUMAN PANETH CELL MODEL USING INTESTINAL ORGANOIDS

Abstract INTRODUCTION Paneth cells are a specialized intestinal epithelial cell subtype whose major function is to produce antimicrobial peptides (AMPs). Alterations in Paneth cell function are associated with Crohn’s disease (CD) but the confounding influences of environmental factors and/or genetic variations makes it difficult to identify the cause of this. Human intestinal organoid (HIO) technology now allows for the generation of intestinal epithelium from any individual but there are relatively few protocols that allow for the enrichment of Paneth cells within organoids. Our goal was to specifically enrich the Paneth cell population in HIOs derived from induced pluripotent stem cells (iPSCs). METHODS iPSCs from a control individual and 2 CD patients were directed to HIOs and then dissociated to generate purified populations of epithelial only-HIOs (eHIOs). eHIOs were passaged weekly in proliferation medium (EGF, Noggin and CHIR99021) and the γ-secretase inhibitor, DAPT, was added to direct towards a Paneth cell fate. Flow cytometry, qPCR and immunocytochemistry were used to determine Paneth cell numbers, and gene and protein expression of various AMPs respectively. RESULTS iPSC-derived eHIOs from the control individual, which were used to develop this enrichment protocol, could be maintained for at least 4 months in proliferation medium. Flow cytometry analysis of the Paneth marker lysozyme revealed that the population of Paneth cells in eHIOs significantly increased from ~1% in proliferation media to ~28% upon treatment with DAPT. qPCR analysis demonstrated that DAPT treatment significantly increased the expression of the Paneth cell associated genes DEFA5, DEFA6, ITLN2, REG3A and PLA2G2A. Immunocytochemistry revealed that DAPT treated eHIOs were enriched for cells possessing granulated lysozyme, and also for additional AMPs such as DEFA5, ITLN2 and REG3A. The presence of the bacterial sensors TLR2, TLR5 and NOD2 were also detected in our Paneth cells. Finally, to confirm the applicability to CD patients, we applied our protocol and found the population of Paneth cells in CD eHIOs significantly increased from ~1% in proliferation media to ~10% upon treatment with DAPT, had significantly increased expression of the Paneth cell related genes and were also were enriched for cells with granulated AMPs. CONCLUSION We have successfully developed a methodology to enrich Paneth cells in iPSC-derived HIOs from both healthy and CD patients. Given that iPSCs can be generated from donor cells stored in well characterized biorepositories or obtained from a small blood draw from any CD patient, this modeling system now opens up a new avenue of research by allowing an examination of how environmental factors (microbes/cytokines) and/or genetic variations influence human Paneth cell function in a personalized manner. Fluorescent image showing the presence of granulated DEFA5 (green) in E-cadherin+ (red) human iPSC-derived intestinal organoids treated with DAPT (X60). qPCR analysis of Paneth cell related genes in proliferation media compared to DAPT treatment. *P < .05, **P < .01, ***P < .001, and ****P < .0001 as compared to gene expression in proliferation media.

Toll receptor ligand Spätzle 4 responses to the highly pathogenic Enterococcus faecalis from Varroa mites in honeybees.

Honeybees play a major role in crop pollination, which supports the agricultural economy and international food supply. The colony health of honeybees is threatened by the parasitic mite Varroa destructor, which inflicts physical injury on the hosts and serves as the vector for variable viruses. Recently, it shows that V. destructor may also transmit bacteria through the feeding wound, yet it remains unclear whether the invading bacteria can exhibit pathogenicity to the honeybees. Here, we incidentally isolate Enterococcus faecalis, one of the most abundant bacteria in Varroa mites, from dead bees during our routine generation of microbiota-free bees in the lab. In vivo tests show that E. faecalis is only pathogenic in Apis mellifera but not in Apis cerana. The expression of antimicrobial peptide genes is elevated following infection in A. cerana. The gene-based molecular evolution analysis identifies positive selection of genes encoding Späetzle 4 (Spz4) in A. cerana, a signaling protein in the Toll pathway. The amino acid sites under positive selection are related to structural changes in Spz4 protein, suggesting improvement of immunity in A. cerana. The knock-down of Spz4 in A. cerana significantly reduces the survival rates under E. faecalis challenge and the expression of antimicrobial peptide genes. Our results indicate that bacteria associated with Varroa mites are pathogenic to adult bees, and the positively selected gene Spz4 in A. cerana is crucial in response to this mite-related pathogen.

Diversity of MrTolls and their regulation of antimicrobial peptides expression during Enterobacter cloacae infection in Macrobrachium rosenbergii

Toll/Toll-like receptor (TLR) is an important pattern recognition receptor that plays an important role in the immunity of animals. Six Toll genes were identified in Macrobrachium rosenbergii, namely, MrToll, MrToll1, MrToll2, MrToll3, MrToll4, and MrToll5. SMART analysis showed that all six Tolls have a transmembrane domain, a TIR domain, and different number of LRR domains. The phylogenetic tree showed that six Tolls were located in six different branches. Among these six Tolls, only MrToll4 contains the QHR motif, which is similar to insect Toll9. MrToll4 belongs to V-type/scc Toll with only one LRRCT domain. MrToll1 and MrToll5 are classical P-type/mcc Toll with two LRRCT domains and an LRRNT. MrTolls were distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. During the infection of Enterobacter cloacae, the expression level of MrToll and MrToll1-4 was upregulated in the intestine of M. rosenbergii. RNA interference experiments showed that the expression of most antimicrobial peptide (AMP) genes was negatively regulated by MrTolls during E. cloacae infection. On the contrary, crustin (Cru) 3 and Cru4 were inhibited after the knockdown of MrToll, and Cru1 and Cru4 were significantly downregulated with the knockdown of MrToll4 during E. cloacae challenge. These results suggest that MrTolls may be involved in the regulation of AMP expression in the intestine during E. cloacae infection.

Effects of the combination of Lactobacillus helveticus and isomalto-oligosaccharide on survival, gut microbiota, and immune function in Apis cerana worker bees.

The adult worker bees were fed sucrose syrup or sucrose syrup supplemented with Lactobacillus helveticus KM7, prebiotic isomalto-oligosaccharide (IMO), or L. helveticus KM7 combined with IMO. Survival rate, gut microbiota, and gene expression of gut antimicrobial peptides in worker honey bees were determined. Administration of L. helveticus KM7 and IMO significantly increased the survival rate in worker bees relative to bees fed sucrose only. Then, higher concentration of both lactic acid bacteria and Bifidobacterium in the gut and lower counts of gut fungi, Enterococcus, and Bacteroides-Porphyromonas-Prevotella were observed in bees fed the combination of KM7 and IMO compared with control bees. The combination of L. helveticus KM7 with IMO showed a greater or comparable modulating effect on those bacteria relative to either KM7 or IMO alone. Furthermore, the combination treatment of L. helveticus KM7 and IMO enhanced mRNA expression of antimicrobial peptide genes, including Abaecin, Defensin, and the gene encoding prophenoloxidase (PPO) in the gut compared with both control bees and those either L. helveticus KM7 or IMO alone. These results suggest that the combination of L. helveticus KM7 and IMO synergistically modifies the gut microbiota and immunity and consequently improves the survival rate of Apis cerana adult workers.

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.

The Development of New Methods to Stimulate the Production of Antimicrobial Peptides in the Larvae of the Black Soldier Fly Hermetia illucens.

(1) The global population is projected to reach a staggering 9.8 billion people by the year 2050, leading to major concerns about food security. The necessity to increase livestock production is inevitable. The black soldier fly (BSF) is known for its ability to consume a wide range of organic waste, and BSF larvae have already been used as a partial substitute for fishmeal. In contrast, the use of antibiotics in livestock feed for growth promotion and prophylaxis poses a severe threat to global health owing to antimicrobial resistance. Insect antimicrobial peptides (AMPs) have shown the potential to rapidly disrupt target bacterial membranes, making bacterial resistance to AMPs a less likely concern. (2) In this study, we explored various methods for stimulating AMP synthesis in BSF larvae and found that thermal injury effectively induced the production of various AMP types. Additionally, we investigated the activation of innate immune response pathways that lead to AMP production following thermal injury. (3) Interestingly, thermal injury treatment, although not involving bacteria, exhibited a similar response to that observed following Gram-positive bacterial infection in eliciting the expression of AMP genes. (4) Our findings offer support for the industrial use of BSF to enhance livestock production and promote environmental health.

Transcription factor STAT enhanced antimicrobial activities in Bombyx mori

STAT, a transcription factor in the JAK/STAT signaling pathway, regulates immune response to pathogens. In the silkworm (Bombyx mori), STAT exists as two split-forms, STAT-S and STAT-L. However, the role of STAT in silkworm immunity remains unclear. Our purpose was to investigate the effect of STAT on the expression of antimicrobial peptide (AMP) genes and resistance against pathogens. The expression levels of STAT-S and STAT-L were significantly up-regulated after induction by pathogenic microorganisms. In BmE cells, lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan stimulated STAT-S and STAT-L to transfer from the cytoplasm to the nucleus. We found that overexpression of STAT-S and STAT-L in cells could promote the expression of AMPs. We generated transgenic silkworm lines overexpressing STAT-L or STAT-S (OE-STAT-S; OE-STAT-L) or interfering with STAT (A4-dsSTAT). Overexpression of STAT-S and STAT-L upregulated the expression of AMP genes in the OE-STAT-S and OE-STAT-L, increased the survival rates of the OE-STAT-S silkworms and lowered the mortality of OE-STAT-L silkworms infected with S. aureus or Beauveria bassiana. By contrast, the death rate of A4-dsSTAT silkworms was higher after infection with these pathogenic microorganisms. These findings may provide insights into the role of STAT in the antimicrobial immune response of silkworms.

Basigin binds bacteria and activates Dorsal signaling to promote antibacterial defense in Penaeus vannamei

The NF-κB pathway plays an important role in immune regulation. Basigin, an immunoglobulin superfamily membrane protein, is involved in the activation of NF-κB. However, its role in NF-κB signaling in response to pathogen infection remains unclear. In this study, we identified the Basigin gene from Pacific white shrimp, Penaeus vannamei, a representative species for studying the innate immune system of invertebrates. Basigin promoted the degradation of the IκB homolog Cactus, facilitated the nuclear translocation of the NF-κB family member Dorsal, and positively regulated the expression of Dorsal pathway downstream antimicrobial peptide genes. Interestingly, recombinant Basigin protein could bind a variety of Gram-positive and Gram-negative bacteria. Silencing of Basigin inhibited the Dorsal signaling activated by V. parahaemolyticus infection and significantly decreased the survival rate of V. parahaemolyticus-infected shrimp. The expression levels of the antimicrobial peptides ALF1 and ALF2 were downregulated, and the phagocytosis of hemocytes was attenuated in Basigin-silenced shrimp. Similar results were observed in shrimp treated with a recombinant extracellular region of the Basigin protein that was able to compete with endogenous Basigin. Therefore, to the best of our knowledge, this study is the first to demonstrate the function of Basigin as a pathogen recognition receptor that activates NF-κB signaling for antibacterial immunity in shrimp.